Target and its application of the PVY-CP as the drug of screening prevention and treatment marmor upsilon
阅读说明：本技术 Pvy-cp作为筛选防治马铃薯y病毒的药物的靶标及其应用 (Target and its application of the PVY-CP as the drug of screening prevention and treatment marmor upsilon ) 是由 李向阳 谢鑫 刘涛 于 2019-08-31 设计创作，主要内容包括：本发明属于基因的功能与应用领域领域,涉及一种PVY-CP作为筛选防治马铃薯Y病毒的药物的靶标及其应用。本发明提供一种筛选防治马铃薯Y病毒的药物的靶标,所述靶标是马铃薯Y病毒外壳蛋白即PVY-CP。本发明还提供一种马铃薯Y病毒外壳蛋白即PVY-CP作为药物靶标在筛选防治马铃薯Y病毒的药物中的应用。本发明进一步提供一种以马铃薯Y病毒外壳蛋白即PVY-CP为靶标的防治马铃薯Y病毒的药物。本发明针对PVY-CP在病毒传播过程中的作用机制,首次将PVY-CP作为潜在抗病毒药剂的作用靶标,对药物活性小分子进行体外筛选,进行药剂小分子在烟草活体植株内的活性检测,结合蛋白免疫印迹法证明了PVY-CP可以作为筛选防治马铃薯Y病毒的药物的靶标。(The invention belongs to the function of gene and application field field, it is related to target and its application of a kind of drug of PVY-CP as screening prevention and treatment marmor upsilon.The present invention provides a kind of target of the drug of screening prevention and treatment marmor upsilon, and the target is marmor upsilon coat protein i.e. PVY-CP.A kind of application the present invention also provides marmor upsilon coat protein, that is, PVY-CP as drug targets in the drug of screening prevention and treatment marmor upsilon.The present invention further provides a kind of using marmor upsilon coat protein, that is, PVY-CP as the drug of the prevention and treatment marmor upsilon of target.The present invention is directed to mechanism of action of PVY-CP during viral transmission, for the first time using PVY-CP as the action target of potential antiviral agent, in-vitro screening is carried out to pharmaceutical activity small molecule, Activity determination of the medicament small molecule in tobacco plants is carried out, binding protein Western blot demonstrates the target that PVY-CP can be used as the drug of screening prevention and treatment marmor upsilon.)
1. a kind of target of the drug of screening prevention and treatment marmor upsilon, which is characterized in that the target is outside marmor upsilon Glutelin, that is, PVY-CP.
2. marmor upsilon coat protein, that is, PVY-CP is as drug targets in the drug of screening prevention and treatment marmor upsilon Using.
3. using marmor upsilon coat protein, that is, PVY-CP as the drug of the prevention and treatment marmor upsilon of target.
4. drug according to claim 3, which is characterized in that the drug is virazole.
Technical field
Target and its application the present invention relates to a kind of PVY-CP as the drug of screening prevention and treatment marmor upsilon, belong to The function and application field of gene.
Background technique
In recent years, viral diseases of plants brings massive losses to agricultural product quality and quantity in agricultural production process, Wherein it is particularly acute with virus disease caused by marmor upsilon (Potato virus Y, i.e. PVY).PVY infects the side of plant Formula is systemic infection.Plant, which has infected, can show different disease symptoms in field after PVY, this and PVY virus strain make The factors such as the weather conditions of article kind and locality are closely bound up.In general, plant at the initial stage of a disease when, it may appear that leaf The phenomenon that piece bright arteries and veins, color can gradually become shallower as between the arteries and veins of subsequent vein, and it is mottled to eventually form system, flower leaf paresthesia occur.And have Plant morbidity when, vein will appear the necrotic tissue of crineous or black, and downright bad part would generally extend to The vein at middle part or stalk part cause crease blade to contract inward curl, show arteries and veins necrosis symptom.When the susceptible situation of plant When serious, the pathogen of downright bad part can also enter the vascular tissue of stem, and at this time the stalk of plant generates brown necrosis group It knits, stalk necrosis symptom occurs.
The difference of the symptom showed on host after plant is infected according to PVY, can be substantially three by PVY points Strain: common strain, arteries and veins necrosis strain and point striped strain.Same strain in different pin main bodies they can also show Different symptom out.After potato is infected by the PVY of different strains, common strain can cause shrinkage, and arteries and veins necrosis strain can draw Rise a degree of mottled, and putting striped strain can then cause allergic reaction.After tobacco is infected, common strain, which will appear, is It unites mottled symptom, arteries and veins necrosis strain will appear the more serious system arteries and veins necrosis symptom of degree, and putting striped strain can then cause The mottled symptom of different degrees of system.
