阅读说明：本技术 稳定的vamp报告子检测 (Stable VAMP report son detection ) 是由 巴兹贝克·达夫列托夫 安德鲁·亚历山大·佩登 亚历山大·米卡埃尔·鲁道夫·鲁斯特 席亚拉·路易 于 2018-02-14 设计创作，主要内容包括：本申请提供了一种多肽,其包含具有荧光素酶活性的N-末端多肽结构域和具有VAMP1、VAMP2或VAMP3活性的C-末端多肽结构域,其中VAMP代表囊泡相关膜蛋白。还提供了相应的核酸分子、表达载体和遗传修饰的细胞。本发明还提供了这些的方法和用途。(This application provides a kind of polypeptides, it includes the N- terminal polypeptide structural domain with uciferase activity and have the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3, wherein VAMP represents vesicle-associated membrane albumen.Additionally provide the cell of corresponding nucleic acid molecules, expression vector and genetic modification.The present invention also provides these method and purposes.)

1. a kind of polypeptide, it includes the N- terminal polypeptide structural domain with uciferase activity and have VAMP1, VAMP2 or The active C- terminal polypeptide structural domain of VAMP3.

2. polypeptide according to claim 1, wherein have the active polypeptide domain of VAMP2 include SEQ ID NO:8 or The amino acid sequence of SEQ ID NO:2 or its conserved amino acid sequence variant.

3. polypeptide according to claim 1, wherein have the active polypeptide domain of VAMP1 include SEQ ID NO:7 or The amino acid sequence of SEQ ID NO:1 or its conserved amino acid sequence variant.

4. polypeptide according to claim 1, wherein have the active polypeptide domain of VAMP3 include SEQ ID NO:9 or The amino acid sequence of SEQ ID NO:3 or its conserved amino acid sequence variant.

5. according to polypeptide described in any preceding claims, wherein the polypeptide domain with uciferase activity includes SEQ The amino acid sequence of ID NO:4 or its conserved amino acid sequence variant.

6. a kind of nucleic acid molecules encode polypeptide according to any one of claim 1 to 5.

7. a kind of expression vector, it includes nucleic acid molecules according to claim 6.

8. a kind of cell of genetic modification, it includes nucleic acid molecules according to claim 6 or according to claim 7 Expression vector.

9. the cell of genetic modification according to claim 8, wherein the cell is selected from the group being made up of: SiMa mind Through blastoma cell, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, immortalized neuronal and primary Neuron.

10. polypeptide according to any one of the preceding claims, nucleic acid molecules, expression vector or gene modification cell use In the detection active purposes of neurotoxin, wherein the neurotoxin activity is selected from the group being made up of: tetanus Nervous toxicity Plain activity, Type B botulic neurotoxin activity, D type botulic neurotoxin activity, F type botulic neurotoxin are living Property, G type botulic neurotoxin activity, or any combination thereof.

11. purposes according to claim 10, wherein including drug products, foodstuff samples, clinical sample or environment sample Neurotoxin activity is detected in the test sample of product or any combination thereof.

12. purposes described in 0 or 11 according to claim 1, wherein comprising tetanus toxoid or Clostridium botulinum toxoid or Neurotoxin activity is detected in the test sample of a combination thereof.

13. purposes described in 0 or 11 according to claim 1, wherein including methods of preparing tetanus, Type B clostridium botulinum nerve Toxin, D type botulic neurotoxin, F type botulic neurotoxin, G type botulic neurotoxin or any combination thereof Test sample in detection neurotoxin activity.

14. a kind of active method of neurotoxin in detection test sample, which comprises

(a) cell of genetic modification according to claim 8 or claim 9, the conditions permit expression are cultivated under the following conditions Comprising the N- terminal polypeptide structural domain with uciferase activity and have the active end C- VAMP1, VAMP2 or VAMP3 more The polypeptide of peptide；

(b) cell of (a) is cultivated in the presence of test sample under the following conditions, the conditions permit includes to have fluorescein The nerve of the N- terminal polypeptide structural domain of enzymatic activity and the polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 The cracking of toxin-induced；With

(c) level for determining the cracking of the neurotoxin induction of the polypeptide, wherein splitting of inducing of the neurotoxin of the polypeptide The detection instruction neurotoxin activity of solution；

Wherein the neurotoxin activity is selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum nerve Neurotoxin active, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type botulic neurotoxin Activity, or any combination thereof.

15. according to the method for claim 14, wherein incubation step (a) and (b) are carried out simultaneously.

16. method according to claim 14 or 15, wherein provide the test sample in the medium.

17. method described in any one of 4 to 16 according to claim 1, wherein by ELISA, immunoblotting or living cells at Cracking as detecting the neurotoxin induction of the polypeptide.

18. method described in any one of 4 to 17 according to claim 1, wherein the test sample includes drug products, food Object sample, clinical sample or environmental sample or any combination thereof.

19. method described in any one of 4 to 18 according to claim 1, wherein the test sample includes tetanus toxoid, Or Clostridium botulinum toxoid, or combinations thereof.

20. method described in any one of 4 to 18 according to claim 1, wherein the test sample includes tetanus Nervous toxicity Element, Type B botulic neurotoxin, D type botulic neurotoxin, F type botulic neurotoxin, G type clostridium botulinum mind Through toxin or any combination thereof.

21. one kind is for detecting the active kit of neurotoxin, the kit includes:

(a) nucleic acid molecules of polypeptide are encoded, the polypeptide includes N- terminal polypeptide structural domain and tool with uciferase activity There is the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3；With

(b) for detecting the active positive control of neurotoxin, wherein the positive control can crack the nucleic acid molecules by (a) The polypeptide of coding.

22. kit according to claim 21, wherein the nucleic acid molecules are a part of expression vector.

23. the kit according to claim 21 or 22, wherein the nucleic acid molecules are in the intracellular of genetic modification.

24. kit according to claim 23, wherein the cell of the genetic modification is selected from the group being made up of: The SiMa neuroblastoma cell of genetic modification, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, forever Biochemical nerve member and primary neuron.

25. the kit according to any one of claim 21 to 24, wherein the kit also includes glimmering for detecting The reagent of light element enzymatic activity.

Background technique

Tetanus is the disease that the acute toxin as caused by clostridium tetani (Clostridium tetani) mediates.Having Under the anaerobic condition of benefit, such as in the wound of necrosis, this ubiquitous bacillus can produce tetanus toxin --- and one The very potent neurotoxin of kind.Methods of preparing tetanus (herein also referred to as " TeNT " or " TNx ") blocks central nervous system In inhibiting nerve transmitting, lead to characteristic muscle rigidity and spasm (1).Even if having modernization Intensive Care Therapy, case fatality rate Very high (up to 80%).

Methods of preparing tetanus and relevant botulic neurotoxin (BoNT) are large-scale Multidomain albumen, mainly by The neuron (1,5) in conjunction with they are with the interaction of neuronal specificity gangliosides and protein and with high affinity. This is combined is mediated by the binding structural domain of 50kDa, which separates in structure with effect part.It is combined in neuron Afterwards, neurotoxin is internalized by into interior endocytic vesicle, and due to acidification, they undergo conformation change in interior endocytic vesicle.This pH draws The structure change of hair make protease (also referred to as light chain) be discharged into proteolysis target where cytosol in.Specific egg Plain boiled water solution target will depend on specific neurotoxin, wherein methods of preparing tetanus, Type B botulic neurotoxin, D type meat Bacillus venenosus neurotoxin, F type botulic neurotoxin and G type botulic neurotoxin homolysis solution vesica related membrane protein (VAMP) 1,2 and 3 (although in different cracking sites).

VAMP1,2 and 3 be for neurotransmission it is required, their cracking leads to the blocking of neurotransmitter secretion, this can It can lead to dead (1,5).For example, the cracking of VAMP2 leads to the potent blocking of neurotransmitter regulator, it is nauseating that Neuromuscular then occurs Numbness.Unfortunately, the VAMP2 of cracking is degraded by mechanism in unknown neuron quickly, therefore almost impossible detects cracking VAMP2 molecule.

In people and the animal supervised, by with contain tetanus toxoid (a kind of TeNT of detoxification form) vaccine It is immunized and carrys out pre- antitetanus.Tetanus toxoid is prepared by TeNT, by, by its detoxification, being immunized without destroying it with formaldehyde Characteristic.The production of tetanus vaccine includes that the production of (i) TeNT, the chemical ablation of (ii) TeNT and (iii) detection are remaining TeNT activity (2).Production method makes toxoid that should not be restored to toxin.

In the production process of tetanus vaccine, the active detection of residual neurotoxin is essential.In decades, Conventional tool for assessing tetanus Product Safety has almost no change, and assessment still relies on rodent biometric Fixed, wherein lethal effect is related to neuromuscular paralysis.According to WHO guide, each new toxoid batch must in cavy or Residual TeNT paralysis Activity determination (2) is carried out in mouse.Animal safety detection is expensive, laborious and time-consuming (3,4).Even if After production of vaccine, will also the storage characteristic to tetanus vaccine carry out other rodent bioassay, can with determination The TeNT activity of energy reverses.In view of WHO announces its target recently: the complete anti-tetanus coverage rate of the mankind reaches 8,000,000,000, needs Will be new, advanced and more effective way produce and assess tetanus toxoid.

Although carrying out the general mankind (pan-human) immune target for having had reached WHO with tetanus toxoid, permitted More domestic animals have carried out routine immunization (6) not only for tetanus but also for D type clostridium botulinum.Clostridium botulinum vaccine is also It must be tested in the process of development, to determine whether there is any remaining neurotoxin activity.For tetanus vaccine, The safety of clostridium botulinum vaccine is assessed usually using rodent bioassay.

New animal is needed to substitute measurement to detect the residual neurotoxin of tetanus toxoid and Clostridium botulinum toxoid Activity.In addition, Type B botulic neurotoxin is a kind of drug ratified, need to detect its effect.Generally recognize For animal substitution measurement should illustrate all three steps of neurotoxin effect: cell surface combination, internalization and substrate (example Such as VAMP2) cracking.So far, two main problems hinder the development of this detection method based on cell: lacking can be with The fast degradation of the continuous cell line and pyrolysis product of methods of preparing tetanus and botulic neurotoxin firm connection (therefore product that neurotoxin effect can not be detected).

Summary of the invention

Inventor develops a kind of novel report molecule, and it includes the N- terminal polypeptide structural domains with uciferase activity With with the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3.This report molecule can be by tetanus Nervous toxicity Element, Type B botulic neurotoxin, D type botulic neurotoxin, F type botulic neurotoxin and/or G type meat poisoning bar Bacterium neurotoxin (and its modified forms, including retain the active chimera of neurotoxin) cracking.Report that the cracking of molecule generates Two kinds of pyrolysis products；First segment, it includes with uciferase activity N- terminal polypeptide structural domain and have VAMP1, A part of the active polypeptide domain of VAMP2 or VAMP3；With the second segment, it includes with VAMP1, VAMP2 or VAMP3 The remainder of active polypeptide domain., it is surprising that the N- terminal polypeptide structural domain with uciferase activity drops Intracellular degradation that is low or preventing the first segment.Advantageously, which thus provides for quantitative measurment nerve The new mechanism of neurotoxin active.

The present invention, the nucleic acid molecule encoding one has been illustrated by generating a kind of novel nucleic acid molecule in the present inventor The novel report molecule of kind, the report molecule includes the N- Terminal fluorescent element enzyme polypeptide connecting with the end C- VAMP2 polypeptide.The core Acid molecule is expressed in the SiMa Human Neuroblastoma Cell Line of genetic modification.By cell in suitable botulic neurotoxin Or it is cultivated in the presence of methods of preparing tetanus, and study the cracking of the neurotoxin induction of the report molecule.It is astonishing , since the degradation of pyrolysis product reduces, the cracking for the novel report molecule that can be induced with quantitative detection neurotoxin.

The function of VAMP1, VAMP2 and VAMP3, structure and neurotoxin cracking site are closely similar and highly conserved.Cause This, although using the novel report comprising the end C- VAMP2 polypeptide domain (that is, with the active polypeptide domain of VAMP2) The present invention has been illustrated in construct, but the present disclosure applies equally to wherein there is the active polypeptide domain of VAMP2 to be replaced For the reporter construct with VAMP1 activity or the active polypeptide domain of VAMP3.

The nucleic acid molecules and expression vector for encoding such polypeptide are also provided herein.Nucleic acid molecules and/or expression vector can To be present in any suitable host cell (that is, cell of genetic modification).

In one aspect, the present invention provides a kind of polypeptides, and it includes the N- terminal polypeptide knots with uciferase activity Structure domain and have the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3.

It suitably, include the amino acid of SEQ ID NO:8 or SEQ ID NO:2 with the active polypeptide domain of VAMP2 Sequence or its conserved amino acid sequence variant.

It suitably, include the amino acid of SEQ ID NO:7 or SEQ ID NO:1 with the active polypeptide domain of VAMP1 Sequence or its conserved amino acid sequence variant.

