阅读说明：本技术 重启l-型细菌中合成噬菌体基因组 (Restart in L-type bacterium and synthesizes phage genome ) 是由 马丁·约翰内斯·勒斯纳 萨穆埃尔·基尔彻 帕特里克·施图德 于 2018-02-08 设计创作，主要内容包括：本发明涉及产生或繁殖工程化噬菌体的方法,包括以下步骤：提供能够感染靶细菌的工程化噬菌体的功能性合成基因组；提供受体细菌；在转化步骤中用所述功能性合成基因组转化所述受体细菌,产生转化的受体细菌；在第一孵育步骤中孵育所述转化的受体细菌,其中所述工程化噬菌体繁殖于所述转化的受体细菌内；和在第二孵育步骤中,用所述靶细菌进一步孵育所述转化的受体细菌或从所述转化的受体细菌中释放出的所述繁殖的工程化噬菌体,其中所述繁殖的工程化噬菌体感染所述靶细菌并在所述靶细菌内进一步繁殖,并且其中所述受体细菌是细胞壁缺陷型细菌。(The present invention relates to the methods generated or breeding is engineered bacteriophage, comprising the following steps: provides the functional synthesis genome for the engineering bacteriophage that can infect target bacteria；Acceptor bacterium is provided；The acceptor bacterium is converted with the functional synthesis genome in step of converting, generates the acceptor bacterium of conversion；The acceptor bacterium of the conversion is incubated in the first incubation step, wherein engineering bacteriophage breeding is in the acceptor bacterium of the conversion；With in the second incubation step, the engineering bacteriophage of the breeding for being further incubated for the acceptor bacterium of the conversion with the target bacteria or being released from the acceptor bacterium of the conversion, it wherein target bacteria described in the engineering phage-infect of the breeding and is further bred in the target bacteria, and wherein the acceptor bacterium is Cell wall deficiency bacterium.)

1. a kind of method of generation or breeding engineering bacteriophage, comprising the following steps:

The functional synthesis genome for the engineering bacteriophage that target bacteria can be infected is provided,

Acceptor bacterium is provided,

The acceptor bacterium is converted with the functional synthesis genome in step of converting, generates the acceptor bacterium of conversion,

The acceptor bacterium of the conversion is incubated in the first incubation step, wherein engineering bacteriophage breeding is in described turn In the acceptor bacterium of change, and

In the second incubation step, the acceptor bacterium of the conversion is further incubated for target bacteria or from the receptor of the conversion Bacterium release breeding the engineering bacteriophage, wherein target bacteria described in the engineering phage-infect bred and It is further bred in the target bacteria,

Wherein the acceptor bacterium is Cell wall deficiency bacterium.

2. according to the method described in claim 1, wherein the acceptor bacterium is gram-positive bacterium or as derived from it Cell wall deficiency variant, specifically listeria (Listeria) bacterium, specifically monocyte hyperplasia Lee This special Salmonella (Listeria monocytogenes) or Cell wall deficiency variant as derived from it.

3. method according to any of the preceding claims, wherein the target bacteria is gram-positive bacterium, specifically Ground is selected from listeria (Listeria), bacillus (Bacillus), enterococcus spp (Enterococcus), streptococcus Belong to (Streptococcus), fusobacterium (Clostridium) or staphylococcus (Staphylococcus), more specifically Listeria monocytogenes (Listeria monocytogenes), Yi Wan Novi Listeria (Listeria Ivanovii), listera innocua (Listeria innocua), bacillus subtilis (Bacillus subtilis), wax-like Bacillus (Bacillus cereus), bacillus thuringiensis (Bacillus thuringiensis) or golden yellow grape Coccus (Staphylococcus aureus).

4. method according to any of the preceding claims, wherein the acceptor bacterium and the target bacteria belong to or spread out It is born from member that is not of the same race or not belonging to.

5. method according to any of the preceding claims, wherein the functional synthesis genome passes through its segment It assembles or is provided by de novo formation method in vitro or in vivo.

6. method according to any of the preceding claims, wherein the functional synthesis genome has at least 10, 000 base-pair, specifically at least 30,000 base-pair, more specifically at least 40, the length of 000 base-pair.