Although being in progress at present to the basic research of marmor upsilon disease, also there is a few medicament energy on the market This kind of disease is effectively administered, but its specific mechanism of action is unclear, research and development is caused to be directed to the special effect agent of the pathogen Difficulty is very big.How effectively to screen anti-PVY drug be prevent and treat Potyvirus disease critical issue, therefore establish one with PVY key protein is the screening model of the drug of potential target, finds the anti-PVY drug of high activity, to prevention and treatment Potyvirus Disease has great importance.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of PVY-CP as screening prevention and treatment potato Y disease The target of the drug of poison and its application.
The technical scheme is that
In a first aspect, the present invention provides a kind of target of the drug of screening prevention and treatment marmor upsilon, the target is Ma Ling Potato Y virus coat protein, that is, PVY-CP.
Second aspect, the present invention provide a kind of i.e. PVY-CP of marmor upsilon coat protein and are screening as drug targets Prevent and treat the application in the drug of marmor upsilon.
The third aspect, the present invention provide a kind of using marmor upsilon coat protein i.e. PVY-CP as the prevention and treatment Ma Ling of target The drug of potato Y virus.
Preferably, the drug is virazole.
PVY-CP plays an important role in the assembly and virus infection overall process of virion.If small point of high activity There is strong interaction between son and target protein, will directly result in protein can not play its corresponding physiological activity, To destroy viral RNA duplication and the assembling process with virion, harm of the virus to plant as a result will be greatly reduced.
The beneficial effects of the present invention are: the present invention is directed to mechanism of action of PVY-CP during viral transmission, for the first time will Action target of the PVY-CP as potential antiviral agent carries out in-vitro screening, the medicine that will be filtered out to pharmaceutical activity small molecule Agent small molecule and PVY-CP find the active binding site of medicament and PVY-CP after carrying out molecular docking, while being situated between using Agrobacterium Target protein is transferred in Ben Shi cigarette by inducing defecation by enema and suppository, carries out Activity determination of the medicament small molecule in tobacco plants, binding protein Western blot demonstrates the target that PVY-CP can be used as the drug of screening prevention and treatment marmor upsilon.
Detailed description of the invention
Fig. 1 is the interaction between MST analysis PVY-CP and antivirotic, and (A) RTC titrates PVY-CP；(B) AOBO drop Determine PVY-CP；
Fig. 2 is the interaction between MST analysis PVY-CP and antivirotic, and (A) COS titrates PVY-CP；(B) BTH drop Determine PVY-CP；
Fig. 3 is the interaction between ITC analysis PVY-CP and antivirotic, (A) blank background；(B) RTC titrates PVY- CP；
Fig. 4 is PVY-CP and WMV-CP Amino acid sequences alignment；
Fig. 5 is molecular docking；
Fig. 6 is the PVY-CP expression of mediated by agriculture bacillus.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
One, experimental material
(1) plasmid and expression bacterial strain
PVY is purchased from Wuhan Virology Institute,Chinan academy of Sciences；
BL21 (DE3) RIL strain is purchased from Takara company；
Ben Shi cigarette, common cigarette K326 can be obtained in the artificial climate hot-house culture of agricultural college, Guizhou University.
(2) for examination albumen and Antiphytoviral small molecule
PVY-CP Protein expression and purification；Diazosulfide and Moroxydine Hydrochloride are provided by Guangxi rural area；Amino-oligosaccharide It is purchased from Beihai Fisheries Base Guangxi Province promulgated by the State Council marine organisms pesticide Co., Ltd；Virazole is purchased from Hangzhou Minsheng Medcine Co., Ltd.