It suitably, include the amino acid of SEQ ID NO:9 or SEQ ID NO:3 with the active polypeptide domain of VAMP3 Sequence or its conserved amino acid sequence variant.

Suitably, the polypeptide domain with uciferase activity includes amino acid sequence or its guarantor of SEQ ID NO:4 Keep amino acid sequence variation.

In one aspect, the present invention provides a kind of nucleic acid molecules, encode polypeptide according to the present invention.

In one aspect, the present invention provides a kind of expression vectors, and it includes nucleic acid molecules according to the present invention.

In one aspect, the present invention provides a kind of cells of genetic modification, and it includes nucleic acid according to the present invention Molecule or expression vector according to the present invention.

Suitably, the cell of the genetic modification is selected from the group being made up of: SiMa neuroblastoma cell, LAN5 Neuroblastoma cell, NG108 neuroblastoma cell, immortalized neuronal and primary neuron.

In one aspect, the present invention provides polypeptide according to the present invention, nucleic acid molecules, expression vector or heredity to repair The cell of decorations is for detecting the active purposes of neurotoxin, wherein the neurotoxin activity is selected from the group being made up of: broken Catching cold, neurotoxin activity, Type B botulic neurotoxin are active, D type botulic neurotoxin is active, F type clostridium botulinum Neurotoxin activity and G type botulic neurotoxin activity, or any combination thereof.

Suitably, in the test specimens comprising drug products, foodstuff samples, clinical sample, environmental sample or any combination thereof Neurotoxin activity is detected in product.

Suitably, nerve is detected in the test sample comprising tetanus toxoid or Clostridium botulinum toxoid or combinations thereof Neurotoxin active.

Suitably, including methods of preparing tetanus, Type B botulic neurotoxin, D type botulic neurotoxin, F Detection neurotoxin is living in the test sample of type botulic neurotoxin or G type botulic neurotoxin or any combination thereof Property.Test sample may include naturally occurring (one or more) neurotoxins and/or its modification (such as it is manually modified, Including chimera) form.

In one aspect, the present invention provides a kind of active method of neurotoxin in detection test sample, the methods Include:

(a) cell of genetic modification according to the present invention, the conditions permit expression packet are cultivated under the following conditions Containing the N- terminal polypeptide structural domain with uciferase activity and there is the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 Polypeptide；

(b) cell of (a) is cultivated in the presence of test sample under the following conditions, the conditions permit includes with glimmering The N- terminal polypeptide structural domain of light element enzymatic activity and polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 The cracking of neurotoxin induction；With

(c) level for determining the cracking of the neurotoxin induction of the polypeptide, wherein the neurotoxin of the polypeptide induces Cracking detection instruction neurotoxin activity；

Wherein the neurotoxin activity is selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum Neurotoxin activity, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type clostridium botulinum nerve Neurotoxin active, or any combination thereof.

Suitably, incubation step (a) and (b) are carried out simultaneously.

Suitably, test sample is provided in the medium.

Suitably, it is split by what the neurotoxin of polypeptide described in ELISA, immunoblotting or living cells image checking induced Solution.

Suitably, test sample includes drug products, foodstuff samples, clinical sample, environmental sample or any combination thereof.

Suitably, test sample includes tetanus toxoid or Clostridium botulinum toxoid, or combinations thereof.

Suitably, test sample includes methods of preparing tetanus, Type B botulic neurotoxin, D type clostridium botulinum nerve Toxin, F type botulic neurotoxin or G type botulic neurotoxin or any combination thereof.Test sample may include natural (such as manually modified, including chimera) form of existing (one or more) neurotoxins and/or its modification.

In one aspect, the present invention provides one kind for detecting the active kit of neurotoxin, the kit packet It includes:

(a) nucleic acid molecules of polypeptide are encoded, the polypeptide includes the N- terminal polypeptide structural domain with uciferase activity With with the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3；With

(b) for detecting the active positive control of neurotoxin, wherein the positive control can crack the nucleic acid by (a) The polypeptide of molecule encoding.

Neurotoxin activity can be selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum Nervous toxicity Plain activity, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type botulic neurotoxin are living Property.

Suitably, nucleic acid molecules are a part of expression vector.

Suitably, nucleic acid molecules are in the intracellular of genetic modification.

Suitably, the cell of the genetic modification is selected from the group being made up of: the SiMa neuroblast of genetic modification Oncocyte, LAN5 neuroblastoma cell, NG108 neuroblastoma cell, immortalized neuronal and primary neuron.

Suitably, the kit also includes the reagent for detecting uciferase activity.

This specification throughout the specification and claims, word "comprising" and " containing " and its variant mean " including but not limited to ", and they are not intended to (and not) and exclude other parts, additive, component, entirety or step.

This specification throughout the specification and claims, unless the context otherwise requires, otherwise singular Include plural form.Particularly, using indefinite article, unless the context otherwise requires, otherwise specification should be by It is interpreted as considering multiple and single (singularity).

Feature, the entirety, feature, compound, chemistry described in conjunction with certain aspects of the present disclosure, embodiment or embodiment Part or group are interpreted as being suitable for any other aspect described herein, embodiment or embodiment, unless incompatible with it.

The patent that is mentioned above, Science and Technology document have been determined to be known obtained by those skilled in the art when submitting Know.The complete disclosure of disclosed patent, disclosure and pending patent application and other herein cited publications is logical It crosses and is incorporated herein by reference, degree is indicated specifically and individually and is incorporated by reference into as each.It is in office why not consistent feelings Under condition, it is subject to the disclosure.

Various aspects of the invention are detailed further below.

Detailed description of the invention

Embodiment of the present invention is further described hereinafter with reference to attached drawing, in which:

Fig. 1 shows figure A: green fluorescent protein (GFP)-VAMP2 construct is inserted into specified viral vectors With infection cell.Figure B: neuroblastoma cell shows strong GFP-VAMP2 expression after puromycin selection.It shows bright Field and GFP fluorescent image.Scheme C: after expressing the tetanus light chain with mCherry protein fusion (bottom right), being positioned at vesica knot The GFP-VAMP2 (top) of structure is releasably into cytosol (lower-left).Figure D: GFP antibody can be used and printed by routine immunization Mark detects the GFP-VAMP2 product of stable cracking.

Fig. 2 shows immortalized mouse neuron is sensitive to D type botulic neurotoxin, the GFP- such as by cracking What the appearance of VAMP2 product was proved.Please note that cracking (trace) and the GFP-VAMP2 of GFP-VAMP2 in toxin sensitive cells Cytosol shift (right figure).

Fig. 3 shows the fusion of VAMP2 and peroxidase APEX2 (APX2) and luciferase (NanoLuc) in disease It is expressed in the SiMa neuroblastoma cell of poison transduction in stable form；Two kinds of constructs carry HA label (for exempting from Epidemic disease trace, left figure).Fusion protein is positioned at cystic structures, such as revealed (right figure) using HA label immunostaining.

Fig. 4 shows the engineered SiMa neuroblastoma cell to carry NanoLuc-VAMP2 report molecule It shows to crack after being handled 48 hours with 10nM Type B and D type botulic neurotoxin.APEX2-VAMP reports molecule pair Clostridium botulinum cracking is resistant.The appearance of the VAMP2 product of cracking is had recorded by immunoblotting using total VAMP2 antibody. Compared with the product of Type B clostridium botulinum cracking, the considerably more rapid migration of the product of D type clostridium botulinum cracking, this and VAMP exist The position Lys59 rather than Gln76 cracking is consistent.

Fig. 5 is shown with the immunoblotting after BoNT B and D (nM) titration, sensibility of the announcement SiMa cell to BoNT/B Higher than BoNT/D.Using anti-VAMP2 antibody detection display BoNT/B cracking down to 1nM, the cracking of BoNT/D is down to 10nM. Detection is carried out using the VAMP2 antibody of the BoNT/B of the high degree of specificity VAMP2 cracked and BoNT/D cracking to show, BoNT/B's Cracking is down to 300pM, and the cracking of BoNT/D is down to 10nM.It is generated respectively for peptide ALQAGASQ and peptide KVLERDQK anti- The VAMP2 antibody of VAMP2 and anti-the BoNT/D cracking of BoNT/B cracking.

Fig. 6 shows the remolding sensitivity between immunoblotting and NanoLuc ELISA measurement compared with having between two kinds of technologies Similar EC₅₀.But NanoLuc measurement shows higher sensitivity, detects the energy down to the BoNT/B cracking VAMP of 30pM Power is higher than baseline (right figure).NanoLuc ELISA measurement is related to the NanoLuc-VAMP product for cracking clostridium botulinum capture and exists On microplate, the microplate carries the antibody of the VAMP2 of the BoNT cracking of high specific, then reads and shines on microplate luminometer Reading.

Fig. 7 shows immunoblotting, NanoLuc measurement is used equally for cracking of the detection tetanus toxin to VAMP2, but It is to be lower than BoNT/B and BoNT/D in the sensibility of SiMa neuroblastoma cell.

Fig. 8 provides the amino acid sequence (SEQ ID NO:1) of people VAMP1.The amino acid 40 to 118 of people VAMP1 indicates Underscore (SEQ ID NO:7).

Fig. 9 provides the amino acid sequence (SEQ ID NO:2) of people VAMP2.The amino acid 38 to 116 of people VAMP2 indicates Underscore (SEQ ID NO:8).

Figure 10 provides the amino acid sequence (SEQ ID NO:3) of people VAMP3.The amino acid 21 to 100 of people VAMP3 indicates Underscore (SEQ ID NO:9).

Figure 11 provides the amino acid sequence (SEQ ID NO:4) of NanoLuc.

Figure 12 provides the nucleic acid sequence (SEQ ID NO:5) of HA-APEX-VAMP2 construct.

Figure 13 provides the nucleic acid sequence (SEQ ID NO:6) of HA-NanoLuc-VAMP2.

The new synthesized form that the cell that Figure 14 shows expression Nanoluc-VAMP2 can be used for distinguishing BoNT/B (herein will It is named as I-IV) effect.Behind BoNT/B preparation I-IV 65 hours of application specified range (nM), immunoblotting is shown Different degrees of Nanoluc-VAMP2 cracking out.Use the antibody of the VAMP2 of cracking (it is generated for peptide ALQAGASQ) It is detected.Anti- SNAP25 antibody is used as loading control.

Figure 15 shows amino acid 40 to 118 (SEQ ID NO:7) (referred to herein as core of VAMP1 of people VAMP1 Heart amino acid sequence).

Figure 16 shows amino acid 38 to 116 (SEQ ID NO:8) (referred to herein as core of VAMP2 of people VAMP2 Heart amino acid sequence).

Figure 17 shows the amino acid 21 to 100 of people VAMP3 (SEQ ID NO:9) (the referred to herein as cores of VAMP3 Heart amino acid sequence).

Specific embodiment

In decades, being strongly desired always to use active to tetanus and/or botulic neurotoxin can own The in vitro test that committed step carries out sensitive assessment substitutes cumbersome animal experiment.Theory thinks that continuous cell line lacks exploitation Sensibility necessary to the measurement of mouse bioassay can be substituted, but simultaneously, using primary neuron or be originated from embryonic cell Neuron be faced with itself challenge because they must be fresh from animal tissue obtain or they need complexity Operating process and several weeks/several months could break up completely.Use SiMa Human Neuroblastoma Cell Line measurement A type clostridium botulinum mind First efficacy assays based on cell of the SNAP25 lytic activity through toxin are better than (hole EC50~1U/) mouse biometric Determine method (11).However, being developed a kind of for measuring tetanus and/or clostridium botulinum due to the fast degradation of VAMP pyrolysis product The equivalent measuring method of the VAMP lytic activity of neurotoxin is challenging.

Inventor have now been developed a kind of novel report molecule, and it includes the N- terminal polypeptide knots with uciferase activity Structure domain and have the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3.Report that molecule can be by tetanus Nervous toxicity Element, Type B botulic neurotoxin, D type botulic neurotoxin, F type botulic neurotoxin and/or G type meat poisoning bar Bacterium neurotoxin (and its modified forms, including retain the active chimera of neurotoxin) cracking.Report that the cracking of molecule generates Two kinds of pyrolysis products；First segment, it includes with uciferase activity N- terminal polypeptide structural domain and have VAMP1, A part of the active polypeptide domain of VAMP2 or VAMP3；With the second segment, it includes with VAMP1, VAMP2 or VAMP3 The remainder of active polypeptide domain., it is surprising that the N- terminal polypeptide structural domain with uciferase activity drops Degradation that is low or preventing the first segment.Advantageously, which thus provides living for quantitative measurment neurotoxin The new mechanism of property.It is expected that using this novel report molecule (for example, using immortalized neuronal cell line based on cell In measuring method) production of clostridium botulinum vaccine and tetanus vaccine can be accelerated, and promote therapeutic botulinum toxin product The diagnostic assays of detection and clostridium botulinum.