7. method according to any of the preceding claims, wherein the step of converting is in polyethylene glycol, specifically Average molecular weight range is 1,000g*mol^-1~30,000g*mol^-1, more specifically 7,000g*mol^-1~20,000g* mol^-1Polyethylene glycol, specifically carried out in the presence of PEG-8000.

8. method according to any of the preceding claims, wherein first incubation step is 4~9h's in range In period, specifically implemented within the period that range is 24~32h.

9. method according to any of the preceding claims, wherein the acceptor bacterium in Cell wall synthesis by interfering Antibiotic is especially selected from beta-Lactam antibiotic, glycopeptide antibiotic, seromycin or phosphonomycin and optional osmotic protection Incubated cell is provided with wall other bacterial in the presence of medium, generates the acceptor bacterium.

10. a kind of method for producing listeria Cell wall deficiency bacterium, comprising the following steps:

It provides with genotype [Δ lmo0584 Δ lmo1653-54 Δ lm01861] or does not include lmo0584, lmo1653-54 The listeria bacterium being characterized with the genome of the functional homologue of lmo01861, specifically monocyte hyperplasia Listeria spp, more specifically Listeria monocytogenes bacterial strain EGD-e, or

The bacterium of listera innocua kind is provided, specifically the bacterium of the bacterial strain 2021 of listera innocua；With

In the presence of Cell wall synthesis interferes antibiotic and optionally the bacterium is cultivated in the presence of osmotic protection medium.

11. according to the method described in claim 10, it includes beta-lactam that wherein the Cell wall synthesis interference antibiotic, which is selected from, Antibiotic, glycopeptide antibiotic, seromycin or phosphonomycin group in, specifically benzyl penicillin.

12. a kind of Cell wall deficiency bacterium of listeria, it is characterised in that genotype [Δ lmo0584 Δ lmo1653- 54 Δ lm01861] or the functional homologue not comprising lmo0584, lmo1653-54 and lmo01861 genome.

13. the Cell wall deficiency bacterium, wherein the bacterium is Listeria monocytogenes, it is specifically single Monocytogenes Listeria bacteria strain EGD-e.

14. Cell wall deficiency bacterium according to claim 12 or 13, can be by described in claim 10 or 11 Method obtains.

15. a kind of method of generation or phage propagation, comprising the following steps:

The functioning gene group that the bacteriophage of target bacteria can be infected is provided,

Acceptor bacterium is provided,

The acceptor bacterium is converted with the functioning gene group in step of converting, generates the acceptor bacterium of conversion,

The acceptor bacterium of the conversion is incubated in the first incubation step, wherein bacteriophage breeding in the conversion by In body bacterium, and

In the second incubation step, the acceptor bacterium of the conversion is further incubated for the target bacteria, wherein that breeds is described Target bacteria described in phage-infect is simultaneously further bred in the target bacteria,

Wherein the acceptor bacterium is Cell wall deficiency bacterium, and the acceptor bacterium and the target bacteria belong to or derive From member that is not of the same race or not belonging to.

Technical field

The present invention relates to for generation or phage propagation, it is specifically engineered the method and apparatus of bacteriophage (mean)。

Background technique

Bacteriophage (Bacteriophage) bacteriophage (phage)) it is special bacterial infection and the disease for constituting its natural enemy Poison.Most of phage-infects give the specific subset of bacterial species bacterial strain without targeting other closely related bacteriums. Due to its special specificity and bacteriolyze potentiality, bacteriophage is used for various biomedical and biotechnology applications.On the one hand, it Be used as quickly and the diagnostic tools of Sensitive Detection bacterial pathogens living.On the other hand, virulent phage is used for specificity The BIOLOGICAL CONTROL of species effectively removes potential pathogen from industrial production chain and food.Further, since antibiotics resistance Property increase, European Union and the U.S. (WHO and CDC) be estimated to be 48,000 death every year, and bacteriophage is as substitution antimicrobial Application be the concerned field risen again, and Phage therapy method shows promising effect.It considers The low discovery rate of conventional antibiotic, and combine the increasingly increase of existing public health threat antibiotic-resistant bacteria, it assumes that phagocytosis The recovery of body treatment continues to be reasonable.Although bacteriophage has high category-and kind-specificity, self-replication and low production Cost, but bacteriophage is faced with many challenges, and these challenges still limit their applications in biomedical environment: due to list The host range of only bacteriophage is limited, and phage mixture needs to be designed all medicine that could cover pathogenic species related Bacterial strain.General to obtain bacteriophage product, specifically the regulatory approval of phage mixture is challenging, and inspection framework It is unclear.In addition, lysogeny (mild) bacteriophage can be integrated into the host genome without inducing cell lysis, And it can even promote the propagation of antibiotic resistant by transduction or bacterial virulence is increased by molten former conversion, effectively arrange In addition to their purposes as biological control agent.In addition, target cell can encode numerous bacteriophage drug resistance mechanisms, including by Body diversification, biofilm formation and CRISPR interference etc..By modifying the genome of bacteriophage, these many limits can be overcome System, and other beneficial characteristics can be included in phage genome.