(3) preparation method of main buffer solution
LB solid medium: 10g tryptone, 5g yeast extract, 10g NaCl, 8g agar powder, secondary water 1000mL, It fullys shake, is uniformly mixed it, adjust PH to 7.4, sterilize 20min under the conditions of 121 DEG C, puts spare to room temperature cooling；
LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, secondary water 1000mL fully shake, It is uniformly mixed it, adjusts PH to 7.4, sterilize 20min under the conditions of 121 DEG C, puts spare to room temperature cooling；
1M Tris-HCl solution: weighing the Tris solid of 121.14g, is added 800mL secondary water, it is to be dissolved completely after make Adjusting PH with hydrochloric acid is 8.0, is finally settled to 1000mL using secondary water；
75% ethanol solution: measuring 750mL ethanol solution, and 250mL secondary water is added, stirs evenly；
0.1M CaCl2Solution: 11.1gCaCl is weighed21000mL secondary water, stirring and dissolving is added in solid；
0.02mM CaCl2Solution: 2.22gCaCl is weighed21000mL secondary water, stirring and dissolving is added in solid；
0.2M PB solution: NaH is weighed2PO4·2H2O solid 10.06g, Na2HPO4·12H2O solid 48.54g is added 800mL secondary water, fullys shake, and is uniformly mixed it, and adjusting PH is 7.4, is settled to 1000mL；
0.01M PB solution: the NaH of 0.2M is measured2PO4The Na of solution 39mL and 0.2M2HPO4Solution 61mL mixing, is added Secondary water dilutes 20 times, fullys shake, so that it is adjusted PH to 7.4 after mixing, be settled to 2000mL；
2 × polyacrylamide gel sample-loading buffer: weighing SDS solid 0.4g, bromophenol blue solid 0.1mg respectively, respectively It measuring 0.5M Tris-HCl 2.5mL (PH 6.8), glycerol 2mL, beta -mercaptoethanol 0.2mL are eventually adding secondary water 5.2mL, It fullys shake, is uniformly mixed it, holding chamber warm standby is used；
5 × electrophoretic buffer: weighing Tris solid 15.1g, glycine 94g, SDS5.0 g respectively, and secondary water is added about 800mL fullys shake, and is uniformly mixed it, is finally settled to 1000mL, room temperature preservation is spare；
In conjunction with buffer solution (buffer A): 50mM Tris, 150mM NaCl, 20mM Imidazole, 10% glycerol, PH 8.0；
Elution buffer solution (buffer B): 50mM Tris-HCl, 150mM NaCl, 300mM Imidazole, 10% Glycerol, PH 8.0；
Crack buffer solution: 50mM Tris-HCl, 150mM NaCl, 1% beta -mercaptoethanol, 10% glycerol, PH 8.0；
Desalination buffer solution: 2mM DTT, 10mM PB, 150mM NaCl, PH 7.5.
PBST solution: measuring the PB solution 1000mL of 0.01M, and 3gNaCl is added, and dissolution is sufficiently stirred i.e. in 3mL Tween-20 It can；
5% skimmed milk power solution (being configured with PBST): weighing 2.5g skimmed milk power, 50mLPBST solution is added, sufficiently It stirs evenly, makes it dissolve.
Two, the vector construction of PVY-CP
(1) extraction of tobacco total serum IgE
1. common cigarette K326 plant is maintained in 24 DEG C of artificial greenhouse, the illumination and 8 that setting condition is 16 hours are small When dark cycle, mature tobacco plant is inoculated with PVY, and after three weeks, the extraction for selecting leaf for RNA is tested；
2. weighing susceptible tobacco greenery about 100mg, liquid nitrogen is added, is ground into powdery, ground powder is shifted Into the 1.5mL centrifuge tube of enzyme deactivation, the RNAiso Plus of 1mL is added, is suspended uniform；
3. suspension is placed in a centrifuge centrifugation 5min, setting condition is 4 DEG C, 12000rpm；
4. supernatant is transferred in 1.5mL RNAase centrifuge tube；
5. the chloroform that volumetric concentration is 20% is added into the supernatant being transferred out of, sufficiently suspending is uniformly mixed it, in 5min is stood under the room temperature of room, until there is liquid layered phenomenon；
6. mixed liquor is placed in a centrifuge centrifugation 15min, condition is 4 DEG C, 12000rpm；
7. supernatant is transferred in 1.5mLRNAase centrifuge tube；
8. isometric isopropanol is added to the supernatant being transferred out of, sufficiently suspending is uniformly mixed it, quiet at -20 DEG C Set 20min；
9. mixed liquor is placed in a centrifuge centrifugation 10min, condition is 4 DEG C, 12000rpm；
10. abandoning supernatant, 75% ethanol solution 1mL is added into precipitating, sufficiently suspending is uniformly mixed it；
11. mixed liquor is placed in a centrifuge centrifugation 5min, condition is 4 DEG C, 12000rpm；
12. abandoning supernatant, 5min is placed under room temperature, remaining ethanol solution is made sufficiently to volatilize；
13. secondary water of the 30 μ l Jing Guo destroy the enzyme treatment is added into residue precipitating.
(2) synthesis of cDNA
Total serum IgE is diluted to the concentration of 10 μ g/ μ L, and is analyzed by 1% agarose gel electrophoresis, according to TaKaRa company CDNA Synthesis System kit synthesize the first chain of cDNA.
Susceptible tobacco RNA about 1g is taken, 5 × Prime ScriptTM Buffer, Prime Script are separately added into TMEnzyme Mix, Oligo dNTPrimer and Random 6mers finally add RNase free H2O is to 20 μ L (reactants Refer to TakaRa kit specification).It is 37 DEG C that instrument condition, which is arranged, and reverse transcription reaction is carried out after 15min；85 are arranged again DEG C, 5s is the condition of the inactivation reaction of reverse transcriptase.