Another example of practicability through the invention shows that the cell for expressing Nanoluc-VAMP2 can be used for distinguishing The effect of the new synthesized form of BoNT/B (referring to Figure 14).

Polypeptide

Provide a kind of polypeptide, it includes with uciferase activity N- terminal polypeptide structural domain and have VAMP1, The active C- terminal polypeptide structural domain of VAMP2 or VAMP3.

Term " peptide ", " protein " and " polypeptide " is used interchangeably herein.

Term polypeptide " structural domain " refers to a part of polypeptide sequence, can independently of polypeptide chain rest part into Change, function and exists.In general, each structural domain in polypeptide can form compact three-dimensional structure, and usually can be with Independently stable and folding.The example of polypeptide " structural domain " in present disclosure be the structural domain with uciferase activity, With the active structural domain of VAMP1, with the active structural domain of VAMP2 or with the active structural domain of VAMP3.

The end N- (N-terminus) (also referred to as amino terminal, NH of protein₂End, the end N- (N-terminal End) or amine end) be with free amino (- NH₂) amino acid terminate protein or polypeptide starting point.According to Convention, peptide sequence are write from the end N- to the end C- (from left to right).The end C- (C-terminus) (also referred to as carboxyl terminal (carboxyl-terminus), carboxyl terminal (carboxy-terminus), C- terminal tail (C-terminal tail), C- End (C-terminal end) or the end COOH-) it is that (protein is more with the amino acid chain of free carboxy (- COOH) termination Peptide) end.

As used herein, term " end N- " and " end C- " are for describing such as opposite position of the structural domain in polypeptide It sets.Therefore, the end the N- end ratio distance C- of the positional distance polypeptide of " end N- " structural domain is closer (in contrast).On the contrary, C- end ratio distance N- end of the position (in contrast) of " end C- " structural domain apart from polypeptide is closer.As used herein, art Language " position (positioned) " refers to site of such as structural domain in the linear amino acid sequence of polypeptide.

Term " end N- " and " end C- " can be used for describing relative position of two or more structural domains in polypeptide. Herein, position ratio " end the C- " structural domain of " end N- " structural domain is closer to the end N- of (in contrast) polypeptide.Phase Instead, position ratio " end the N- " structural domain of " end C- " structural domain is closer to the end C- of (in contrast) polypeptide.

" end N- " structural domain can with but not necessarily have to be located at polypeptide the end N- (that is, it can with but it is different Surely it must be positioned at the starting point of the polypeptide terminated with the amino acid with free amino).In other words, the of N- terminal domains One amino acid needs not be first amino acid of (but can be) polypeptide.It means that the end N- and " N- in polypeptide There may be other amino acid, polypeptide domains (such as label, such as HA label) etc. between the starting point of end " structural domain As long as (end the N- end ratio distance C- of the positional distance polypeptide of the structural domain is closer；Alternatively, when being used to describe two or more When the relative position of a structural domain, as long as position ratio " end the C- " structural domain of the structural domain is closer to the end N-).

Equally, " end C- " structural domain can with but not necessarily have to be located at polypeptide the end C- (that is, it can with but Not necessarily have to be located at the end of the polypeptide terminated with the arbitrary amino acid with free carboxy).In other words, C- end structure The last one amino acid in domain needs not be the last one amino acid of (but can be) polypeptide.It means that in polypeptide Other amino acid, polypeptide domain etc. (such as label) is likely present between the end C- and the end of " end C- " structural domain As long as (end the C- end ratio distance N- of the positional distance polypeptide of the structural domain is closer；Alternatively, when being used to describe two or more When the relative position of a structural domain, as long as position ratio " end the N- " structural domain of the structural domain is closer to the end C-).

Polypeptide comprising N- terminal polypeptide structural domain (A) and C- terminal polypeptide structural domain (B) is usually written as A-B, i.e., from The end N- to the end C- (from left to right).For example, it is tied comprising the end N- enzyme luciferase (such as NanoLuc or " Nluc ") The polypeptide of structure domain and the end C- VAMP2 structural domain, which is usually write, is Nluc-VAMP2 (or NlucVAMP2).

For example, polypeptide of the invention can include HA in the end N- of the polypeptide domain with uciferase activity Label.Other suitable labels are well known in the art, and can additionally or alternatively be used.

In the structural domain with uciferase activity and have between the active structural domain of VAMP1, VAMP2 or VAMP3 also There may be connector (linker), and proline glycine linlcers can be used for example.Other suitable connectors are in the art It is well known that and can additionally or alternatively use.

Polypeptide provided herein includes the N- terminal polypeptide structural domain with enzymatic uciferase activity.

Polypeptide domain with " uciferase activity " refers to the polypeptide structure for remaining the functional activity of luciferase Domain, that is, its can by oxidation photon transmitting substrate (such as fluorescein and furimazine) generate bioluminescence [ACS Chem Biol.2012Nov 16；7(11):1848–1857‘Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate ' Hall et al.].Such as this paper institute With, the polypeptide with " luciferase activity " include from luciferase classification oxidizing ferment (its by oxyluciferin or Furimazine and generate bioluminescence) any polypeptide (i.e. comprising any functional luciferase).Those skilled in the art Member readily appreciates that how to identify the polypeptide domain with uciferase activity using routine experiment known in the art. [Beyond D-luciferin:Expanding the Scope of Bioluminescence Imaging in vivo.Spencer T.Adams,Jr.,Stephen C.Miller Curr Opin Chem Biol.2014；0:112–120] In summarize and be suitable for identifying the experiment of functional luciferase.

In one embodiment, the polypeptide domain with luciferase activity includes shown in SEQ ID NO:4 Amino acid sequence or its functional variety (or function fragment).Such variant can be the naturally occurring function of SEQ ID NO:4 The functional variety of variant (for example, allele functional variety), the functional variety of synthesis or Improved synthesis.Term " variant " is also contained Lid homologue.

Functional variety usually only comprising SEQ ID NO:4 one or more amino acid conservative substitution or albumen it is non- Substitution, missing or the insertion of non-critical amino-acid in key area.Therefore, the functional variety of SEQ ID NO:4 can be SEQ The conserved amino acid sequence variant of ID NO:4, wherein the variant has luciferase activity.

Non-functional variant is the amino acid sequence variation of the SEQ ID NO:4 without luciferase activity.Non-functional change Body usually contains non-conservative substitutions, missing or the insertion of the amino acid sequence of SEQ ID NO:4 or truncates (premature too early ) or the substitution of key amino acid or key area, insertion or missing truncation.For identifying that functional and non-functional becomes The method of body (such as functional and non-functional allelic variant) is well known to those of ordinary skill in the art.

[Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.Hall MP,Unch J,Binkowski BF,Valley MP, Butler BL,Wood MG,Otto P,Zimmerman K,Vidugiris G,Machleidt T,Robers MB,Benink HA,Eggers CT,Slater MR,Meisenheimer PL,Klaubert DH,Fan F,Encell LP,Wood KV.ACS Chem Biol.2012,7 (11): 1848-57] it is provided in crucial and non-critical amino-acid total in luciferase Knot.Therefore, will easily identify can be with substituted amino acid to provide SEQ ID NO:4's by those skilled in the art Functional variety (or function fragment), such as conserved amino acid sequence variant.Those of ordinary skill in the art can also use standard Alignment programs are readily determined the homologue of SEQ ID NO:4.

Polypeptide with uciferase activity may include and the amino acid sequence of SEQ ID NO:4 or part thereof or segment With at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the amino acid sequence of 98%, 99% or 100% identity.Suitably, homogeneity percentage may be calculated and refer to sequence Arrange the percentage of the identity of the whole length of (such as SEQ ID NO:4) or part thereof or segment.

Amino acid sequence shown in SEQ ID NO:4 is the amino acid of luciferase NanoLuc (being sold by Promega) Sequence.Term " NanoLuc " and " NLuc " are used interchangeably herein.More details about NanoLuc luciferase can See Hall et al., page 2012,7,1848 to page 1857 of ACS chem Biol.

The alternate example of suitable luciferase includes the light of firefly luciferin from firefly Photinus pyralis Enzyme (FLuc；EC 1.13.12.7).Fluc is a kind of globulin, can use ATP and molecular oxygen to be catalyzed The oxygenation of luciferin, to generate oxyluciferin (oxyluciferin), this is a kind of highly unstable singlet Compound is excited, can be shone in relaxation to ground state.Various other biologies use different luciferins in various luminescence-producing reactions Enzyme generates (such as pumpkin lamp mushroom Omphalotus olearius, several marine organisms such as sea pansy (sea to adjust its light Pansy) (Renilla reniformis has 2 monooxygenase (Renilla-luciferin of luciferase sea pansy fluorescein 2-monooxygenase)；RLuc) and the luciferase of Flagellatae).Other examples of luciferase include the light of firefly of modification Firefly luciferase (Ultra-Glo；From Photuris pennysylvanica), click beetle luciferase (CBLuc；It is originated from Pyrophorus plagiophthalamus), copepod luciferase (GLuc；From Gaussia ) and Ostracoda crustacean luciferase (CLuc princeps；From Cypridina noctiluca).In this field often Visible Thorne of the summary of different luciferases et al., Chem Biol.2010Jun 25；17(6)；646-657.

Polypeptide provided herein also includes with the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3.

As used herein, " with VAMP1, VAMP2 or VAMP3 activity " polypeptide domain be refer to as VAMP1, The polypeptide structure portion that VAMP2 or VAMP3 albumen works.

VAMP covers the protein families characterized by the end C- conformity membrane (integral membrane) structural domain.N- End (amino acid 1-90 or more depends on isotype and species) includes SNARE motif towards cytosol, is used for It interacts with SNAP-25 and syntaxin (syntaxin).All VAMP are involved in film fusion process.VAMP passes through it The end N- chain and syntaxin and SNAP-25 family member interact, and form fusion core or SNARE compound, make For the necessary condition of exocytosis.When analyzing VAMP knock-out animal model, it has been demonstrated that VAMP is to the basic of Vesicle fusion Effect.

VAMP1 and VAMP2 is referred to as Small Synaptic Vesicles albumen (synaptobrevin), and in brain, spinal cord and periphery mind Through being expressed in member.They are the constituents of synaptic versicle, they participate in the release of neurotransmitter in synaptic versicle.VAMP3 (also referred to as cellule vacuolar protein (cellubrevin)) is generally expressed, and the composition as secretory granules and Secretory vesicles Ingredient participates in adjustment type exocytosis and composing type exocytosis [Nature Reviews Molecular Cell Biology 2,98-106(February 2001)‘SNARE-mediated membrane fusion’Y.A.Chen& R.H.Scheller]。

VAMP1, VAMP2 and VAMP3 are methods of preparing tetanus (TeNT), Type B botulic neurotoxin (BoNT/ B), D type botulic neurotoxin (BoNT/D), F type botulic neurotoxin (BoNT/F) and G type clostridium botulinum Nervous toxicity The known substrate of plain (BoNT/G).Neurotoxin cracks the bottom VAMP1, VAMP2 and VAMP3 at conservative cracking site sequence Object, as shown in table 1 below.

Table 1

Polypeptide domain with " VAMP1 activity " refers to a kind of polypeptide domain, (i) remains the function of VAMP1 Activity, that is to say, that it is present in vesica, and is capable of forming SNARE compound, and (ii) can be by tetanus nerve Toxin (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type meat poisoning bar At least one of bacterium neurotoxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, at least three Kind, at least four or at least all cracking.

Those skilled in the art readily appreciate that how to identify with vesica correlation using routine experiment known in the art The active polypeptide domain of VAMP1.[Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly.Fasshauer D, Eliason WK,Brünger AT,Jahn R.Biochemistry.1998Jul 21；37 (29): 10354-62.] and [Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion.Hu K,Carroll J,Fedorovich S,Rickman C,Sukhodub A,Davletov B.Nature.2002Feb 7；415 (6872): 646-50] in summarize and be suitable for identifying the experiment of functionality VAMP1.

Those skilled in the art also easily know how that identification can be broken using routine experiment known in the art Wind neurotoxin (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type At least one of botulic neurotoxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, extremely Few three kinds, at least four or the polypeptide domain that at least all cracks.[Specificity of botulinum protease for human VAMP family proteins.Yamamoto H, et al. Microbiol Immunol, 2012Apr.PMID 22289120] experiment for being suitable for identifying the polypeptide domain that can be cleaved as described above is summarized in.In addition, being mentioned in table 1 The abundant guidance of the appropriate cracking site sequence of VAMP1 is supplied.There are one (or multiple), these are conservative in polypeptide domain Cracking site sequence may also indicate that it can be by methods of preparing tetanus (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type botulic neurotoxin (BoNT/F) and/or G type meat poisoning Bacillus neurotoxin (BoNT/G) cracks in due course.