Unfortunately, the genome projectization of virulent phage is mostly difficult and the process of work intensive type.Big In most cases, phage genome during infection by with carry homologous sequence and it is required heredity change plasmid it is homologous Recombination is modified.Because phage replication quickly and recombination fraction usually very low (10^-4~10^-10), screen recombinant phage It is labor-intensive, and needs to introduce reporter gene.Recently, researcher proposes a kind of essence for modifying phage genome U.S. synthetic method: yeast artificial chromosome (YAC) and overlapping phage genome segment be assembled in yeast cells in vivo from And generate the complete recombination group being trapped in YAC.Then, YAC- phage DNA is converted into Escherichia coli to restart (reboot) these bacteriophages.Up to the present, this method is limited to infect gram-negative cells and has host's dependent/non-dependent The T7 family viral of copy feature.Whether similar method is suitable for different bacteriophage family and/or infection Gram-positive The bacteriophage of cell, it is current or unknown.

Therefore, the letter for being also applied for being suitble to the complete recombinant phage genome of bacteriophage of Gram-positive host is generated Single effective ways, still deposit needs.

Summary of the invention

It based on the above background, specifically include infection gram the object of the present invention is to provide phage propagation is used for The simple and effective method and apparatus of the bacteriophage of positive host cell.

The purpose is by according to claim 1 or method for claim 10 and Cell Wall Deficient according to claim 12 Type bacterium and realize.

Accordingly, the first aspect of the present invention is related to for phage propagation, the method for the bacteriophage being specifically engineered. Method includes the following steps:

The functioning gene group that the bacteriophage of target bacteria can be infected is provided,

Acceptor bacterium is provided,

The acceptor bacterium is converted with functional genome in step of converting, generates the acceptor bacterium of conversion,

The acceptor bacterium of (incubate) described conversion is incubated in first incubation (incubation) step, wherein institute Bacteriophage breeding is stated in the bacterium of the conversion, and

The acceptor bacterium of the conversion is further incubated for the target bacteria in the second incubation step, wherein described numerous Target bacteria described in the phage-infect grown simultaneously further is bred in the target bacteria,

Wherein the acceptor bacterium is Cell wall deficiency bacterium.

In the context of the present specification, term " Cell wall deficiency " bacterium refers to the culture medium in seepage stability In with other Cell wall deficiency variants with wall bacterium of Cell Wall Deficient state active proliferation.They lack the multilayer peptide Glycan coating (envelope), usually limitation for example convert gram-positive bacterium with big DNA molecular.Cell wall deficiency is thin Bacterium is also referred to as L-type bacterium, L- phase (phase) bacterium or L- phase variant.

Therefore, this Cell wall deficiency bacterium specifically metabolic active cells wall deficiency bacterium and/or can The Cell wall deficiency bacterium of active proliferation.Specifically, this Cell wall deficiency bacterium can be it is temporary or permanent Cell Wall Deficient.

In the context of the present specification, the term " synthesis genome " specifically refers to artificial or non-naturally-occurring Genome.Equally, in the context of the present specification, the term " bacteriophage of engineering " specifically refer to it is artificial or Non-naturally occurring bacteriophage, the bacteriophage especially characterized by synthesizing genome.

Specifically, the functioning gene group is in the form of a kind of " naked " nucleic acid or a variety of " naked " nucleic acid, for example, not having Protein coat or coating, are converted.

Alternatively, the bacteriophage of the breeding described in second incubation step is incubated for the target bacteria, Described in breeding bacteriophage from the acceptor bacterium of the conversion, in particular by the acceptor bacterium for cracking the conversion, For example, being released by osmotic shock (osmotic shock) method.