(3) PCR amplification
Primer needed for expanding PVY-CP according to the sequence design submitted in NCBI, and be sent to the raw work biology in Shanghai and closed At as shown in the table:
PVC-CP-F:CGggatccATGCATGCCCGGTGGTAGCAGC；
PVC-CP-R:CCGctcgagCTCGAGCATATTTTTCACACCCAGC；
Above-mentioned primer is respectively as shown in SEQ ID NO.1,2
Identify that specific primers carry out PCR amplifications by template and 2 couples of PVY-CP of the cDNA of synthesis, and it is separately added into 10 × PCR Bufffer、MgCl2(25mmol/L), dNTP (10mmol/L) and Taq enzyme (5U/ μ L) finally add deionized water to 25 μ L.Amplification condition are as follows: the annealing temperature of 94 DEG C of 3min, 20 circulations, 94 DEG C of 50s, 65 DEG C of 50s, 72 DEG C of 50s, each circulation reduce 1℃.Then 25 circulation 50s are carried out at 94 DEG C, 55 DEG C of progress 50s, 72 DEG C of progress 50s, final incubation time is 10min. After reaction, 5 μ L pcr amplification products is taken to carry out 0.8% polyacrylamide gel electrophoresis.
DNA molecular amount is that standard judges whether the segment of target gene expands.PCR product is recycled, is purified, it will be pure PCR product after change is sent to Dalian treasured biotech firm and is sequenced, and compares software using the online BLSAT of NCBI and carry out sequence point Analysis.
(4) T4 ase enzyme connection PVY-CP gene and pET28a plasmid
The PVY-CP gene of double digestion is subjected to enzyme with T4 ase enzyme with pET28a plasmid and connects reaction, while being carried out without mesh Gene blank control group.
1 PVY-CP gene of table and pET28a plasmid enzyme disjunctor system
(5) double digestion PVY-CP gene and pET28a plasmid
PET28a plasmid and the PVY-CP target gene of recycling carry out the double enzyme digestion reaction of two kinds of BamH I, Xho I enzymes.This Two kinds of restriction enzymes are purchased in precious biotech firm.After being separately added into various reactants according to following table, under the conditions of 37 DEG C Carry out endonuclease reaction, about 2-3h, in 60 DEG C of inactivation 15min after having reacted.
2 double digestion PVY-CP gene of table and pET28a plasmid
Three, the expression and purification of PVY-CP
(1) preparation of competent cell
1. the picking monoclonal colonies from the plate newly activated are inoculated into the clean centrifuge tube containing 100mL LB culture medium In, in cultivating 4h or so on 37 DEG C, the shaking table of 200rpm condition, until OD 600 is 0.45；
2. sample is placed in about 10min on ice；
3. bacterium solution is placed in a centrifuge centrifugation 15min, condition is 4 DEG C, 4000rpm；
4. abandoning supernatant, the 0.1M CaCl of 30mL pre-cooling is added2Solution, sufficiently resuspension cell；
5. mixed liquor is placed in a centrifuge centrifugation 4min, condition is 4 DEG C, 5000rpm；
6. abandoning supernatant, 4 DEG C of 0.02M CaCl being pre-chilled in advance are added again2-0.08M MgCl2Solution 2mL, again Cell is resuspended；
7. the cell suspension of resuspension is dispensed, and hasten to freeze using liquid nitrogen, be placed under -80 DEG C of refrigerated conditions It saves backup.
(2) conversion of pET28a-His-PVY-CP recombinant plasmid
1. taking (DE 3) RIL of competent cell BL 21 of 100 μ L to be added 2 μ L's under superclean bench aseptic condition PET28a plasmid；
2. sample is placed in about 30min on ice；
3. set 42 DEG C for water-bath, after temperature to after 42 DEG C by sample heat shock 80s；
4. sample is placed in about 2min on ice；
5. the LB liquid medium that 900 μ L contain that antibiotic of card is added into the centrifuge tube equipped with sample；
6. bacterium solution is placed in shaking table and cultivates 45min, instrument is set as 150rpm；
7. bacterium solution is placed in a centrifuge centrifugation 5min, instrument is set as 4000rpm, is abandoned on 900 μ L after centrifugation Liquid is clear, sufficiently resuspension thallus, draws 100 μ L bacterium solutions with pipettor and is applied on LB solid plate；
It is incubated overnight 8. culture dish is put into 37 DEG C of incubators.
