CN104673758A - Duck Tembusu virus infectious clone strain, and preparation method and application thereof - Google Patents
Duck Tembusu virus infectious clone strain, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a duck Tembusu virus infectious clone strain. The infectious clone strain is a clone strain of a parent toxic duck Tembusu virus strong strain FX2010. The clone strain has the complete genome sequence disclosed as SEQ ID NO.1. The infectious clone strain is prepared by a reverse genetic manipulation process. The invention also discloses a preparation method and application of the duck Tembusu virus infectious clone strain. The duck Tembusu virus infectious clone strain can be used for developing a novel duck Tembusu virus vaccine, can be used as a virus vector for expressing an exogenous gene, and is an important tool for molecular biology research of duck Tembusu virus.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of duck tembusu virus infections clone strain and its preparation method and application.
Background technology
Duck tembusu virus (Duck Tembusu virus, DTMUV) is a kind of flavivirus causing duck egg drop reduction, growth retardation and death.Since spring in 2010, the ground such as Shanghai City, Jiangsu Province, Zhejiang Province, Anhui Province break out a kind of a kind of neopathy causing the underproduction of egg duck, meat duck growth retardation and death in succession, and research proves to cause the cause of disease of this disease to be tembusu virus (Tembusu virus).This virus all has virulence to egg duck and meat duck, and sick duck main manifestations is high heat, dyskinesia, appetite decline, and even useless exhausted, egg drop reduction even stops, and seriously can cause death, mortality ratio can reach 5%-10%.It is reported that the sick financial loss caused of duck tembusu virus is more than 5,000,000,000 yuan.
Duck tembusu virus interior syndrome state continent occurs first, and this disease has hyperinfection, and throughout the year all can be popular, brings great difficulty to the prevention and control of duck tembusu virus disease, and at present the most effective means of this disease of prevention is vaccine immunity.But, still lack the vaccine of the prevention and control duck tembusu virus disease of highly effective and safe at present.
Summary of the invention
The present invention will solve the technical problem of the prevention and control duck tembusu virus disease vaccine lacking highly effective and safe at present, a kind of duck tembusu virus infections clone strain is provided, this duck tembusu virus infections clone strain is the infections clone strain of the duck tembusu virus virulent strain FX2010 utilizing reverse genetics manipulation technology to obtain, it can not only be used for development of new duck tembusu virus disease vaccine, and can be used as virus vector expression alien gene.
In addition, the preparation method and application that a kind of above-mentioned duck tembusu virus infections clone strain is provided also are needed.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of duck tembusu virus infections clone strain, this infections clone strain is the clonal strain of parent's poison duck tembusu virus virulent strain FX2010, it has the complete genome sequence shown in SEQ ID NO.1, and this infections clone strain is obtained by reverse genetic manipulation method.
Preferably, described reverse genetic manipulation method comprises the following steps:
By segmentation amplification, cDNA clones and fusion DNA vaccine, obtain the full-length cDNA of the duck tembusu virus virulent strain FX2010 containing transcriptional promoter sequence;
Described cDNA is obtained RNA through in-vitro transcription, then this RNA transfection cell is rescued from cell the infections clone strain obtaining duck tembusu virus virulent strain FX2010.
Preferably, described transcripting promoter is T7 promotor.
In another aspect of this invention, provide a kind of recombinant vectors, comprise the partial nucleic acid fragment in following duck tembusu virus virulent strain FX2010 whole genome sequence: 3656-6353 position nucleic acid fragment, 6216-9775 position nucleic acid fragment, 9753-10991 position nucleic acid fragment, 3656-9775 position nucleic acid fragment or 3656-10991 position nucleic acid fragment in SEQ ID NO.1 sequence.
In another aspect of this invention, additionally provide a kind of nucleic acid molecule, comprise the complete genome sequence that the 1-1512 position nucleic acid fragment of the FX2010 of duck tembusu virus virulent strain shown in SEQ ID NO.1 whole genome sequence, 1397-2896 position nucleic acid fragment, 2643-4020 position nucleic acid fragment or SEQ ID NO.1 sequence 5 ' end is also equipped with transcripting promoter.
In another aspect of this invention, additionally provide a kind of preparation method of duck tembusu virus infections clone strain, comprise the following steps:
Each DNA fragmentation of segmentation amplification duck tembusu virus full-length genome;
Part DNA fragmentation being cloned into carrier respectively, then obtaining the recombinant vectors containing covering duck tembusu virus genome 3656-10991 position nucleotide sequence through subclone;
The full-length cDNA of the duck tembusu virus containing transcriptional promoter sequence is obtained by fusion DNA vaccine;
Described cDNA is obtained RNA through in-vitro transcription, then this RNA transfection cell is rescued from cell the infections clone strain obtaining duck tembusu virus.