The method described in the present invention is a kind of for recombinating or production, breeding, reactivation or the engineering of natural bacteriophage New method, it is compared with prior art, faster and more reliable.This method does not need to screen suitable recombinant or introduce and can examine The reporter gene of survey.In addition, it is suitable for infecting the very wide range of bacteriophage of gram-positive organism.This method is to life At the major step stepped with required biomedical and biotechnology characteristic customization bacteriophage.Advantageously, institute of the invention The method of stating overcomes the limitation of known method, and is therefore widely used in the bacteriophage of infection gram-positive organism.

Specifically, the step of converting and/or first incubation step are implemented in osmotic protection medium.

In the context of the present specification, term " osmotic protection medium (osmoprotective the medium) " tool The culture medium for making the Cell wall deficiency bacteria living and/or growth is referred to for body, and is further included especially Non-toxic water soluble infiltration with the osmotic pressure between the Cell wall deficiency bacterium and the culture medium lower than the concentration of threshold value Reactive compound, higher than the rupture that Cell wall deficiency bacterium will occur for the threshold value.The non-limiting example of such compound Including non-toxic organic acid or its salt, such as succinate, carbohydrate or nontoxic salts, such as ammonium sulfate or sodium chloride.

Specifically, second incubation step is implemented in the case where osmotic protection medium is not present.

In some embodiments, the step of conversion is implemented in liquid medium.In certain embodiments In, first incubation step is implemented in liquid medium.In some embodiments, second incubation step exists Implemented in fluid nutrient medium.

In some embodiments, the osmotic protection medium include be specifically 0.075mol*L with concentration range^-1 ~0.5mol*L^-1Succinate.In some embodiments, the osmotic protection medium includes monosaccharide, disaccharides or trisaccharide, tool Glucose or sucrose for body.In some embodiments, the osmotic protection medium includes glycerol.In certain embodiments In, the osmotic protection medium includes that concentration range is 0.25mol*L^-1~0.75mol*L^-1, specifically 0.5mol*L^-1's Sucrose.

In some embodiments, the acceptor bacterium is the Cell wall deficiency variant or derivative of gram-positive bacterium From the Cell wall deficiency variant of gram-positive bacterium.

In the context of the present specification, the term " being derived from " specifically refers to corresponding bacterium, for example, parent is thin Born of the same parents with wall gram-positive bacterium, by, for example, when such as there is Cell wall synthesis interference antibiotic under condition of culture and/or The process of Cell wall deficiency bacterium is converted in the presence of osmotic protection medium.

In some embodiments, the acceptor bacterium is that the Cell wall deficiency of listeria (Listeria) is thin Bacterium.In some embodiments, the acceptor bacterium is Listeria Monocytogenes (Listeria Monocytogenes Cell wall deficiency variant) or the Cell wall deficiency derived from Listeria monocytogenes become Body.In some embodiments, the acceptor bacterium is Listeria monocytogenes EGD-e (Listeria Monocytogenes EGD-e) Cell wall deficiency variant or cell derived from Listeria monocytogenes EGD-e Wall deficiency variant.In some embodiments, the acceptor bacterium is listera innocua (Listeria innocua) Cell Wall Deficient variant or Cell wall deficiency variant derived from listera innocua.In some embodiments, it is described by Body bacterium is listera innocua 2021 (Listeria innocua 2021), specifically, listera innocua bacterial strain SLCC 5639 (Listeria innocua strain SLCC 5639) (extraordinary Listeria culture collection (Special Listeria Culture Collection), University of Wuerzburg (Univ.of Wurzburg), Germany) Cell Wall Deficient Type variant, or Cell wall deficiency variant as derived from it.

In some embodiments, the target bacteria is gram-positive bacterium.In some embodiments, the target is thin Bacterium is selected from listeria (Listeria), bacillus (Bacillus), enterococcus (Enterococcus), streptococcus (Streptococcus), fusobacterium (Clostridium) or staphylococcus (Staphylococcus).In certain embodiment party In formula, the target bacteria is selected from Listeria monocytogenes (Listeria monocytogenes), Yi Wannuo common vetch Li Si Special bacterium (Listeria ivanovii), listera innocua (Listeria innocua), bacillus subtilis (Bacillus Subtilis), Bacillus cercus (Bacillus cereus), bacillus thuringiensis (Bacillus ) or staphylococcus aureus (Staphylococcos aureus) thuringiensis.