(3) expression of PVY-CP
1. 10mL is added into the 50mL centrifuge tube of sterilizing in the picking monoclonal on the plate for grow monoclonal colonies LB liquid medium and 30ng/mL card that antibiotic, in overnight incubation on 37 DEG C, the shaking table of 200rpm；
2. 1M IPTG is added, is placed in 16 DEG C later, the shaking table of 200rpm when the OD 600 for surveying bacterium solution reaches 0.65 Upper culture about 14h；
3. bacterium solution is placed in a centrifuge centrifugation 25min, instrument is set as 5000rpm, abandons supernatant, collects thallus；
4. being placed in about 10min on ice, cracking buffer solution about 40mL is added into centrifuge tube, is placed in and is resuspended on ice 10min；
5. carrying out clasmatosis, ultrasound condition setting using ultrasonic cell disintegration instrument are as follows: 40%power, ultrasonic 4s stop 6s, Continuous ultrasound 35min；
6. the good suspension of ultrasound is fitted into the centrifuge tube of sterilizing, in 4 DEG C, in the centrifuge under the conditions of 12,000rpm It is centrifuged 30min, retains supernatant, 20 μ L supernatants are separately subjected to 12%SDS-PAGE detected through gel electrophoresis.
(4) purifying of PVY-CP
1. the solution prepared in advance (combination buffer and elution buffer) is taken out with 0.22 μm of water system miillpore filter Filter out solid impurity, and ultrasonic bubble removing；
2. cleaning peristaltic pump about 10mL with 20% ethanol solution, then with secondary water cleaning pump about 10mL, use after having cleaned BufferA rinses Ni column about 20mL, and the supernatant that centrifugation obtains is crossed Ni column about 2h；
3. carrying out gradient elution to Ni column with albumen affinity chromatography system；
4. after having eluted, successively cleaning purification system, about 50mL using 20% ethanol solution and secondary water；
5. the albumen efflux at peak position out is carried out 12%SDS-PAGE detected through gel electrophoresis, screening target protein is deposited Efflux；
6. destination protein is concentrated with 10KDa super filter tube；
7. carrying out de-salting operation to the destination protein after concentration using desalting column:
8. obtained albumen is carried out 12%SDS-PAGE detected through gel electrophoresis again, and destination protein is fast with liquid nitrogen Freeze, is placed in -80 DEG C of refrigerators and saves.
Four, the verifying of PVY-CP
(1) PVY-CP is verified by polyacrylamide gel electrophoresis
Gel verifying is carried out to the protein sample that purifying obtains using PAGE gel electrophoresis, the formula of running gel is such as Table 3.
Electrophoresis step is as follows:
1. the protein sample of 20mL is added into 2 × SDS sample-loading buffer of 20mL, it is sufficiently mixed uniformly；
2. water-bath 10min under the conditions of mixed liquor is placed in 100 DEG C；
3. the sample after 15 μ L denaturation is added into the sample cell of gel, deposition condition is arranged are as follows: concentration glue 90V, 20min；Separation gel 120V, about 80min；
4. taking out gel after the completion of electrophoresis, dyeing processing is carried out to gel with Coomassie Brilliant Blue dye, is examined after the completion of dyeing Mas bright blue destainer carries out decolorization, after the completion of decolourizing, observes protein band.
The preparation of 3 12%SDS-PAGE running gel of table
(2) PVY-CP destination protein is identified by high-resolution mass spectrometer
1. PAGE gel block to be cut into the blob of viscose of 1mm 3 or so, it is fitted into clean 10mL centrifuge tube；
2. slowly rinsing blob of viscose about with 50% acetonitrile solution and NH 4 HCO 3 (pH 8.0) solution of 100mM 10min repeated washing 3 times, abandons cleaning solution；
3. the liquid on blob of viscose surface is blotted with Speed Vac；
4. by albumen blob of viscose soaking in the DTT solution of 10mM and the NH of 50mM4HCO3In (PH 8.0) mixed liquor, slowly Ground is gradually heated to 56 DEG C, blots the liquid on blob of viscose surface again later；
5. albumen blob of viscose to be immersed in the imidazole solution of 55mM and the NH of 50mM4HCO3In (PH 8.0) mixed liquor, black Dark place soaking about 30min at room temperature, blots the liquid on blob of viscose surface again later；
6. by the 10mM NH of blob of viscose 100mL4HCO3Solution slowly cleans about 10min, and blob of viscose surface is blotted after having cleaned Solution；
7. slowly washing about 10min with the acetonitrile solution of 100mL, blob of viscose surface solution is blotted after having cleaned；
8. blob of viscose surface liquid is sufficiently drained with Speed Vac, about 5min；
9. the Promega trypsin enzyme solutions after 5mL dilution are added into the centrifuge tube equipped with blob of viscose, keep enzyme solutions complete Full submergence blob of viscose；
10. a large amount of 4 HCO of 10mM NH, 3 solution about 20mL, the blob of viscose for making it be enough to cover swelling is added；
11. soaking is stayed overnight under conditions of 37 DEG C；
12. isometric 60%ACN and 5% formic acid water mixed solution, sonic oscillation about 10min is added；
13. being centrifuged about 40min under conditions of 20000g, supernatant is retained；
14. the 60%ACN/5% aqueous formic acid of about 80mL, sonic oscillation about 10min are added to remaining blob of viscose；
15. being centrifuged about 40min under conditions of 20000g, the supernatant retained with the last time is mixed；
16. after the supernatant of Collection and conservation is drained, residual filter residue is dissolved in the aqueous formic acid of 30 μ L 0.1%, on Mass spectrometer is detected.