In one embodiment, having the active polypeptide domain of VAMP1 includes amino shown in SEQ ID NO:1 Acid sequence or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety of SEQ ID NO:1 The functional variety of (for example, allele functional variety), the functional variety of synthesis or Improved synthesis.Term " variant " also covers together Source object.

In one embodiment, with the active polypeptide domain of VAMP1 include SEQ ID NO:1 amino acid 40 to 118 or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety (example of SEQ ID NO:1 Such as, allele become functional variety), synthesis functional variety or Improved synthesis functional variety.Term " variant " also covers together Source object.

The amino acid 40 to 118 of SEQ ID NO:1 represents the core VAMP1 amino acid for interacting and cracking for BoNT (amino acid 40 to 118 is referred to herein as SEQ ID NO:7 to sequence, and is shown in Fig. 8 (only marking the amino acid of underscore) In Figure 15).VAMP1 amino acid sequence, BoNT interaction and cracking site be it is well known in the art (see, for example, Yamamoto H,Ida T,Tsutsuki H,Mori M,Matsumoto T,Kohda T,Mukamoto M,Goshima N, Kozaki S,Ihara H.Microbiol Immunol.2012Apr；56(4):245-53).Therefore, those skilled in the art It can also be readily determined with other active suitable polypeptide domains of VAMP1.

Functional variety usually only comprising SEQ ID NO:1 one or more amino acid conservative substitution or albumen it is non- Substitution, missing or the insertion of non-critical amino-acid in key area.Therefore, the functional variety of SEQ ID NO:1 can be SEQ The conserved amino acid sequence variant of ID NO:1, wherein the variant has VAMP1 activity.

Non-functional variant is the amino acid sequence variation without the active SEQ ID NO:1 of VAMP1.Non-functional variant is logical Non-conservative substitutions, missing or the insertion of amino acid sequence often containing SEQ ID NO:1 truncate (premature too early ) or the substitution of key amino acid or key area, insertion or missing truncation.For identifying that functional and non-functional becomes The method of body (such as functional and non-functional allelic variant) is well known to those of ordinary skill in the art.

[Proc Natl Acad Sci U S A.1998Dec 22；95(26):15781-6.Conserved structural features of the synaptic fusion complex:SNARE proteins Reclassified as Q-and R-SNAREs.Fasshauer D, Sutton RB, Brunger AT, Jahn R] in provide Crucial and non-critical amino-acid general introduction in VAMP1.Therefore, will easily identify can be by by those skilled in the art Substituted amino acid is to provide the functional variety (or function fragment) of SEQ ID NO:1, such as conserved amino acid sequence variant.This Field those of ordinary skill can also easily identify the homologue of SEQ ID NO:1 using standard sequence alignment programs.

It may include having with the amino acid sequence of SEQ ID NO:1 or part thereof or segment with the active polypeptide of VAMP1 Have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the amino acid sequence of 98%, 99% or 100% identity.Suitably, homogeneity percentage may be calculated and refer to sequence Arrange the percentage of the identity of the whole length of (such as SEQ ID NO:1) or part thereof or segment.

Amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of naturally occurring people VAMP1.About people Visible [the Specificity of botulinum protease for human VAMP family of the more details of VAMP1 Proteins.Yamamoto H, et al. Microbiol Immunol, 2012Apr.PMID22289120].

Polypeptide domain with " VAMP2 activity " refers to a kind of polypeptide domain, (i) remains the function of VAMP2 Activity, that is, it is present in vesica, and is capable of forming SNARE compound, and (ii) can be by methods of preparing tetanus (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type clostridium botulinum mind Through at least one of toxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, at least three kinds, extremely Few four kinds or at least all cracking.

Those skilled in the art readily appreciate that how to identify with vesica correlation using routine experiment known in the art The active polypeptide domain of VAMP2.J Biol Chem.1999May 28；274(22):15440-6.Mixed and non- cognate SNARE complexes.Characterization of assembly and biophysical Properties.Fasshauer D, Antonin W, Margittai M, Pabst S, Jahn R.] and [Nature.2002.Feb 7；415(6872):646-50.Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion.Hu K,Carroll J,Fedorovich S, Rickman C, Sukhodub A, Daveltov B.] in summarize and be suitable for identifying the experiment of functionality VAMP2.

Those skilled in the art also easily know how that identification can be broken using routine experiment known in the art Wind neurotoxin (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type At least one of botulic neurotoxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, extremely Few three kinds, at least four or the polypeptide domain that at least all cracks.[Specificity of botulinum protease For human VAMP family proteins.Yamamoto H, et al. Microbiol Immunol, 2012Apr.PMID 22289120] Suitable assays for identifying the polypeptide domain that can be cleaved as described above are summarized in.In addition, in table 1 Provide the abundant guidance of the appropriate cracking site sequence of VAMP2.There are one (or multiple) these guarantors in polypeptide domain The cracking site sequence kept may also indicate that it can be by methods of preparing tetanus (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type botulic neurotoxin (BoNT/F) and/or G type meat poisoning Bacillus neurotoxin (BoNT/G) (depending on the circumstances) cracking.

In one embodiment, having the active polypeptide domain of VAMP2 includes amino shown in SEQ ID NO:2 Acid sequence or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety of SEQ ID NO:2 The functional variety of (for example, allele functional variety), the functional variety of synthesis or Improved synthesis.Term " variant " also covers together Source object.

In one embodiment, with the active polypeptide domain of VAMP2 include SEQ ID NO:2 amino acid 38 to 116 or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety (example of SEQ ID NO:2 Such as, allele functional variety), synthesis functional variety or Improved synthesis functional variety.Term " variant " also covers homologous Object.

The amino acid 38 to 116 of SEQ ID NO:2 represents the core VAMP2 amino acid for interacting and cracking for BoNT (amino acid 38 to 116 is referred to herein as SEQ ID NO:8 to sequence, and is shown in Fig. 9 (only marking the amino acid of underscore) In Figure 16).VAMP2 amino acid sequence, BoNT interaction and cracking site be it is well known in the art (see, for example, Yamamoto H,Ida T,Tsutsuki H,Mori M,Matsumoto T,Kohda T,Mukamoto M,Goshima N, Kozaki S,Ihara H.Microbiol Immunol.2012Apr；56(4):245-53).Therefore, those skilled in the art It can also be readily determined with other active suitable polypeptide domains of VAMP2.

Functional variety usually only comprising SEQ ID NO:2 one or more amino acid conservative substitution or albumen it is non- Substitution, missing or the insertion of non-critical amino-acid in key area.Therefore, the functional variety of SEQ ID NO:2 can be SEQ The conserved amino acid sequence variant of ID NO:2, wherein the variant has VAMP2 activity.

Non-functional variant is the amino acid sequence variation without the active SEQ ID NO:2 of VAMP2.Non-functional variant is logical Non-conservative substitutions, missing or the insertion of amino acid sequence often containing SEQ ID NO:2 truncate (premature too early ) or the substitution of key amino acid or key area, insertion or missing truncation.For identifying that functional and non-functional becomes The method of body (such as functional and non-functional allelic variant) is well known to those of ordinary skill in the art.

[Proc Natl Acad Sci U S A.1998Dec 22；95(26):15781-6.Conserved structural features of the synaptic fusion complex:SNARE proteins reclassified as Q-and R-SNAREs.Fasshauer D,Sutton RB,Brunger AT,Jahn R] and [Proc Natl Acad Sci U S A.2006May 30；103(22):8378-83.Conformation of the Synaptobrevin transmembrane domain.Bowen M, Brunger AT.] in provide vesica correlation VAMP2 Middle crucial and non-critical amino-acid summary.Therefore, will easily identify can be substituted by those skilled in the art Amino acid is to provide the functional variety (or function fragment) of SEQ ID NO:2, such as conserved amino acid sequence variant.This field is general Logical technical staff can also easily identify the homologue of SEQ ID NO:2 using standard sequence alignment programs.

It may include having with the amino acid sequence of SEQ ID NO:2 or part thereof or segment with the active polypeptide of VAMP2 Have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the amino acid sequence of 98%, 99% or 100% identity.Suitably, homogeneity percentage may be calculated and refer to sequence Arrange the percentage of the identity of the whole length of (such as SEQ ID NO:2) or part thereof or segment.

Amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence of naturally occurring people VAMP2.About people Visible [the Substrate recognition of VAMP-2by botulinum neurotoxin B of the more details of VAMP2 And tetanus neurotoxin.Chen S, et al. J Biol Chem, 2008Jul 25.PMID 18511417].

Polypeptide domain with " VAMP3 activity " refers to a kind of polypeptide domain, (i) remains the function of VAMP3 Activity, that is to say, that it is present in vesica, and is capable of forming SNARE compound, and (ii) can be by tetanus nerve Toxin (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type meat poisoning bar At least one of bacterium neurotoxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, at least three Kind, at least four or at least all cracking.

Those skilled in the art readily appreciate that how to identify with VAMP3 activity using routine experiment known in the art Polypeptide domain.[J Biol Chem.1999May 28；274(22):15440-6.Mixed and non-cognate SNARE complexes.Characterization of assembly and biophysical Properties.Fasshauer D, Antonin W, Margittai M, Pabst S, Jahn R.] in summarize be suitable for mirror The experiment of fixed functionality VAMP3.

Those skilled in the art also easily know how that identification can be broken using routine experiment known in the art Wind neurotoxin (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type At least one of botulic neurotoxin (BoNT/F) and G type botulic neurotoxin (BoNT/G), at least two, extremely Few three kinds, at least four or the polypeptide domain that at least all cracks.[Specificity of botulinum protease for human VAMP family proteins.Yamamoto H, et al. Microbiol Immunol, 2012Apr.PMID 22289120] experiment for being suitable for identifying the polypeptide domain that can be cleaved as described above is summarized in.In addition, being mentioned in table 1 The abundant guidance of the appropriate cracking site sequence of VAMP3 is supplied.There are one (or multiple), these are conservative in polypeptide domain Cracking site sequence may also indicate that it can be by methods of preparing tetanus (TeNT), Type B botulic neurotoxin (BoNT/B), D type botulic neurotoxin (BoNT/D), F type botulic neurotoxin (BoNT/F) and/or G type meat poisoning Bacillus neurotoxin (BoNT/G) (depending on the circumstances) cracking.

In one embodiment, having the active polypeptide domain of VAMP3 includes amino shown in SEQ ID NO:3 Acid sequence or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety of SEQ ID NO:3 The functional variety of (for example, allele functional variety), the functional variety of synthesis or Improved synthesis.Term " variant " also covers together Source object.

In one embodiment, with the active polypeptide domain of VAMP3 include SEQ ID NO:3 amino acid 21 to 100 or its functional variety (or function fragment).Such variant can be the naturally occurring functional variety (example of SEQ ID NO:3 Such as, allele functional variety), synthesis functional variety or Improved synthesis functional variety.Term " variant " also covers homologous Object.

The amino acid 21 to 100 of SEQ ID NO:3 represents the core VAMP3 amino acid for interacting and cracking for BoNT (amino acid 21 to 100 is referred to herein as SEQ ID NO:9 to sequence, and is shown in Figure 10 and (only marks the amino of underscore Acid) and Figure 17 in).VAMP3 amino acid sequence, BoNT interaction and cracking site be it is well known in the art (see, for example, Yamamoto H,Ida T,Tsutsuki H,Mori M,Matsumoto T,Kohda T,Mukamoto M,Goshima N, Kozaki S,Ihara H.Microbiol Immunol.2012Apr；56(4):245-53).Therefore, those skilled in the art It can also be readily determined with other active suitable polypeptide domains of VAMP3.

Functional variety usually only comprising SEQ ID NO:3 one or more amino acid conservative substitution or albumen it is non- Substitution, missing or the insertion of non-critical amino-acid in key area.Therefore, the functional variety of SEQ ID NO:3 can be SEQ The conserved amino acid sequence variant of ID NO:3, wherein the variant has VAMP3 activity.

Non-functional variant is the amino acid sequence variation without the active SEQ ID NO:3 of VAMP3.Non-functional variant is logical Non-conservative substitutions, missing or the insertion of amino acid sequence often containing SEQ ID NO:3 truncate (premature too early ) or the substitution of key amino acid or key area, insertion or missing truncation.For identifying that functional and non-functional becomes The method of body (such as functional and non-functional allelic variant) is well known to those of ordinary skill in the art.

[Proc Natl Acad Sci U S A.1998Dec 22；95(26):15781-6.Conserved structural features of the synaptic fusion complex:SNARE proteins Reclassified as Q-and R-SNAREs.Fasshauer D, Sutton RB, Brunger AT, Jahn R] in provide Crucial and non-critical amino-acid general introduction in VAMP3.Therefore, will easily identify can be by by those skilled in the art Substituted amino acid is to provide the functional variety (or function fragment) of SEQ ID NO:3, such as conserved amino acid sequence variant.This Field those of ordinary skill can also easily identify the homologue of SEQ ID NO:3 using standard sequence alignment programs.