In some embodiments, the acceptor bacterium and the target bacteria belong to or derived from not of the same race.In certain realities It applies in mode, the acceptor bacterium and the target bacteria belong to or derived from the member not belonged to.

In some embodiments, the acceptor bacterium is derived from the member of listeria, and the target bacteria belongs to Selected from bacillus (Bacillus), enterococcus spp (Enterococcus) streptococcus (Streptococcus), fusobacterium (Clostridium) or the category of staphylococcus (Staphylococcus).

In some embodiments, the functioning gene group is naturally occurring phage genome.In certain implementations In mode, the functioning gene group is synthesis or artificial phage genome, specifically engineering bacteriophage.

In the context of the present specification, the term " synthesis or artificial phage genome " specifically refers to artificial Non-natural nucleic acid construct.

This synthesis or artificial phage genome can be originated from natural phage genome, wherein introducing a kind of or more Kind of foreign genetic element such as gene, regulating element (for example, promoter), operon or open reading frame and/or naturally occurring Genetic elements have been replaced, have modified and/or have deleted.This synthesis phage genome is also possible to be originated from a variety of different biologies The chimera (mosaic) of a variety of genetic elements.

In some embodiments, the functioning gene group, specifically functional synthesis or artificial gene group, lead to It crosses and assembles its segment in vitro or in vivo and provide.Specifically, the segment of the functioning gene group can pass through de novo formation Method, PCR cloning PCR or amplification and provide, wherein provided by segment may then pass through methods known in the art and be assembled in institute It states in functioning gene group.The non-limiting example of assembled in vitro is the Ji Busen (Gibson) assembling, wherein described above-mentioned Segment shares overlap when assembling to it.The non-limiting example assembled in vivo is the yeast assembling, wherein ferment Mother cell is converted with the above-mentioned also segment comprising overlap, and the segment is assembled in the yeast cells.

In some embodiments, the functioning gene group, the specifically functional synthesis or artificial gene group, It is provided by de novo formation method.

In some embodiments, the synthesis or artificial phage genome are originated from temperate bacteriophage, wherein the conjunction At or artificial phage genome lack the cracking circulation or the lysogeny control zone repressor gene.

In some embodiments, the functioning gene group is linear or circular nucleic acid molecules.In certain embodiments In, the functioning gene group is single-stranded or double-stranded RNA or DNA molecular.

In some embodiments, the functioning gene group has at least 10, the length of 000 base-pair.Certain In embodiment, the functioning gene group has the length of at least 30.000 base-pairs.In some embodiments, described Functioning gene group has the length of at least 40.000 base-pairs.In some embodiments, the functioning gene group tool There is the length of at least 120,000 base-pairs.In some embodiments, the functioning gene group has 10,000 base The length range of right~160,000 base-pairs.In some embodiments, the functioning gene group has 35,000 alkali Base is right~length ranges of 160,000 base-pairs.In some embodiments, the functioning gene group has 40,000 The length range of base-pair~130,000 base-pair.

In some embodiments, the functioning gene group is originated from siphovirus (Siphovirus), specifically grows Tail virus TP21-L, siphovirus 2638A, siphovirus P35, siphovirus B025, siphovirus B035, siphovirus B056, siphovirus PSA or siphovirus P70 or myovirus (Myovirus), specifically myovirus A511, flesh tail Viral P100, myovirus Bastille (Myovirus Bastille) or bacteriophage K or podovirus (podovirus).

In some embodiments, the step of converting is implemented in the presence of polyethylene glycol.In certain embodiments In, the polyethylene glycol has 1,000g*mol^-1~30,000g*mol^-1Average molecular weight.In some embodiments, institute Polyethylene glycol is stated with 7,000g*mol^-1~20,000g*mol^-1Average molecular weight.In some embodiments, described poly- Ethylene glycol is PEG-8000.In some embodiments, the step of converting is about 6% (w/v)~about 36% in concentration range (w/v) implemented in the presence of polyethylene glycol.In some embodiments, the step of converting is about 24% (w/ in concentration V) implemented in the presence of polyethylene glycol.