Five, the repercussion study of PVY-CP and antiviral agent
(1) ITC studies the interaction of PVY-CP albumen and antiviral small molecule
Sample preparation: if antiviral small molecule is water soluble compound, can directly be dissolved with PBS buffer solution, if anti- Viral small molecule is insoluble chemical compound, then should first be dissolved with DMSO, then be diluted to required concentration with PBS buffer solution.Note that To be always ensured that the final volumetric concentration of DMSO is not higher than the 5% of medical fluid total volume when solvent medicament；And the buffer of albumen Same solution is necessary for the buffer of dissolution small molecule.The instrument condition of ITC should be tentatively arranged are as follows: referring to 5 μ of power Cal/s, 25 DEG C, the total frequency injection of agitator speed 750rpm/min 20 times.
(2) MST studies the interaction of PVY-CP albumen and antiviral small molecule
This experiment will move instrument to virazole (RTC), Moroxydine Hydrochloride using the micro thermophoresis of Monolith NT.115 (AOBO), the interaction of the antiviral small molecule such as amino-oligosacchride (COS) and diazosulfide (BTH) and PVY-CP are ground Study carefully.
Before experiment starts, it should which the dilution that drug is carried out to concentration gradient is formulated as the medical fluid of 16 various concentrations (500、250、125、62.5、31.25、15.63、7.81、3.91、1.95、0.98、0.49、0.24、0.12、0.06、0.03、 0.015 μM), the PVY-CP of prepared drug and fluorochrome label carries out the incubation under dark, about 30min.
The instrument parameter of MST should be arranged are as follows: LED power 40%, Laser power 30%, Red excitation
Six, the internal living body verifying of high activity medicament
(1) preparation of Agrobacterium competence
The preparation of Agrobacterium competent cell
1. the pYBA1132 bacterium solution of -80 DEG C of preservations is taken to cross on the LB solid plate of rifampin and kanamycins, it is placed in Overnight incubation in 28 DEG C of incubators；
2. picking single colonie is inoculated in sterile centrifugation tube after growing single colonie, 5mL LB liquid medium is added, in 12~14h is cultivated in shaking table under the conditions of 28 DEG C of 200rpm；
3. drawing 2mL bacterium solution in sterile centrifugation tube, and 100mL LB liquid medium is added, in 28 DEG C of 220rpm conditions Under shaking table in cultivate about 5h, until OD 600 be 0.5；
4. bacterium solution is completely transferred in sterile 50mL centrifuge tube, it is placed in about 15-30min on ice；
5. in the centrifuge under the conditions of 4 DEG C, being centrifuged about 5min in 3,000rpm, abandoning supernatant；
6. CaCl precooled in advance is added into the precipitating of reservation2Solution 10mL, slowly gently suspending cell；
7. by suspension in placing about 20min on ice；
8. in the centrifuge under the conditions of 5000rpm, being centrifuged about 5min in 4 DEG C, abandoning supernatant；
9. the CaCl that 2mL is pre-chilled in advance is added into 50mL suspension2Solution, slowly gently suspending cell；
10. agrobacterium suspension is sub-packed in sterile enzyme deactivation centrifuge tube, about 100 μ L of every pipe is stored under the conditions of -80 DEG C It is spare.
(2) Agrobacterium-mediated Transformation
Electric shocking method converts Agrobacterium
1. placing ice chest in clean station, taking 1ul plasmid to be added in 100 μ l competent cells, gently mix It is even；
2. sample is transferred to after mixing in the electric shock cup completely to sterilize, it is electroporated；
3. the about 300 μ l of LB liquid medium of antibiotic-free is added in electric shock cup, gently mix；
4. the bacterium solution after mixing is transferred into the centrifuge tube of the 1.5mL of sterilizing；
5. cultivating about 4h in the shaking table under the conditions of 180rpm in 28 DEG C；
6. the about 100 μ l of thallus suspension after culture is taken to be coated on the LB solid plate containing rifampin and kanamycins On；
7. being put into after culture dish is inverted in 28 DEG C of incubator and cultivating 3d.