In another aspect of this invention, additionally provide a kind of above-mentioned duck tembusu virus infections clone strain prevent in preparation or treat the application in the vaccine of duck tembusu virus disease.
In another aspect of this invention, the application of a kind of above-mentioned duck tembusu virus infections clone strain as virus vector is additionally provided.
In another aspect of this invention, additionally provide a kind of restructuring duck tembusu virus, this recombinant virus the E gene of above-mentioned duck tembusu virus infections clone strain is replaced with the E gene of duck tembusu virus low virulent strain and the restructuring duck tembusu virus that is built into.
Duck tembusu virus infections clone strain of the present invention, development of new duck tembusu virus disease vaccine can not only be used for, and can virus vector expression alien gene be used as, simultaneously also for study further the genome structure of duck tembusu virus, function and and virus host interaction mechanism important research tool is provided.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the FX2010 pnca gene group Full-length cDNA Construction strategy schematic diagram of the embodiment of the present invention 1;
Fig. 2 is the FX2010 strain point fragment amplification gene group electrophorogram of the embodiment of the present invention 1;
Fig. 3 is fusion total length 3 ' (A) and 5 ' (B) two halves end electrophorogram of the embodiment of the present invention 1;
Fig. 4 is the FX2010 full-length cDNA amplification electrophorogram of the embodiment of the present invention 1;
Fig. 5 is 72 hours DF-1 cellular change figure after the embodiment of the present invention 2 transfection;
Fig. 6 is the RT-PCR qualification result figure of the embodiment of the present invention 2;
Fig. 7 is the embodiment of the present invention 2 Revive virus cells infected IFA detected result figure;
Fig. 8 is the embodiment of the present invention 2 inoculated into chick embryo pathology figure;
Fig. 9 is the embodiment of the present invention 3 recombinant virus Full-length cDNA Construction strategy schematic diagram;
Figure 10 is the electrophoresis result figure of the embodiment of the present invention 3 recombinant virus genomes DNA cloning;
Figure 11 is 80 hours DF-1 cellular change figure after the embodiment of the present invention 3 transfection.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, usual condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the chief editors such as J.G. Sai Deman, Ma Xuejun, Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.
The present invention utilizes the methods such as segmented-PCR, Partial Fragment clone, fusion DNA vaccine to obtain duck tembusu virus FX2010 strain full-length cDNA containing T7 promoter sequence, successfully save out duck tembusu virus FX2010 strain by the method such as in-vitro transcription, transfection, establish the reverse genetic operating system (also known as infections clone) of this virus.First extract the RNA of duck tembusu virus FX2010 strain, utilize RT-PCR method, divide fragment amplification viral full-length genome, the end sequence of adjacent segment is overlapped.Fragment before viral genome 4020 is increased by the mode of fusion DNA vaccine and obtains, as the first half section of full-length gene; According to the position of restriction enzyme site single in viral genome, by the sequence clone of virus genomic 3656 to 10991 on pSIMPLE18 blunt vector, thus obtain covering duck tembusu virus second half section genomic plasmid 3-N-pSIMPLE.Introduce T7 promotor at 5 ' end, cut 3-N-pSIMPLE with ApaLI enzyme and obtain the second half section, utilize the method for fusion DNA vaccine to obtain the total length PCR primer of this virus.Then utilize the method for in-vitro transcription to prepare and there is infective viral cell-free transcription folder, after purifying, to DF-1 cell, transfection notices that observation of cell changes, compared with the control, the cell of transfected virus RNA starts to occur pathology after 48 hours in transfection, and within 72 hours, pathology is obvious.Through the multiple method qualification such as RT-PCR, indirect immunofluorescence (IFA), prove virus rescue success.Through sequence alignment, the sequence of Revive virus and the sequence of duck tembusu virus FX2010 strain completely the same.
The present invention successfully utilizes the reverse genetic operating system of structure, PCR method is adopted to be replaced by the E gene of the E gene of virulent strain FX2010 with low virulent strain FX2010-180P, amplification contains the recombinant virus PCR primer of weak malicious E gene under obtaining strong malicious background, after in-vitro transcription, by the RNA transfection of acquisition to DF-1 cell, a strain restructuring duck tembusu virus is saved in success, and through order-checking qualification, the genome sequence of gained strain sequence and expection is completely the same.