Specifically refer to the molecule of the corresponding polyethylene glycol about the term " average molecular weight " of polyethylene glycol Measure the arithmetic average or intermediate value of distribution.This average molecular weight can by methods known to those skilled in the art, for example, logical Either statically or dynamically light scattering (SLS, DLS), size exclusion chromatography or gel electrophoresis is crossed to be measured.

In some embodiments, first incubation step carries out within a period of time of 4 hours to 96 hours ranges Implement.In some embodiments, first incubation step carries out real within a period of time of 24 hours to 32 hours ranges It applies.

In some embodiments, first incubation step 15 DEG C to 37 DEG C at a temperature of, specifically at 20 DEG C Implemented at a temperature of to 32 DEG C.In some embodiments, second incubation step 15 DEG C to 37 DEG C at a temperature of, Specifically 20 DEG C to 32 DEG C at a temperature of implemented.In some embodiments, the step of converting is at 15 DEG C to 37 Within the temperature range of DEG C, specifically implemented within the temperature range of 20 DEG C to 37 DEG C.

In some embodiments, the acceptor bacterium in the presence of Cell wall synthesis interferes antibiotic by incubating Hatching cell is provided with wall other bacterial (cell walled precursor bacterium), to generate the recipient cell Bacterium.Specifically, the other bacterial is incubated in the presence of Cell wall synthesis interferes antibiotic is greater than other bacterial in the present invention Under the conditions of doubling time a period of time.In some embodiments, the other bacterial interferes antibiosis in Cell wall synthesis It is incubated in the presence of element 2 days to 5 days, specifically 3 days to 4 days.

In some embodiments, the Cell wall synthesis interference antibiotic is selected from beta-Lactam antibiotic, glycopeptide antibiosis Element, phosphonomycin (fosfomycin) or seromycin.

In some embodiments, the beta-Lactam antibiotic is selected from cephalosporins (cephalosporins), carbon Cephalosporanic olefinic (carbacephems), monobactam class or penicillins.

In some embodiments, the Cell wall synthesis interference antibiotic is benzyl penicillin.

Alternatively, the acceptor bacterium can by cell with the protein level or genome water in wall other bacterial It is flat upper to inhibit albumen relevant to Cell wall synthesis, or by the presence of cell wall degradation or lyases such as lysozyme and permeating It is incubated for the cell band wall other bacterial in the presence of protection medium and provides.

According to another aspect of the present invention, it provides a kind of for producing the Cell wall deficiency bacterium of listeria Method.Method includes the following steps:

There is provided with genotype [Δ lmo0584 Δ lmo1653-54 Δ lm01861] or do not include lmo0584, The listeria bacterium that the genome of the functional homologue of lmo1653-54 and lmo01861 is characterized, or

The bacterium of listera innocua kind is provided, specifically the bacterium of listera innocua bacterial strain 2021, it is more special specific For listera innocua bacterial strain SLCC 5639 (extraordinary Listeria culture collection, WüRzburg, Germany university, moral State)；With

It in the presence of Cell wall synthesis interferes antibiotic and is optionally cultivated in the presence of osmotic protection medium described thin Bacterium.

The term " lmo0584 " refer in Listeria monocytogenes EGD-e gene (gene I/D: 984661, NCBI gene database).

The term " lmo1653-54 " refer in Listeria monocytogenes EGD-e two genes (gene I/D: 985674 and gene I/D: 985673, NCBI gene databases).

The term " lmo01861 " refer in Listeria monocytogenes EGD-e gene (gene I/D: 985831, NCBI gene databases).

The term " functional homologue " refers to identical function and the nucleic acid in the context of the present specification The different gene of sequence.

In some embodiments, the listeria bacterium by make gene lmo0584, lmo1653-54 and Lmo01861 or its homologue are inactivated and are provided.In some embodiments, described gene lmo0584, lmo1653-54 and The deletion of lmo01861 or its homologue inactivated through the gene or its homologue, in the gene or its homologue Base replace, missing, insertion or sequence inversion and implemented.

In some embodiments, the Cell wall synthesis interference antibiotic is selected from beta-Lactam antibiotic, specifically It is cephalosporins, carbacephems, monobactam class or penicillins, glycopeptide antibiotic class, phosphonomycin or circumfili ammonia Acid.