It may include having with the amino acid sequence of SEQ ID NO:3 or part thereof or segment with the active polypeptide of VAMP3 Have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the amino acid sequence of 98%, 99% or 100% identity.Suitably, homogeneity percentage may be calculated and refer to sequence Arrange the percentage of the identity of the whole length of (such as SEQ ID NO:3) or part thereof or segment.

Amino acid sequence shown in SEQ ID NO:3 is the amino acid sequence of naturally occurring people VAMP3.About people Visible [the Nature Reviews Molecular Cell Biology 2,98-106 (February of the more details of VAMP3 2001)SNARE-mediated membrane fusion’Y.A.Chen&R.H.Scheller]。

" nonessential " or " non-key " amino acid residue is can be relative to wild-type sequence (for example, SEQ ID NO:1 is extremely 4 sequence) change, without eliminating, or it is highly preferred that not substantially change the residue of biological activity, and " required " amino acid Residue leads to this change.For example, the conservative amino acid residues in prediction polypeptide of the present invention are particularly unsuited for changing, in addition to cross-film Amino acid residue in structural domain can usually be replaced with other hydrophobic residues of approximately equivalent, without significantly changing Activity.

" conserved amino acid substitution " is that wherein amino acid residue is had the case where amino acid residue of similar side chain replaces. This field has determined that the amino acid residue families with similar side chain.These families include the amino acid with basic side chain (such as lysine, arginine, histidine), the amino acid (such as aspartic acid, glutamic acid) with acid side-chain, have without Amino acid (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, half Guang of the polar side chain of charge Propylhomoserin), amino acid with non-polar sidechain (such as alanine, valine, leucine, isoleucine, proline, phenylpropyl alcohol ammonia Acid, methionine, tryptophan), the amino acid (such as threonine, valine, isoleucine) with β branched side chains and have fragrance The amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) of race's side chain.Therefore, the nonessential amino acid in protein Residue is preferably replaced by another amino acid residue from identical side chain family.It, can be with alternatively, in another embodiment It is randomly incorporated into mutation on all or part of coded sequence, such as by saturation mutagenesis method, and gained mutant can be screened Biological activity to identify the mutant of retentive activity.After mutagenesis SEQ ID NO:1 to 4, coding can be recombinantly expressed Protein, and can determine the biological activity of protein.

As used herein, " biologically-active moiety " of protein or the protein portion with " bioactivity " includes participating in The protein fragments to interact between molecule and non-molecule.The biologically-active moiety of protein includes peptide, the peptide include with The amino acid sequence (for example, amino acid sequence shown in SEQ ID NO:1 to 4) of the protein is homologous enough or as derived from it Amino acid sequence comprising amino acid more less than full length protein, and show at least one of encoded protein Activity.In general, biologically-active moiety includes the active structural domain of at least one or motif with protein, for example, biology is living Property part can optionally retain one of following activity；Luciferase, VAMP1, VAMP2 or VAMP3 activity.

The biologically-active moiety of protein can be polypeptide, the polypeptide be, for example, SEQ ID NO:1 into 4 length 50, 100,150,200,250,300,350,400,450,500 or more amino acid.The biologically-active moiety of protein can be used It acts on exploitation and adjusts the target for being mediated the agent of active (such as bioactivity as described herein).

The meter of the following sequence homology carried out between sequence or identity (two terms are used interchangeably herein) It calculates.

In order to determine the homogeneity percentage of two amino acid sequences or two nucleic acid sequences, in order to best omparison purpose, Sequence is compared (for example, in order to omparison purpose, it can be in first and second amino acid sequence or nucleotide sequence One or two in introduce vacancy, to carry out optimal comparison, and nonhomologous sequence can be ignored).It is preferred real at one It applies in scheme, the length of the reference sequences compared in order to omparison purpose is at least the 30% of reference sequences length, preferably at least 40%, more preferably at least 50%, even more desirably at least 60%, even more desirably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Then the amino acid residue or nucleotide of more corresponding amino acid position or nucleotide position.When first sequence When a position in column is occupied by amino acid residue identical with corresponding position in second sequence or nucleotide, which exists The position is identical (as used herein, amino acid or nucleic acid identity " be equal to amino acid or nucleic acid " homology ").Two Homogeneity percentage between sequence is the function of the quantity of the shared same position of sequence, and it considers vacancy (to need to draw Enter these vacancy to carry out the optimal comparison of two sequences) quantity and each vacancy length.

Mathematical algorithm can be used to complete in the determination of homogeneity percentage between the comparison of sequence and two sequences.One In a preferred embodiment, using Needleman et al. ((1970) J.Mol.Biol.48:444-453) algorithm ( In the GAP program being incorporated into GCG software package) (can be obtained from http://www.gcg.com), using 62 matrix of BLOSUM or PAM250 matrix, and with 16,14,12,10,8,6 or 4 gap weight (gap weight) and 1,2,3,4,5 or 6 length power (length weight) is weighed to measure the percentage identity between two amino acid sequences.In another preferred embodiment party In case, using the GAP program (can obtain from http://www.gcg.com) in GCG software package, NWSgapdna.CMP square is used Battle array, and two nucleotide sequences are measured with the Length Weight of 40,50,60,70 or 80 gap weight and 1,2,3,4,5 or 6 Between percentage identity.Particularly preferred one group of parameter is (if operator is uncertain which parameter to determine one using A molecule should then use this group of parameter whether in sequence identity of the invention or homology limitation) it is that BLOSUM 62 scores Matrix, gap penalty 12, gap extension penalties 4, frameshift gap point penalty 5.

Alternatively, the algorithm of Meyers et al. ((1989) CABIOS 4:11-17) can be used, (it is already incorporated into ALIGN In program (2.0 editions)), using PAM120 weight residue table, GAP LENGTH PENALTY 12 and gap penalty 4, to determine two amino Homogeneity percentage between acid or nucleotide sequence.

Nucleic acid and protein sequence as described herein may be used as " search sequence " to scan for public database, with Such as identify other family members or correlated series.Altschul et al. ((1990) J.Mol.Biol.215:403- can be used 410) NBLAST and XBLAST program (2.0 editions)) carry out this search.BLAST nucleotide search can use NBLAST program, Score=100, word length=12 carry out, to obtain the nucleotide sequence with nucleic acid molecule homologous of the invention.BLAST protein is searched Rope can use XBLAST program, score=50, and word length=3 carry out, to obtain the amino homologous with protein molecule of the invention Acid sequence.It compares to obtain vacancy to be compared, it can be such as Altschul et al. (1997, Nucl.Acids Res.25:3389-3402 vacancy BLAST is utilized described in).When using BLAST and blank blast program, it can be used each The default parameters of a program (such as XBLAST and NBLAST).Referring to<http://www.ncbi.nlm.nih.gov>.

Polypeptide as described herein can have identical enough or substantially the same as the amino acid sequence of SEQ ID NO:1 to 4 Amino acid sequence.Terms used herein " identical enough " or " substantially the same " refer to the first amino acid or nucleotide sequence Comprising with the second amino acid or nucleotide sequence is enough or minimal number it is identical or equivalent (such as with similar side chain) Amino acid residue or nucleotide, so that the first and second amino acid or nucleotide sequence have common structural domain or common function It can activity.For example, containing having at least about 60% or 65% identity, may 75% identity, be more likely to 85%, 90%, 91%, the amino acid sequence of the common structural domain of 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or Nucleotide sequence is defined herein as identical enough or substantially the same.

Nucleic acid molecules

Additionally provide the nucleic acid molecules for encoding polypeptide described herein.

The nucleic acid molecules of coding polypeptide can be synthetically prepared by established standard method, such as Beucage S.L. etc. Phosphoamidite method or Matthes of people (1981) Tetrahedron Letters 22, p 1859-1869 description et al. (1984) EMBO J.3, p 801-805 description method.In phosphoamidite method, oligonucleotides is for example synthesized in automated DNA It synthesizes, purify, anneal, connect in instrument and be cloned into suitable carrier.

Nucleotide sequence can be the genomic source and synthesis source of mixing, the synthesis source of mixing and the source cDNA or Mixed genomic source and the source cDNA, according to standard technique by synthesis source, genomic source or the source cDNA (by suitable When) segment connection and prepare.The segment of each connection corresponds to the various pieces of entire nucleotide sequence.DNA sequence dna Specific primer can be used to prepare by polymerase chain reaction (PCR), such as US 4,683,202 or Saiki R et al. Described in (Science (1988) 239, pp 487-491).

According to standard technique, nucleotide sequence can be the mixture of genomic source and external source source.For example, nucleotide The gene editing technology that such as CRISPR/Cas9 can be used in sequence generates, wherein coding to be had to the knot of luciferase activity The nucleic acid sequence in structure domain introduces the upstream that coding has the native sequence nucleic acid of the active structural domain of VAMP1, VAMP2 or VAMP3.

As used herein, term " nucleic acid molecules " or " nucleotide sequence " refer to oligonucleotide sequence or polynucleotides sequence Column and its variant, homologue, segment and derivative (such as its part).Nucleotide sequence can be genomic source or synthesis Source or recombinant sources can be double-strand or single-stranded, no matter represent positive-sense strand or antisense strand.It is related to the present invention Term " nucleotide sequence " includes genomic DNA, cDNA, synthetic DNA and RNA (such as mRNA) and for example by using nucleotide Analog and the analog of DNA or RNA generated.

Nucleic acid molecules can be single-stranded or double-stranded, but preferably double-stranded DNA, the more preferably cDNA of coded sequence.In In one preferred embodiment, encoding, there is the nucleotide sequence of polypeptide of specific feature as herein defined itself not cover Native nucleotide sequence of the lid in its natural surroundings native nucleotide sequence and is similarly in it in its natural environment The natural relevant sequence connection of native nucleotide sequence in natural surroundings.We are preferred real by this for ease of reference, It applies scheme and is known as " non-native nucleotide sequence " or " non-naturally occurring sequence ".In this regard, term " natural nucleotide Sequence " or " naturally occurring sequence " refer to the whole nucleotide sequence in its natural surroundings, and with it is natural with it When relevant entire promoter is operably connected, promoter is also in its natural surroundings.Therefore, polypeptide of the invention can be with By the nucleotide sequence expression in its native organism, but wherein the nucleotide sequence is not by day therewith in the organism The control of right relevant promoter.

Preferably, the polypeptide is not natural polypeptides.In this regard, term " natural polypeptides " or " naturally occurring Polypeptide " refers to the complete polypeptide in its natural surroundings, and is expressed by its native nucleotide sequence.In general, There is the nucleotides sequence of the polypeptide of special properties as herein defined using recombinant DNA technology (that is, recombinant DNA) preparation coding Column.However, in another embodiment of the present invention, it is all or part of that chemical method well-known in the art can be used Synthesizing ribonucleotide sequence is (referring to Caruthers MH et al. (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al. (1980) Nuc Acids Res Symp Ser 225-232).

As used herein, term " recombination " refers to biomolecule, such as gene or protein, and (1) has been moved away from it Naturally occurring (natural) environment, all or part of nucleic acid molecules or albumen qualitative correlation when (2) are not in native form with it, (3) it is operably connected with polynucleotides or polypeptide, is not connect in nature with this polynucleotides or polypeptide, or (4) It is not present in nature.

For example, comprising with uciferase activity N- terminal polypeptide structural domain and have VAMP1, VAMP2 or The polypeptide of the active C- terminal polypeptide structural domain of VAMP3 be considered as recombinate biomolecule (polypeptide as provided herein it is all Specific embodiment is the same).Equally, the nucleic acid molecules for encoding polypeptide provided herein are also " recombination ".

Hybridization

The invention also includes the sequence complementary with sequence of the invention or can with sequence of the invention or be complementary The purposes of the sequence of sequence hybridization.

The term as used herein " hybridization " should include " nucleic acid chains pass through process of the base pairing in conjunction with complementary strand ", and The amplification procedure carried out in polymerase chain reaction (PCR) technology.

Hybridization conditions are the melting temperatures (Tm) based on nucleic acid binding complex, as Berger and Immel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology,Vol.152,Academic Press, San Diego CA) taught in, and determining " stringency (stringency) " is imparted, as described below.

Maximum stringency usually occurs at about Tm-5 DEG C (5 DEG C lower than the Tm of probe)；High stringency degree occurs be lower than Tm About 5 DEG C to 10 DEG C；Medium stringency occurs be lower than about 10 DEG C to 20 DEG C of Tm；Degree low strict occurs be lower than about 20 DEG C to 25 of Tm ℃.As it will appreciated by a person of ordinary skill, maximum stringency hybridization can be used for identifying or detecting identical nucleotide sequence, and Medium (or low) stringency hybridization can be used for identifying or detecting similar or related polynucleotide sequence.

Preferably, the present invention include can be in medium stringency condition (for example, 50 DEG C and O.2xSSC) or high stringency conditions Under (for example, 65 DEG C and O.1xSSC { lxSSC-0.15M NaCl, 0.015M sodium citrate pH7.0 }) with nucleosides defined herein The purposes of the sequence of acid sequence (or its complementary series) hybridization.