The structure of embodiment 1 duck tembusu virus FX2010 pnca gene group full length cDNA clone
1. materials and methods
1.1 material
1.1.1 virus and cell
Duck tembusu virus FX-2010 strain, DF-1 cell are preserved by China Agriculture Academe Shanghai Veterinary Institute.
1.2 method
1.2.1FX2010 strain duck tembusu virus nucleic acid extraction
The RNAiso Plus reagent of Takara company is used to extract viral RNA, operate according to the prompting on product description, concrete grammar is as follows: draw 300 μ l virus liquids in 1.5ml without in RNA enzyme centrifuge tube, adds 700 μ l RNAiso plus Reagent and blows and beats mixing room temperatures and leave standstill 5 minutes; Then in above-mentioned mixed solution, add 200 μ l chloroforms, use forced oscillation 15s, room temperature leaves standstill 12000rpm4 DEG C of centrifugal 10min after 2min.Get supernatant liquor and be transferred to that another is clean without in the centrifuge tube of RNase, in supernatant liquor, add the mixing of isopyknic Virahol, room temperature to leave standstill after 10 minutes 12000rpm4 DEG C centrifugal 10 minutes.Supernatant discarded, the ethanol 1ml adding 75% along centrifugal tube wall turns upside down gently and shakes up for several times.12000rpm 4 DEG C of centrifugal 10min, abandon clean liquid, pellet dried at room temperature 5-10 minute, after 75% ethanol all volatilizees, add the RNase free water dissolution precipitation of 20 μ l.
1.2.2 reverse transcription
7 the specific reverse transcription primer (table 1) that can cover total length utilizing this lab design to synthesize, according to M-MLV RTase cDNA Synthesis Kit (Code D6130) specification sheets requirement, with the RNA of said extracted for template carries out reverse transcription.Concrete operations are as follows: get 5 μ l RNA templates, and 4 μ l mixing specific reverse transcription primers, add in the PCR reaction tubes without RNase, to be placed in PCR instrument 70 DEG C, put rapidly and 2min on ice after 10min.4 μ l 5 × first Strand Synthesis Buffer are added again, 2 μ l 10mM dNTP, 1 μ l RNase Inhibitor, 1 μ l M-MLV Reverse Transcriptase, 3 μ l DEPC H in above-mentioned process mixed solution
2o, to be then placed in PCR instrument 30 DEG C, 10min; 42 DEG C, 1h; 75 DEG C, 10min.In the cDNA that-70 DEG C of preservations obtain.
Table 1 FX2010-180P reverse transcription primer
1.2.3 point fragment design of primers and the Strategies For The Cloning of full length sequence
1.2.3.1 point fragment design of primers of full length sequence
According to the gene order of the duck tembusu virus FX2010 strain that this laboratory records, utilize the specific amplimer of Primer5 software design, synthesized by Invitrogen company, concrete primer is as table 2, the restriction enzyme site sequence that line expression sequence own contains, line and overstriking represent the restriction enzyme site sequence of introducing.
Table 2 FX2010 strain full-length gene group amplimer
1.2.3.2 point fragment design of primers and the Strategies For The Cloning of full length sequence
In-vitro transcription will be carried out, so zero position need be held to introduce T7 promoter sequence (the black bolded section in upper table in FX-S-T7-1F primer sequence is T7 promoter sequence) and protectiveness base at full length sequence 5 ' after obtaining full-length infectious CDNA.Then the mapdraw software in DNASTAR is utilized, restriction enzyme site analysis is carried out to the genome sequence of FX2010 strain, according to single restriction enzyme site, full-length genome is divided into 6 fragments to increase, and rear 3 fragments (nucleotide position is from 3656-10991) are utilized single restriction enzyme site EcoRV successively, AgeI carries out subclone, latter half genome sequence is connected to identical carrier (Fig. 1).According to above restriction enzyme site position, design primer, makes adjacent two sections of sequence fragments all cover the position at restriction enzyme site place.
1.2.4 total length divides fragment pcr amplification
Use wherein 6 pairs of primers of design in table 2, according to the design shown in Fig. 1, with viral cDNA for template, point six sections of amplifications.
Be added in PCR reaction tubes by sample by above-mentioned system, be placed in PCR instrument reaction amplification, each fragment amplification reaction conditions is as follows:
Amplified fragments 1-1512, uses primers F X-S-T7-1F, FX-1512R; Amplified fragments 1397-2896, uses primers F X-1397F, FX-2896R; Amplified fragments 2643-4020, uses primers F X-2643F (XhoI), FX-4020R; Reaction conditions: 94 DEG C, 2min; (94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 1.5min) × 30 circulations, 68 DEG C, 10min; 4 DEG C of preservations.