In some embodiments, the listeria bacterium is Listeria Monocytogenes.In certain realities It applies in mode, the listeria bacterium is Listeria monocytogenes bacterial strain EGD-e

In some embodiments, the bacterium is cultivated at a temperature of range is 20~32 DEG C.

Alternatively, the Cell wall deficiency bacterium can pass through inhibition and institute on protein level or genomic level The relevant albumen of Cell wall synthesis is stated, or by being situated between in the presence of cell wall degradation or lyases such as lysozyme and in osmotic protection Band wall bacterium is incubated in the presence of matter and is produced.

According to another aspect of the present invention, a kind of Cell wall deficiency bacterium of listeria is provided, wherein described Bacterium is characterized in that the genotype [Δ lmo0584 Δ lmo1653-54 Δ lm01861], or do not include lmo0584, The genome of the functional homologue of lmo1653-54 and lmo01861.

In some embodiments, the Cell wall deficiency bacterium is Listeria monocytogenes.In certain realities It applies in mode, the Cell wall deficiency bacterium is Listeria monocytogenes bacterial strain EGD-e.

In some embodiments, the Cell wall deficiency bacterium is the method energy by above-mentioned aspect according to the present invention Method enough obtain or by above-mentioned aspect according to the present invention obtains.

Anywhere listed herein using the substitute of single detachable feature as " embodiment ", it should be understood that Be each embodiment that these substitutes could be freely combined and be formed present invention disclosed herein.

The present invention is further illustrated by following embodiment and attached drawing, by these embodiment and attached drawing it can be concluded that into The embodiment and advantage of one step.These embodiments are intended to illustrate the present invention, rather than limit its range.

Detailed description of the invention

Fig. 1 shows restarting with the Listeria phage genome of L-type bacterial strain Rev2L.(A-B) restart L-type bacterium The ability of the Listeria bacteriophage of strain Rev2L is carried out as described in (A) using the genomic DNA of Listeria bacteriophage P35 Evaluation.L-type conversion reaction is prepared as shown in (B), at 32 DEG C be incubated for and in post-conversion 24 hours when test the finger Show that the bacterial plaque on bacterial strain forms (plaque formation).DNAseI indicates P35 gDNA using 30 minutes of DNAseI in advance Digestion.(C) efficiency for converting and restarting is measured using the dilution series of P35 gDNA.(D) other seven Listerias are bitten The group of thallus is restarted in Rev2L using 500~1000ng gDNA, and uses Listeria monocytogenes or Yi Wannuo Dimension Listeria (Listeria ivanovii) is detected as indicator strain.Bacteriophage attribute is listed in a tabular form. (E) dynamics is generated in the bacteriophage of 96 hours inner evaluations, three kinds of bacteriophage L-types.Data are average value ± SD (n=3).Φ= Bacteriophage, PEG=polyethylene glycol, gDNA=genomic DNA, tr=terminal redundancy (terminal redundancy), cp=ring Shape arranges (circular permutation), and cos=cohesive end sites, nd=is not detected.

Fig. 2 shows (A) bacillus of Listeria L-type and restarting for (B) staphylophage genome. Rev2L cell is converted with 1~5 μ g bacteriophage gDNA, is incubated at 32 DEG C for 24 hours, and measures the phagocytosis on its corresponding indicator strain Body production.Lack the gDNA or L-type transformation reaction is used as reference material.Φ=bacteriophage.

Fig. 3 shows restarting for the synthesis assembled in vitro phage genome of Listeria L-type.Synthesis is restarted in Rev2L The general work process of genome is as shown in (A).Genome phage DNA is from Listeria monocytogenes bacteriophage In P35, listera innocua bacteriophage B025 and Bacillus cercus bacteriophage (Bacillus cereus phage) TP21-L It extracts and purifies.Unless otherwise indicated, the overlapping PCR fragments for covering full-length genome are generated using exo+ polymerase (B, D, F), And it carries out assembled in vitro and generates ring molecule.Group reaction cartridge converts and restarts in Rev2L cell, and uses incomplete group Dress body is plated on corresponding indicator strain as control (C, E, G).It is small 24 using indicator strain for bacteriophage TP21-L When after detection restart reaction bacteriophage production, alternatively, 10 μ L were consolidated in 6 hours in post-conversion in the case where instruction (amplification) Determine the bacteriophage production (G) when restarting in reaction and measure for 24 hours described in the addition of HER1399 culture.