(3) Agrobacterium is injected
1. pYBA1132-PVY-CP (wt) Agrobacterium single bacterium after picking conversion is fallen in sterile centrifugation tube, 5mL is added The rifampin and kanamycins of LB liquid medium and 50 μ g/mL are cultivated about in the shaking table under the conditions of 180rpm in 28 DEG C 2d；
2. the rifampin and Ka Na of the MES solution of 10mM, the acetosyringone of 40 μ L, 50 μ g/mL are added with 1% ratio The LB liquid medium of mycin and 10mL cultivates about 16h in 28 DEG C in the shaking table under the conditions of 180rpm；
3. bacterium solution is placed in 20 DEG C, it is centrifuged about 10min in the centrifuge under the conditions of 4000rpm, abandons supernatant；
4. the MgCl of 10mM is added into precipitating2And MES solution, suspension thalline, adjust OD 600 to 1,5.
5. the agrobacterium suspension concentration OD 600 containing P19 is adjusted to 1.5 using same operational means；
6. after above two agrobacterium suspension is mixed in equal volume, being placed in about 3h at dark at room temperature；
7. drawing agrobacterium suspension with syringe to be injected into tobacco piece, leaf growth situation is observed.
(4) it sprays medicament and carries out WB analysis
1. choosing fresh blade after 3d to the fresh blade spraying Candidate Agents virazole for showing illness, WB is carried out.
2. taking a small amount of leaf tissue, liquid nitrogen is added, is fully ground broken；
3. 0.5mL, which is added, contains the protein extract of PMSF, fully shake, be uniformly mixed it, after be placed in it is quiet on ice Set about 10min；
4. mixed liquor is placed in a centrifuge centrifugation 15min, condition is 4 DEG C, 15000g；
5. supernatant is transferred in the centrifuge tube of clean enzyme deactivation, it is placed on ice；
6. the protein extract that 0.5mL contains PMSF is added again into precipitating, fully shake, is uniformly mixed it, after It is placed in and stands about 10min on ice；
7. mixed liquor is placed in a centrifuge centrifugation 15min, condition is 4 DEG C, 15000g；
8. being transferred to the centrifuge tube containing the supernatant obtained for the first time for obtained supernatant is centrifuged for the second time
In, the summation of supernatant is the total protein extracted twice；
9. taking a certain amount of protein sample, the identification of SDS-PAGE running gel is carried out；
10. removing glass plate after the completion of electrophoresis, gel is taken out, by extra glue (including bromophenol blue band and blank glue) water Truncation removes；
11. the length and width of the glue after measuring excision with ruler, cut with a paper cutter and cut 6 slightly bigger filter paper and one Open pvdf membrane；
12. the pvdf membrane reduced methanol soaking 30s is used secondary water soaking 3-5min after taking-up, it is finally putting into and turns It is saved backup in film buffer；
13. blob of viscose and the filter paper cut are totally placed in soaking about 5min in transferring film buffer, save backup；
14. in the black plate face of plastic clip plate successively smooth bubble-free spread sponge, three layers of filter paper, blob of viscose, pvdf membrane, Three layers of filter paper, operation must guarantee that filter paper and film edge are neatly smooth；
15. rubber plate clamp is put into electrophoretic blotting groove, transferring film buffer is added into electrophoresis tank until flooding blob of viscose；
16. electrophoresis tank is placed in condensation cabinet, condensation cabinet temperature setting is 4 DEG C；
17. starting transferring film operation, 75V, 60mA, 12h are set by electrophoresis apparatus；
18. after electrophoresis, taking out pvdf membrane, being placed in clean kit, Ponceaux staining solution is added, soaking is big About 30s is slowly rinsed twice after taking-up with the secondary water after sterilizing；
19. pvdf membrane is placed in the centrifuge tube of 50mL sterilizing, 5% skimmed milk power solution 50mL is added, is placed in 4 DEG C condensation cabinet in about 12h；
20. overnight solution is abandoned, 3% 3 μ L of skimmed milk power solution 3mL and primary antibody is added, places at room temperature, incubates Educate 2h；
21. slowly rinse pvdf membrane using PBST solution, about 3 times are repeated, every time about 5min；
22. after the solution of flushing is abandoned, 3% 3 μ L of skimmed milk power solution 3mL and secondary antibody is added, place at room temperature, It is incubated for 2h；
23. slowly rinse pvdf membrane using PBST solution, about 3 times are repeated, every time about 5min；
24. after the solution of flushing is abandoned, the chemiluminescent substrate of HRP is added, about 5min is stood；
25. detecting luminous signal with X- mating plate in darkroom.
Seven, result verification
(1) verifying of pET28a-PVY-CP recombinant vector
PET28a-PVY-CP recombinant vector is verified with double digestion, is produced and the sizable item of PVY-CP after double digestion Band and empty plasmid band, it can be seen that illustrate the success of pET28a-PVY-CP (wt) construction of recombinant vector.