Expression vector

As used herein, term " carrier " or " construct " are another seed nucleus for referring to transhipment and being operably connected with it The nucleic acid molecules of acid.Term " carrier " and " construct " are used interchangeably herein.Carrier can independently replicate or it can To be integrated into host DNA.Carrier may include the restriction enzyme sites for being inserted into recombinant DNA, and may include it is a kind of or Multiple choices label.Carrier can be the nucleic acid molecules of plasmid, bacteriophage or clay (cosmid) form.Preferably, carrier is suitable In the expression (that is, carrier is " expression vector ") in cell.Preferably, expression vector is suitable for thin in neuronal cell or neuron It is expressed in born of the same parents system.Most preferably, carrier be suitable for Human Neuroblastoma Cell Line (such as SiMa neuroblastoma cell, LAN5 neuroblastoma cell or NG108 neuroblastoma cells), immortalized neuronal, primary neuron or heredity repairs It is expressed in the organism of decorations.

Preferably, (expression) carrier can breed in host cell and steadily pass to offspring.

As used herein, it " is operably connected " and refers to single in following control elements or combination and coded sequence with function Can relationship each other together, such as with the relationship of connection, so as to guide the expression of coded sequence.

As used herein, " regulating and controlling sequence " is DNA the or RNA element for referring to control gene expression.Expression control sequence Example include promoter, enhancer, silencer, Shine Dalgarno sequence, TATA box, internal ribosome entry site (IRES), transcription factor attachment site, transcription terminator, site of polyadenylation, rna transport signal or to ultraviolet light mediate The important sequence of Gene response.Preferably, carrier includes one or more regulating and controlling sequences, can be grasped with nucleic acid sequence to be expressed Make ground connection.Regulating and controlling sequence include instruct constitutive expression sequence and Tissue-specific regulatory sequence and/or induction sequence Column.

As used herein, " promoter " refers to the nucleotides sequence for starting transcription in DNA or RNA in conjunction with RNA polymerase Column.Promoter can be inducible expression's or constructive expression's.Alternatively, promoter is in the control of repressor or stimulates the protein Under system.Preferably, promoter be selected from SV40, CMV, Actin, EF1 alpha, UB, RCV, PGK, CAG, MMLV-LTR or CMV-LTR hybrid promoter.

As used herein, " transcription terminator " refers to a kind of DNA element, terminates the RNA for being responsible for that DNA is transcribed into RNA The function of polymerase.Preferred transcription terminator is characterized in a succession of T residue, is followed by the Double Symmetry region rich in GC.

As used herein, " translation control element " refers to DNA the or RNA element of control mRNA translation.Preferred translation control Element processed is ribosome bind site.Preferably, translation control element is from the system homologous with promoter, for example, promoter and Its relevant ribozyme binding site.Preferred ribosome bind site is T7 or T3 ribosome bind site.

" restriction enzyme recognition site " used herein refers to the motif identified on DNA by restriction enzyme.

As used herein, " selected marker " refers to the albumen for assigning phenotype on cell when expressing in host cell Matter, the phenotype allow the cell for selecting to express the selectable marker gene.In general, this may be to confer to fight raw element (for example, ammonia Parasiticin, kanamycins, chloramphenicol, tetracycline, hygromycin, neomycin or methotrexate (MTX)) resistance protein.Antibiotic Other examples be penicillin；Hydrochloric acid ampicillin, ampicillin sodium, Amoxicillin Sodium, carbapen, benzyl penicillin, head Spore rhzomorph, Cefotaxime Sodium, cefalexin hydrochloride, vancomycin, seromycin.Other examples include antibacterial inhibitor, such as: Chloramphenicol, erythromycin, lincomycin, tetracycline, Spectinomycin Sulfate, Clindamycin Hydrochloride, Chlortetracycline.

The design of expression vector depends on selection, the expression of required protein etc. of host cell to be transformed Factor.Expression vector of the invention can be introduced into host cell, to generate the albumen by nucleic acid encode as described herein Matter or polypeptide (including fusion protein or polypeptide) are (for example, include N- terminal polypeptide structural domain and tool with luciferase activity There is the polypeptide of the active C- terminal domains of VAMP1, VAMP2 or VAMP3).

Preferably, carrier includes those genetic elements necessary to host cell expression polypeptide described herein.It is thin in host Element needed for transcription and translation includes promoter, the code area of destination protein and transcription terminator in born of the same parents.

Expression vector of the invention can be standard expression vectors, such as pCDNA3, pIRES-NEO or retrovirus vector Body, such as pQCXIP, pQCXIN, pQCXIG, pLXIN, pBMN, pBABE-hygro and pBABE-puro.

Term " expression vector ", " expression construct ", " construct " and " carrier " is used interchangeably herein.

Preferably, expression vector is high copy number expression vector；Alternatively, expression vector is low copy number expression vector.

The preparation of expression vector

It will be appreciated by those skilled in the art that can be used for preparing the molecular engineering of expression vector.

It can be by using (mutually priming) oligonucleotides mutually started and nucleic acid sequence as described herein Synthetic nucleic acid molecule prepares the nucleic acid molecules being used for incorporation into expression vector present invention as described above.

Many molecular engineerings have been developed DNA to be operably connected with carrier by complementary cohesive tennini.One In a embodiment, complementary homopolymer segment can be added to being inserted into the nucleic acid molecules in carrier DNA.Then, carrier And nucleic acid molecules pass through the Hydrogenbond between complementary homopolymer tail portion, form recombinant DNA molecules.

In an alternative embodiment, using the synthetic linker containing one or more restriction sites by nucleic acid molecules with Expression vector is operably connected.In one embodiment, it is digested by restriction endonuclease and generates nucleic acid molecules.It is excellent Selection of land handles nucleic acid molecules with bacteriophage T4 archaeal dna polymerase or e. coli dna polymerase I, these enzymes can utilize its 3'- 5'- exonuclease activity removes protrusion --- 3'- single stranded end, and the recessed end 3'- is filled using its polymerization activity, To generate flat terminal DNA fragments.Then in the enzyme for the connection that can be catalyzed flat end DNA molecular (such as bacteriophage T4 DNA Ligase) in the presence of, flat terminal fragment and the linkers of big molar excess are incubated with.Therefore, reaction product be Its end has the nucleic acid molecules of polymeric joint sequence.Then by these nucleic acid molecules limitation enzymatic lysis appropriate, and even It is connected to the expression vector by enzymatic lysis, which generates the end with the compatible ends of nucleic acid molecules.

Alternatively, the carrier comprising connection dependent/non-dependent clone site (LIC) can be used.It then can be by required PCR Amplifier nucleic acid molecule is cloned into LIC carrier, without carry out restricted digestion or connection (Aslanidis and de Jong, Nucl.Acid.Res.18,6069-6074, (1990), Haun, et al., Biotechniques 13,515-518 (1992)).

In order to separate and/or modify the target nucleic acid molecules for being inserted into selected plasmid, it is preferable to use PCR.It can be with Appropriate primer prepared by the PCR designed for sequence adds restriction nuclease inscribe to separate required nucleic acid molecule encoding area Enzyme or the site LIC, code area are placed in required reading frame.

In a preferred embodiment, by using such as Saiki et al. ((1988) Science 239,487-491) Disclosed in polymerase chain reaction be used for incorporation into expression of the invention using Oligonucleolide primers appropriate to prepare Nucleic acid molecules in carrier.Code area is amplified, and primer itself is impregnated in the sequence product of amplification.It is preferred real at one It applies in scheme, amplimer includes restriction endonuclease recognition site, allows the sequence product of amplification being cloned into conjunction In suitable carrier.

Preferably, nucleic acid molecules are obtained by PCR, and is digested using restriction endonuclease and connects that (technology is It is well-known in the art) it is introduced into expression vector.It is highly preferred that nucleic acid molecules are introduced into expression vector, the table Up to carrier such as pCDNA3, pIRES-NEO or retroviral vector such as pQCXIP, pQCXIN, pQCXIG, pLXIN, pBMN, PBABE-hygro or pBABE-puro.

Expression vector of the invention may include single copy of aforementioned nucleic acid molecules or the multicopy of aforementioned nucleic acid molecules.

Nucleic acid molecules and/or expression vector as described herein can reside in any suitable host cell (to produce The cell of raw genetic modification).

Host cell

It include comprising nucleotide sequence according to the present invention or coding tool about term " host cell " of the invention There is any cell of the nucleotide sequence of the polypeptide of specific feature as herein defined.Preferably, nucleotide sequence is mixed Into the genome of cell.

Term " host cell ", " cell of gene modification " and " recombinant host cell " are used interchangeably.These terms are not (in natural surroundings, natural nucleus glycoside coding sequences are at it for the natural nucleus glycoside coding sequences for covering in its natural surroundings Under the control of natural promoter (being also in its natural surroundings)), and refer to that (for example, conversion or transfection) of genetic change is thin Born of the same parents.The term refers to specific subject cell, also refers to the offspring or potential offspring of this cell.Because due to mutation or environment shadow It rings, certain modifications may occur in offspring, so this offspring actually may be different from parental cell, but be included in this In the range of term used in text.

The cell of genetic modification can be eukaryocyte or prokaryotic cell.Preferably, the cell of gene modification is that clostridium is quick Perceptual cell is (for example, clostridial neurotoxins can be in conjunction with the cell (such as clone/cell line) of simultaneously cross-cell membrane transhipment.In this way The example of cell be well-known, and technical staff will for example pass through test methods of preparing tetanus and/or meat Whether bacillus venenosus neurotoxin (for example, Type B BoNT, D type BoNT, F type BoNT and/or G type BoNT) can be in conjunction with simultaneously across cell Film is transported to determine such cell.Routine experiment for testing this point is well-known, including following embodiment part Described in some experiments.Suitable cell includes the cell of Human Neuroblastoma Cell Line (for example, SiMa neuroblastoma Cell, LAN5 neuroblastoma cell, NG108 neuroblastoma cell), immortal neuron, primary neuron or heredity The biology of modification.

Any suitable means (such as gene editing technology) can be used to change host cell gene group to generate this The nucleic acid sequence of the nucleic acid sequence of invention, protein structure domain of the gene editing technology by coding with luciferase activity is drawn Entering coding has the upstream of natural (endogenous) nucleic acid sequence of the active protein domain of VAMP1, VAMP2 or VAMP3.It can replace For ground or additionally, standard technique known in the art expression vector conversion of the invention, infection or transfection place can be used Chief cell.

Transformation of host cells

It can be converted with expression vector (it includes foregoing nucleic acid molecules) of the invention, the host of infection or transfection Cell generates (express) polypeptide provided herein.

Expression vector of the invention can be introduced into cell by conventional conversion, transfection or transduction techniques." turn Change ", " transfection " and " transduction " refer to the technology for being introduced into exogenous nucleic acid in cell.The specific method used generally depends on The type of carrier and cell.The technology includes but is not limited to turn that calcium phosphate or calcium chloride co-percipitation, DEAE- glucan mediate Dye, lipofection, chemical poration or electroporation.

It is as known in the art for converting, transfecting or the technology of transducer cell is disclosed in such as Sambrook et al. (1989)Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y；Ausubel et al. (1987) Current Protocols in Molecular Biology,John Wiley and Sons,Inc.,NY；Cohen et al. (1972) Proc.Natl.Acad.Sci.USA 69, 2110；Luchansky et al. (1988) Mol.Microbiol.2,637-646.

Successful conversion, transfection or the cell of transduction (or cell of genetic modification), i.e., containing expression vector of the invention or Those of nucleic acid molecules cell can be identified by technology well known in the art.For example, can cultivate with table of the invention The cell transfected up to carrier is to generate with luciferase activity and the active protein of VAMP1, VAMP2 or VAMP3.It can lead to Cross the presence of expression vector dna in check-up cell well-known in the art.Alternatively, the antibody being hybrid with it can be used To detect the presence of polypeptide or part thereof He segment.

In a preferred embodiment, the present invention includes the culture of the cell of conversion.Preferably, culture is gram Grand homogeneous.

Cell may include the single copy of previously described expression vector, alternatively, multiple copies of expression vector.

Purposes and method

Inventor has surprisingly observed that can be used for detecting neurotoxin active (especially tetanus, Type B clostridium botulinum, D Type clostridium botulinum, F type clostridium botulinum and/or G type botulic neurotoxin activity) novel report molecule.Report molecule quilt After specific neurotoxin cracking, two crack fragments are generated；First segment, it includes the end N- with uciferase activity is more Peptide domain and a part with the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3；With the second segment, Include the remainder with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3.Advantageously, the first segment is sufficiently stable It is active (that is, cracking of examining report molecule) so as to detect neurotoxin.