Fig. 4 shows mild Listeria bacteriophage B025 by mildly converting to the life style of toxicity.Outer-gene group Packaging strategy is used to generate mild Lee for lacking and integrating inhibiting factor (Δ rep) or complete lysogeny control zone (Δ LCR) The mutant of this special bacterium bacteriophage B025.Workflow is as shown in (A).The segment three of the wild type assembly contains molten original Property control gene, and be divided into omission only two overlapping fragments of repressor or complete LCR, to generate for genome assembling Five PCR fragments (B).Recombination group is restarted, and the obtained bacteriophage mutants are purified, and using PCR and Sequencing detects suitable genotype (C).Yi Wan Novi Listeria WSLC3009 uses the soft agar soverlay technique, using number Cumulative wild type, Δ rep and Δ LCR bacteriophage is measured to be infected.Primer pair shown in use tests surviving bacteria lawn (lawn) (B025wt) or single colonie (B025 Δ rep and B025 Δ LCR) whether there is B025 prophage (E).The molten original of LCR= Property control zone, PFU=plaque formation unit, the bacterial attachment site of attB=prophage integration, attL=WSLC3009:: Left side prophage flanking region in B025.

Fig. 5 also shows restarting for the Listeria phage genome in L-type bacterial strain Rev2L.For restarting bacteriophage The detailed operation process of genome is shown in (A), wherein each parameter is by single optimization.The parameter of optimization includes: conversion The time (B) of preceding Rev2L growth, the adjusting optical density (600nm) (C) of L-type culture when conversion, before DM3 culture medium is added The DM3 volume (E) into the DNA/L- type/PEG- mixture and used is added in final PEG concentration (D) before being incubated for The average molecular weight (F) of PEG chain.Selected optimum condition is instructed to (asterisk).The quality for restarting phage DNA used makes (G) is evaluated with pulsed field gel electrophoresis.Using the dilution series of phage DNA, the weight of bacteriophage P35 and A511 is compared Open efficiency (H).Ability of the various Listeria phage-infects with wall Rev2 cell containing Rev2 or bacteriophage by indicating Point sample (spotting) bacteriophage dilution is evaluated on the soft agar covering of bacterial strain.(I) PenG=benzyl penicillin, OD= Optical density, PEG=polyethylene glycol.Data are average value ± SD (n=3).

Fig. 6 shows restarting for the synthesis assembled in vitro phage genome of Listeria L-type.Using genomic DNA and The genome of assembled in vitro carries out the efficiency that genome is restarted as input material and compares: using 4 times of P35 gDNA Dilution series or Ji Busen (Gibson) group reaction cartridge restarts in Rev2L, with 2 μ g and 125ng input materials start respectively into Row gDNA and group reaction cartridge.Total pfu of each conversion reaction is quantified and is compared.Data are average value ± SD (n=3).

Fig. 7 shows the missing of TP21-L lysogeny control gene.It is whole that shortage is generated using outer-gene group packaging strategy Close the mild Bacillus bacteriophage TP21-L mutant of repressor (Δ rep) or complete lysogeny control zone (Δ LCR). Workflow is as shown in (A).The segment three of the wild type assembly controls gene containing the lysogeny, and is divided into province Slightly only two overlapping fragments of repressor or complete LCR, to generate five PCR fragments for genome assembling.Recombinate base Because group is restarted, the obtained bacteriophage mutants are purified, and test suitable genotype using PCR and sequencing (B).LCR=lysogeny control zone.

Fig. 8 shows L-type bacterial strain Rev2 (Listeria Monocytogenes) and by non-pathogenic listera innocua Liszt's phage genome in reversible L-type that 2021 (SLCC 5639) are obtained is restarted.Described two L-type cultures Each converted with the genomic DNA (1 μ g) of Listeria monocytogenes bacteriophage P70, be incubated at 32 DEG C For 24 hours, and Listeria monocytogenes L99 is used to be formed as indicator strain test bacterial plaque.Soft-agar overlay is (after infection 24 hours) bacterial plaque be revealed.

Specific embodiment