The recombinant plasmid that verifying reassembles into function after double digestion is sent to Shanghai Sheng Gong biotech firm and carries out gene sequencing, Sequence submitted in obtained sequencing result and NCBI is compared.Comparison result shows the PVY-CP of inspection The gene order (GenBank accession number AAV63981.1) that sequence has been filed on other has 98% gene similitude.All knots Fruit is provable, and pET28a-PVY-CP (wt) recombinant plasmid of building has succeeded.
(2) identification of PVY-CP albumen
When taking purifying out the albumen efflux of peak position and the sample after desalination carry out it is denatured by boiling after, use 12%SDS- PAGE electrophoresis obtains obvious band by the affine column purification of Ni-NAT, and size meets the size 36KDa of target protein.Desalination Albumen efflux afterwards, size also correspond to the size 36KDa of target protein, the target protein that can tentatively obtain purifying It is determined as PVY-CP.And it by the way that peptide fragment is compared, send and is covered in the peptide fragment sample of identification with reported PVY-CP Lid rate is up to 62.7%, the results showed that the albumen of purifying is PVY-CP.
(3) antiviral agent and the external interaction research of PVY-CP
1, MST studies antiviral agent and the external interaction of PVY-CP
With MST technical research virazole (RTC), Moroxydine Hydrochloride (AOBO), amino-oligosacchride (COS) and diazosulfide (BTH) interaction between PVY-CP.Studying discovery antivirotic virazole by MST has strong phase interaction to PVY-CP With.Dissociation constant between RTC and PVY-CP is Kd=4.56 μM.Antivirotic Moroxydine Hydrochloride has weak phase interaction to PVY-CP With the dissociation constant between AOBO and PVY-CP is Kd=77.5 μM (Fig. 1).Therefore the subsequent experimental of this experiment will use disease-resistant Positive control of the toxic agent virazole as Chemicals.
Linear fit can not be carried out by the experimental result that MST studies discovery antivirotic COS and BTH and PVY-CP, because This infers COS and BTH and PVY-CP without interaction (Fig. 2).
2, ITC studies antiviral agent and the external interaction of PVY-CP
Discovery virazole is studied by MST and PVY-CP has interaction in vitro, therefore will use ITC research verifying medicament Interaction between small molecule and albumen.PVY-CP is subjected to ITC titrimetry with the solvent of medical fluid and RTC medical fluid respectively, As a result as shown in Figure 3.After being analyzed and processed to obtained data result, binding constant Ka=2.47 × 10 are obtained3, the result It is consistent with MST result, comprehensive experimental data twice is it can be shown that RTC and PVY-CP have interaction in vitro.
3, Medicine small molecule and PVY-CP molecular docking situation
Using the method for Blast search, the simulation building of PVY-CP structure is carried out.By compare find, PVY-CP with Watermelon mosaic virus (WMV) CP homology is 66.41% (Fig. 4), therefore constructs PVY-CP according to WMV-CP Three-dimensional structure.
The model of foundation and active small molecular are subjected to molecular docking, molecular docking situation is shown in Fig. 5.The active group of RTC The equal amino acid residues of arginine (R) 153, arginine (R) 179, aspartic acid (D) 197 and lysine (K) 217 are deep into be formed Active pocket in, tentatively infer and exist simultaneously different degrees of binding force between the Medicine small molecule and this four amino acid.
(4) the internal living body verifying of high activity medicament
Expression of the PVY-CP in Ben Shi cigarette plants is probed into using agrobacterium-mediated transformation and in conjunction with WB means, it is real Test the following Fig. 6 of result.The results show that as the spraying concentration of antivirotic virazole gradually increases, the protein band of PVY-CP It is gradually dimmed, and control group is without significant change.This is the experimental results showed that in plants, expression of the virazole to PVY-CP There is certain inhibiting effect.
Nucleotide series table:
<110>Guizhou University
<120>target and its application of the PVY-CP as the drug of screening prevention and treatment marmor upsilon
<160>2
<210>1
<211>30
<212>DNA
<213>artificial sequence
<400>1
CGggatccAT GCATGCCCGG TGGTAGCAGC 30
<210>2
<211>34
<212>DNA
<213>artificial sequence
<400>2
CCGctcgagC TCGAGCATAT TTTTCACACC CAGC 34
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>target and its application of the PVY-CP as the drug of screening prevention and treatment marmor upsilon
<160> 2
<210>1
<211>30
<212> DNA
<213>artificial sequence
<400>1
CGggatccAT GCATGCCCGG TGGTAGCAGC 30
<210>2
<211>34
<212> DNA
<213>artificial sequence
<400>2
CCGctcgagC TCGAGCATAT TTTTCACACC CAGC 34