Therefore, the cell of polypeptide of the invention, nucleic acid molecules, expression vector or genetic modification can be used for detecting neurotoxin Activity, wherein the neurotoxin activity is selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum mind Through neurotoxin active, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type clostridium botulinum Nervous toxicity Plain activity.

As used herein, " neurotoxin activity " refers to naturally occurring tetanus, Type B clostridium botulinum, D type meat poisoning bar It (in this case, is to have that any one of bacterium, F type clostridium botulinum and G type botulic neurotoxin, which crack its substrate, The active polypeptide domain of VAMP1, VAMP or VAMP3) ability.Naturally occurring neurotoxin and its respective neurotoxin Activity is well known in the present art, and is more fully described elsewhere (referring to table 1 and, for example, Botulinum Neurotoxins.Editors:Rummel,Andreas,Binz,Thomas(Eds.)Springer,Current Topics in Microbiology and Immunology,2013)。

For example, " methods of preparing tetanus activity " refers in cracking site GASQ⁷⁸VAMP1 is cracked at FESS (or to be had The active polypeptide domain of VAMP1), in cracking site GASQ⁷⁶At FETS crack VAMP2 (or have the active polypeptide knot of VAMP2 Structure domain) and/or in cracking site GASQ⁵⁹VAMP3 (or there is the active polypeptide domain of VAMP3) is cracked at FETS.

For example, " Type B botulic neurotoxin activity " refers in cracking site GASQ⁷⁸At FESS crack VAMP1 (or tool Have the active polypeptide domain of VAMP1), in cracking site GASQ⁷⁶At FETS crack VAMP2 (or have the active polypeptide of VAMP2 Structural domain) and/or in cracking site GASQ⁵⁹VAMP3 (or there is the active polypeptide domain of VAMP3) is cracked at FETS.

For example, " D type botulic neurotoxin activity " refers in cracking site RDQK⁶¹At LSEL crack VAMP1 (or tool Have the active polypeptide domain of VAMP1), in cracking site RDQK⁵⁹At LSEL crack VAMP2 (or have the active polypeptide of VAMP2 Structural domain) and/or in cracking site RDQK⁴²VAMP3 (or there is the active polypeptide domain of VAMP3) is cracked at LSEL.

For example, " F type botulic neurotoxin activity " refers in cracking site ERDQ⁶⁰At KLSE crack VAMP1 (or tool Have the active polypeptide domain of VAMP1), in cracking site ERDQ⁵⁸At KLSE crack VAMP2 (or have the active polypeptide of VAMP2 Structural domain) and/or in cracking site ERDQ⁴¹VAMP3 (or there is the active polypeptide domain of VAMP3) is cracked at KLSE.

For example, " G type botulic neurotoxin activity " refers in cracking site ESSA⁸³At AKLK crack VAMP1 (or tool Have the active polypeptide domain of VAMP1), in cracking site ETSA⁸¹At AKLK crack VAMP2 (or have the active polypeptide of VAMP2 Structural domain) and/or in cracking site ETSA⁶⁴VAMP3 (or there is the active polypeptide domain of VAMP3) is cracked at AKLK.

Tetanus, Type B clostridium botulinum, D type clostridium botulinum, F type clostridium botulinum and/or G type botulic neurotoxin Modified forms (such as manually modified, recombinant, chimera, toxoid etc.) may retain " neurotoxin activity " (i.e. it The ability of VAMP1, VAMP2 and/or VAMP3 is cracked in a manner of shown in table 1).Therefore, the present invention can be used for detecting this Whether the neurotoxin of modification retains " neurotoxin activity ".The present invention includes these purposes and method.

The diversity of botulic neurotoxin be summarized in for example [Research in Microbiology, Volume166,Issue 4,May 2015,Pages 303–317,Genomes,neurotoxins and biology of Clostridium botulinum Group I and Group II, Andrew T.Carter, Michael W.Peck] in.

Therefore, the neurotoxin activity that the present invention can be used for detecting the naturally occurring form of these neurotoxins (and/or is deposited ) and its chimera or the neurotoxin of manually modified form activity.Advantageously, this invention therefore provides for detecting The means that the neurotoxin activity of these neurotoxin modified forms increases or decreases.

It can be used and be adapted to detect for the active any means of neurotoxin.Several standard technology can be used to detect herein The cracking of the polypeptide.Example technique includes but is not limited to immunoblotting (also referred to as Western blotting), sandwich ELISA survey The imaging of fixed or living cells.These methods or details that is conventional and respectively how carrying out in this field are well-known (ginseng See such as [Specificity of botulinum protease for human VAMP family Proteins.Yamamoto H, et al. Microbiol Immunol, 2012Apr.PMID 22289120；Substrate recognition of VAMP-2by botulinum neurotoxin B and tetanus neurotoxin.Chen S, Et al. J Biol Chem, 2008Jul 25.PMID 18511417]).

In the case where the cell of not genetic modification, the active detection of neurotoxin can carry out in vitro.For example, can So that polypeptide of the invention is contacted with neurotoxin (or test sample as described below), and it can determine depositing for pyrolysis product (such as passing through ELISA or Western blotting).The exploitation of this detection method is completely in the conventional energy of those skilled in the art Within the scope of power.

It is alternatively possible to which the cell using genetic modification carries out the active detection of neurotoxin, such as elsewhere herein institute It states.Advantageously, the cell of genetic modification provide for test neurotoxin effect all three stages means, described three A stage is i.e.: being delivered in cell cytosol and neurotoxin induction in conjunction with cell surface, by neurotoxin peptase The cracking of its substrate (in this case, being polypeptide of the invention).It can determine that the presence of pyrolysis product (such as passes through cell The ELISA or Western blotting of lysate).Also living cells imaging can be used to detect neurotoxin activity, because of Nervous toxicity " the first segment " that element cracking polypeptide generates is (i.e. comprising having the N- terminal polypeptide structural domain of uciferase activity and having The segment of a part of the active C- terminal domains of VAMP1, VAMP2 or VAMP3) it will will be deposited no longer in conjunction with vesica It is in cell cytosol.The exploitation of this detection method is completely within the scope of the conventional capability of those skilled in the art.

The cracking of neurotoxin induction can also be detected using the uciferase activity of the polypeptide after cracking.This field is public That knows is related to the exploitation of the standard technique and these methods of luciferase detection completely in the conventional capability of those skilled in the art In range.The summary of luciferase detection method is found in [Application of enzymebioluminescence for medical diagnostics.Frank LA,Krasitskaya VV.Adv Biochem Eng Biotechnol.2014； 144:175-97；Current advanced bioluminescence technology in drug discovery.Hoshino H.Expert Opin Drug Discov.2009Apr；4(4):373-89].

The special antibody in cracking end to polypeptide can also be used to detect the cracking of neurotoxin induction.Using to more One advantage of the special antibody in cracking end of peptide is that they can be used for determining which kind of specific neurotoxin is (such as broken Wind/Type B BoNT, D type BoNT, F type BoNT or G type BoNT --- it is shown in Table and 1) is responsible for the cracking of polypeptide and (and thereby determines that for example such as There are which kind of neurotoxins in the lower test sample).

For example, the antibody of the VAMP2 specificity of VAMP2 or the BoNT/D cracking to BoNT/B cracking can be generated (antibody is generated for peptide ALQAGASQ and peptide KVLERDQK respectively --- referring to Fig. 5 and corresponding legend).

The present invention can be used for detecting the activity of the neurotoxin in test sample.

As used herein, " test sample " can be the active existing any sample of neurotoxin to be tested and (have The known composition in known, unknown or part), wherein the neurotoxin activity is selected from the group being made up of: tetanus Neurotoxin activity, Type B botulic neurotoxin activity, D type botulic neurotoxin activity, F type clostridium botulinum nerve Neurotoxin active and G type botulic neurotoxin activity, or any combination thereof.

" test sample " may include drug products (such as the vaccine for being applied to subject, such as toxoid, wherein Toxoid be from pathogenic microorganism (such as tetanus or clostridium botulinum) chemical modification toxin), be no longer it is toxic, But still there is antigenicity, it can be used as vaccine.In this case, the present invention can be used for detecting (undesired) residual Nervous toxicity Plain activity.

As used herein, " subject " refers to human or animal (such as human or animal of vaccine to be seeded).Usual animal is Vertebrate, such as primate, rodent, domestic animal or wild animal.Primate includes chimpanzee, food crab Monkey, Ateles and macaque, such as rhesus macaque (Rhesus).Rodent includes mouse, rat, marmot, ferret, rabbit and storehouse Mouse.Domestic animal and wild animal include ox, horse, pig, deer, wild ox, buffalo, felid such as domestic cat, canid such as dog, fox Leopard cat, wolf, poultry such as chicken, emu (emu), ostrich and fish such as trout, catfish and salmon.In certain realities of aspect described herein It applies in scheme, subject is mammal, such as primate, such as people.Subject can be male or female.Subject It can be the subject (for example, adult) developed completely or be undergoing the subject of growth course (for example, children, Ying Erhuo Fetus).

Preferably, subject is mammal.Mammal can be people, non-human primates, mouse, rat, dog, cat, Horse or ox, but it is not limited to these examples.

Suitable test sample can also include foodstuff samples, clinical sample or environmental sample (wherein foodstuff samples, clinic Sample or environmental sample may pollute and/or may contain neurotoxin by clostridium), or any combination thereof.In such case Under, the present invention can be used for detecting the presence of pollution.

Suitable test sample further includes methods of preparing tetanus, Type B botulic neurotoxin, D type clostridium botulinum mind Through toxin, F type botulic neurotoxin and/or G type botulic neurotoxin (for example, the Nervous toxicity from known batch The test sample of element, optionally, the neurotoxin of the known batch are produced for therapeutic or non-therapeutic use), or Any combination thereof.In this case, the present invention can be used for detecting or determining the effect of neurotoxin.To avoid doubt, test Neurotoxin in sample can be naturally occurring neurotoxin, or can be (such as manually modified, the packet of its modification Include chimera) form.

In a preferred method, using the cell detection of genetic modification of the invention (or measurement) neurotoxin activity, (a) Cultivate cell under the following conditions, the conditions permit expression comprising with uciferase activity N- terminal polypeptide structural domain and Polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3.In this embodiment it is possible to make the thin of genetic modification Born of the same parents contact with neurotoxin (or test sample), and the presence of pyrolysis product can be determined (such as by ELISA, protein Trace or living cells imaging).The exploitation of these methods is also completely within the scope of the conventional capability of those skilled in the art.

It thus provides a kind of active method of neurotoxin in detection test sample, which comprises

(a) cell of genetic modification according to the present invention, the conditions permit expression packet are cultivated under the following conditions Containing the N- terminal polypeptide structural domain with uciferase activity and there is the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 Polypeptide；

(b) cell of (a) is cultivated in the presence of test sample under the following conditions, the conditions permit includes with glimmering The N- terminal polypeptide structural domain of light element enzymatic activity and polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 The cracking of neurotoxin induction；With

(c) determine that the cracking of the neurotoxin induction of the polypeptide is horizontal, wherein what the neurotoxin of the polypeptide induced The detection instruction neurotoxin activity of cracking；

Wherein the neurotoxin activity is selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum Neurotoxin activity, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type clostridium botulinum nerve Neurotoxin active, or any combination thereof.

Condition of culture in step (a) should allow express comprising with uciferase activity N- terminal polypeptide structural domain and Polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3.Optimum culture condition is to allow polypeptide expression to exist completely Within the scope of the conventional capability of those skilled in the art.In addition, identifying whether selected condition of culture allows polypeptide expression to be also conventional , and standard technique such as ELISA or Western blotting can be used to detect the amount of polypeptide expression.

Depending on host cell, cell is made to grow or cultivate cell in a manner of known to technical staff.Culture to be used Base (culture medium) (also referred herein as " growth medium (growth medium) ", " culture medium (medium) " or " culture medium (media) ") it must suitably meet the requirement of discussed cell.Preferably, culture medium is enough to support that host is thin The growth of born of the same parents.The description of culture medium suitable for various cells is found in textbook " Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications,Sixth Edition,R.Ian Freshney,2010John Wiley&Sons,Inc.".Only for example, SiMa neuroblastoma cell is preferably following Under the conditions of cultivate: keep SiMa cell (collecting from DSMZ cell) raw in the RPMI culture medium for being supplemented with 10% fetal calf serum It is long.In order to break up, with 10 μ g/ml laminin precoating plates.By SiMa cell with every hole 1 × 10⁴The density of a cell It is inoculated in 96 orifice plates or with every hole 2 × 10⁴The density of a cell is inoculated in 48 orifice plates, and differential medium (RPMI, B27 or GS21 replenishers, 1mM HEPES and the 1%NEAA containing 10 μM of AT- retinoic acids) middle incubation 72 hours.

The cell of genetic modification can be grown in liquid medium, the fluid nutrient medium include it is one of following or It is a variety of: carbon source (usually sugared form), nitrogen source (usually in the form of organic nitrogen source, such as yeast extract) or salt (such as sulphur The salt of sour ammonium, inorganic salts, microelement such as iron, manganese and magnesium) and vitamin (if applicable) preferably exist at 0 DEG C to 100 DEG C Within the temperature range of 25 DEG C to 40 DEG C, in carbon dioxide.

Preferred carbon source is sugar, such as monosaccharide, disaccharides or polysaccharide.The example of carbon source is glucose, carbon dioxide, bicarbonate Sodium, bicarbonate, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose, gossypose, shallow lake Powder or cellulose.Preferably, carbon source is carbon dioxide.Alternatively, carbon source is bicarbonate.The mixture for adding several kinds of carbon source can also It can be advantageous.

Nitrogen source is usually organic or inorganic nitrogen compound or the material comprising these compounds.The example of nitrogen source includes liquid Or gaseous ammonia or ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrate, urea, amino acid or multiple Close nitrogen source such as corn steep liquor, Soybean Meal, soybean protein, yeast extract, meat extract etc..Nitrogen source can be used alone or mix It closes and uses.

The inorganic salt compound that may be present in culture medium includes the chlorine of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron Salt, microcosmic salt and sulfate.

Inorganic sulfocompound (such as sulfate, sulphite, dithionite, four sulfate, thiosulfate, Sulfide) or organosulfur compound (such as mercaptan (" mercaptan " and " thiol ")) can be used as production sulfur-bearing it is fine The source of the sulphur of chemicals especially methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the salt containing sodium can be used as the source of phosphorus accordingly.

Chelating agent can be added into culture medium, and metal ion is kept in the solution.Specially suitable chelating agent packet Include dihydric phenol, such as catechol or protocatechuic acid ester and organic acid, such as citric acid.

Culture medium used according to the invention can also include other growth factors, such as vitamin or growth promoter, It includes such as biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid (panthothenate) and pyridoxol.Growth factor With salt usually from complex media components, such as yeast extract, syrup, corn pulp etc..Furthermore, it is possible to add into culture medium Add suitable precursor.The definite composition of culture based compound depends greatly on specific experiment, and for every kind of tool Body situation determines respectively.Information in relation to medium optimization is found in textbook " Applied Microbiol.Physiology, A Practical Approach " (editor P.M.Rhodes, P.F.Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 963577 3).Growth medium can also be obtained from commercial supplier, for example, Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) etc..

The pH of fluid nutrient medium can keep constant and (adjust in the training period) or not keep constant.

The general introduction of known cultural method is found in textbook (the Bioproze β technik 1.Einf ü hrung of Chmiel in die Bioverfahrenstechnik[Bioprocess technology 1.Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas religion section Book (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment](Vieweg Verlag,Brunswick/Wiesbaden,1994))。

It goes out by heating (20 minutes at 1.5 bars and 121 DEG C) or by filtration sterilization to all nutrient media components Bacterium.Component can be sterilized together, or if desired, can individually sterilized.As needed, all nutrient media components can be Culture exists when starting, or continuous or be added portionwise.

Cultivation temperature will change with host cell according to specific experiments.Cultivation temperature usually between 15 DEG C to 45 DEG C, It is preferred that more preferably between 25 to 37 DEG C, and can keep constant or can change during the experiment between 25 DEG C to 40 DEG C Become.Only for example, for SiMa neuroblastoma cell, temperature is preferably 25 to 40 DEG C, and more preferably 37 DEG C.

The pH of culture medium should in the range of 5 to 8.5, preferably from about 7.0.It in the training period can be by adding alkaline chemical combination Object (such as sodium hydroxide, potassium hydroxide, ammonia and ammonium hydroxide) or acid compound (such as phosphoric acid or sulfuric acid) control the pH of culture.It can be with Foaming is controlled by using defoaming agent (such as fatty acid polyethylene glycol ester).It, can be to culture in order to keep the stability of carrier The suitable substance with selection index system, such as antibiotic are added in base.It is mixed by introducing oxygen or oxygen-containing gas into culture Object (such as surrounding air) is closed to maintain aerobic conditions.Cultivation temperature is usually 20 DEG C to 45 DEG C, and preferably 25 DEG C to 40 DEG C.It holds Continuous culture is until occur polypeptide expression.This target is usually realized in 6 to 96 hours.

Condition of culture in step (b) is in the presence of test sample, and the condition of culture allows comprising having The N- terminal polypeptide structural domain of uciferase activity and polypeptide with the active C- terminal polypeptide of VAMP1, VAMP2 or VAMP3 Neurotoxin induction cracking.In other words, condition of culture is such that if there are functional nerve poison in test sample The cracking of the neurotoxin induction of some polypeptides then can at least occur for element.Optimum culture condition is to allow the neurotoxin of polypeptide The cracking of induction is completely within the scope of the conventional capability of those skilled in the art.In addition, identifying whether selected condition of culture allows The cracking of the neurotoxin induction of polypeptide is also conventional, and standard technique such as ELISA or Western blotting inspection can be used (as discussed elsewhere) amount of the cracking of the neurotoxin induction of detection polypeptide.

In one embodiment, incubation step (a) and (b) are carried out simultaneously.In other words, can start in cell culture When test sample is added in the cell of genetic modification, or can be added after polypeptide expression has begun.Optimization is being surveyed The time point of the cell of genetic modification is cultivated in the presence of test agent completely in the conventional capability range of those of ordinary skill in the art It is interior.

Optionally, test sample is provided in the medium.

As described in elsewhere, test sample may include drug products, foodstuff samples, clinical sample or environmental sample or Any combination thereof.Alternatively, test sample may include tetanus toxoid or Clostridium botulinum toxoid, or combinations thereof.

Test sample may include methods of preparing tetanus, Type B botulic neurotoxin, D type clostridium botulinum Nervous toxicity Element, F type botulic neurotoxin, G type botulic neurotoxin or any combination thereof.To avoid doubt, in test sample Neurotoxin can be naturally occurring neurotoxin, or can be its modification (such as manually modified, including chimera) Form.

Method provided herein includes step (c), that is, the level of the cracking of the neurotoxin induction of polypeptide is determined, wherein more The detection instruction neurotoxin activity of the cracking of the neurotoxin induction of peptide；And wherein the neurotoxin activity be selected from by with The group of lower composition: methods of preparing tetanus activity, Type B botulic neurotoxin activity, D type botulic neurotoxin are living Property, F type botulic neurotoxin activity and G type botulic neurotoxin activity, or any combination thereof.

Any suitable standard technique (such as ELISA, Western blotting, the work of elsewhere herein discussion can be used Cell imaging etc.) determine that the cracking of the polypeptide of (or detection) neurotoxin induction is horizontal.

As used herein, neurotoxin induction cracking " detection " cover it is detectable (such as it is visible, can quantify Etc.) cracking of any horizontal or amount neurotoxin induction.For example, this is included in test point (wherein test point can be with That test sample is for example added to 6 hours or longer time after cell) at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% substrate (this hair Bright polypeptide) cracking.Suitable time point after test sample to be added to cell is (for example, as used above " test point ") other examples include at least 24 hours, 48 hours or 72 hours.

" cracking of neurotoxin induction " refers to the cracking of polypeptide of the invention, wherein the cracking is due to neurotoxin Activity caused by (that is, this does not include due to for example non-specific polypeptide degradation of other mechanism or by other non-nerves The cracking of polypeptide caused by toxin enzymatic lysis).The size of standard technique such as ELISA or pyrolysis product of this field can be used (such as on Western blotting) identifies the cracking of neurotoxin induction, to confirm that the cracking of neurotoxin induction has occurred and that. The detection instruction neurotoxin activity of the cracking of the neurotoxin induction of polypeptide is (for example, the level or quantity and test specimens of cracking The active horizontal or quantity of neurotoxin is proportional in product).In the context of the present invention, term " neurotoxin induction split Solution " and " neurotoxin activity " may be used interchangeably.

In certain embodiments of the invention, it may be advantageous using negative control (as described above).For example, can be with Determine that the cracking of the polypeptide of neurotoxin induction is horizontal, it is then that it is horizontal with the polypeptide cleavage in negative control sample or with it is more The predetermined reference level of peptide cracking is compared, wherein surveying compared with control sample or compared with predetermined reference level existing The increase of cracking level shows that there are the cracking that neurotoxin induces in the case where test agent.Negative control, which is included in, is not present test In the case where sample or in the presence of the active sample of known shortage neurotoxin (such as heat-inactivated test sample), in phase Same cell is cultivated under the conditions of.

Additionally, or alternatively, may be advantageous using positive control (such as to ensure when test toxoid False negative is not generated when remaining neurotoxin activity).The example of positive control can be known comprising functional nerve toxin Test sample in the presence of, cultivate same cell under the same conditions.

It in certain embodiments, the use of there is the antibody of specificity to target nerve toxin pyrolysis product may be advantageous 's.The example of this kind of antibody is (seeing also Fig. 5 and corresponding legend) described elsewhere herein.

The exploitation and use of other positive controls and negative control appropriate are completely in the conventional energy of those skilled in the art Within the scope of power.

Kit

It is also provided herein for detecting the active kit of neurotoxin, wherein the kit includes

(a) nucleic acid molecules of polypeptide are encoded, the polypeptide includes the N- terminal polypeptide structural domain with uciferase activity With with the active C- terminal polypeptide structural domain of VAMP1, VAMP2 or VAMP3；With

(b) for detecting the active positive control of neurotoxin, wherein the positive control can crack the nucleic acid by (a) The polypeptide of molecule encoding.

Neurotoxin activity can be selected from the group being made up of: methods of preparing tetanus activity, Type B clostridium botulinum Nervous toxicity Plain activity, D type botulic neurotoxin activity, F type botulic neurotoxin activity and G type botulic neurotoxin are living Property, or any combination thereof.

(a) nucleic acid molecules (and encoded polypeptide) are described in detail in elsewhere herein.

Suitably, nucleic acid molecules can be a part of expression vector and/or (such as can lose in the cell of genetic modification Pass the SiMa neuroblastoma cell of modification) it is interior (for example, forming part of it；It is contained therein).It is also well described herein The cell of expression vector comprising such nucleic acid molecules and genetic modification.

Kit provided herein also includes for detecting the active positive control of neurotoxin, wherein the positive control It can crack by the polypeptide of the nucleic acid molecule encoding of (a).

In one embodiment, positive control may include the methods of preparing tetanus of appropriate amount, Type B clostridium botulinum nerve At least one in toxin, D type botulic neurotoxin, F type botulic neurotoxin and G type botulic neurotoxin Kind.As described elsewhere herein, neurotoxin can be naturally occurring or its modified forms, and wherein modified forms retain mind Through neurotoxin active.

Optionally, kit also includes the reagent for detecting uciferase activity.Suitable reagent is in the art It is well known that including but not limited to luciferin, furimazine, coelenterazine (coelenterazine), ATP and other known to Luciferase substrate [Beyond D-luciferin:expanding the scope of bioluminescence imaging in vivo.Adams ST Jr,Miller SC.Curr Opin Chem Biol.2014Aug；21:112-20].

Kit may include following any or whole: the polypeptide of measurement reagent, buffer and one or more cracking The selective binding companion of segment, it is all these to may be housed in the container for being suitble to transport.Selective binding companion can be with Antibody including one of polypeptide fragment with cracking selective binding.The example of such antibody (is gone back described elsewhere herein Referring to Fig. 5 and corresponding legend).

In some embodiments, kit includes selective binding companion on the continuous surface of solids.Illustratively Kit includes equipped with the container for detecting binding partners and for detecting the active positive control of neurotoxin.

In addition, kit may include guiding material, the guiding material includes the finger using the material provided in kit Lead (i.e. scheme).Although guiding material generally includes written or printing material, can store it is such guidance and by its It is communicated in any medium of end user and guiding material is provided.Suitable medium includes but is not limited to electronic storage medium (example Such as disk, tape (tape), cassette tape (cartridge), chip) and optical medium (such as CD ROM).Medium can wrap It includes and the address of the internet sites of guiding material is provided.Such guidance can be consistent with any method as described herein or purposes.

Unless being defined otherwise herein, otherwise all technical and scientific terms used herein has and fields of the present invention The identical meanings that are generally understood of those of ordinary skill.For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons, NY (1 94)；And Hale And Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) are Those skilled in the art provide the universaling dictionary of many terms used in the present invention.Although with those described herein method Or the similar or equivalent any method and material of material can be used in practice of the invention, but this document describes preferred sides Method and material.Therefore, term defined below is described more fully in reference book on the whole below.In addition, as herein Used, unless the context is clearly stated, otherwise singular references " one ", "one" and " described " include plural.Unless It is otherwise noted, otherwise nucleic acid is write from left to right with 5' to the direction 3'；Amino acid sequence with amino to carboxyl direction from left to right It writes.It should be understood that the present invention is not limited to described ad hoc approach, scheme and reagents, because they can be according to this field skill Environment that art personnel use and change.

Following non-limiting embodiment demonstrates each aspect of the present invention.