Use primers F X-3656F (ApaLI)-SmaI, FX-6353R-AgeI amplified fragments 3656-6353; Reaction conditions: 94 DEG C, 2min; (94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 3min) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.
Use primers F X-6216F (EcoRV), FX-9775R (AgeI) amplified fragments 6216-9775; Reaction conditions: 94 DEG C, 2min; (94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 3min30s) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.
Use primers F X-9753F (AgeI), FX-10991R-NotI amplified fragments 9753-10991; Reaction conditions: 94 DEG C, 2min; 94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 1min30s) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.
PCR primer need be identified through 0.8% agarose gel electrophoresis, and reclaims purifying.Concrete operations are as follows:
Preparation 50 × TAE Buffer, compound method: weigh Tris242g, Na2EDTA2H2O37.2g in 1L beaker; In beaker, add about 800ml deionized water, stir; Add the glacial acetic acid of 57.1ml, fully dissolve; Add after deionized water is settled to 1L, room temperature preservation.Then 0.5 × TAE Buffer is diluted to for subsequent use.
Prepare 0.8% sepharose, fill a prescription as follows: 0.8g agarose+100ml 0.5 × TAE damping fluid+5 μ l goldview, mixing, microwave boils, and glue is poured into the glue groove being plugged comb in advance.10 × loading the buffer adding 5 μ l in PCR primer respectively mixes, being added by all samples with pipettor is placed in the loading wells of 0.8% sepharose filled it up with in the tiselius apparatus of 0.5 × TAE, carry out electrophoresis, after about 30min, gel is taken out, band is observed in ultraviolet imagery system, with pocket knife, object band is cut, put into centrifuge tube, prepare to reclaim.
Reclaim test kit specification sheets according to the nucleic acid gel of Axygen and reclaim DNA fragmentation.After ultraviolet lamp incision glue, calculated weight, this weight is as a gel volume V (100mg=100 μ l volume), and add the Buffer DE-A of 3V, 75 DEG C of water-baths, melt completely to glue, about 5min.Add the Buffer DE-B of 0.5 Buffer DE-A volume, and mix; Mixed solution is transferred to DNA and prepare (as in two milliliters of centrifuge tubes) 12000rpm in pipe, centrifugal 1 minute, abandon filtrate.Preparation pipe (pillar) put back in two milliliters of centrifuge tubes, add 500 μ l Buffer W1,1200rpm, centrifugal 30 seconds, abandons filtrate.Preparation pipe (pillar) put back in 2ml centrifuge tube, add 700 μ l Buffer W2, centrifugal 30 seconds of 12000rpm, abandons filtrate, washs a 12000rpm, centrifugal 1 minute again with same method with 700 μ l Buffer W2.2ml centrifuge tube is put back, 12000rpm by preparing pipe, empty from 1 minute.To prepare pipe and be placed in the 1.5ml centrifuge tube of cleaning (new), add 25 μ l deionized waters preparing film central authorities, room temperature leaves standstill 1 minute, and 12000rpm centrifugal 1 minute eluted dna, saves backup recovery product.
1.2.5 structure is cloned
According to the specification sheets of the T4PNK enzyme of NEB company, by the PCR primer 3656-6353 reclaimed, 6216-9775,9753-10991 carry out phosphatizing treatment.Then ligation is carried out.
Adopt Tss legal system for competent cell:
Getting competent cell seed JM109 and DH5 α is inoculated in the test tube of 2ml LB, and 37 DEG C of shaking culture are spent the night; Get above-mentioned nutrient solution LB to dilute by 1:100,37 DEG C of shaking culture are about 1-3h, OD value 0.5.After culture is placed on cooled on ice, 4000r/m, 4 DEG C, supernatant discarded after centrifugal 10min, collects thalline.The LB substratum of 1 × Tss20ml:19ml is got out, 0.2033g MgCl before using
2.6H2O, the PEG4000 of 1.9g.Degerming by filtering (0.22 μ l filter membrane) after LB and PEG4000 mixing, then add DMSO1ml and joined and be placed on ice.The thalline collected is resuspended in 1 × Tss (every 100ml culture is dissolved in the 1 × Tss of 10ml), and resuspended mixing operation is carried out on ice always, is sub-packed in the Eppendorf pipe of 1.5mL with 100 μ l/ pipes.Finally be placed in-70 DEG C of cryogenic refrigerators to preserve.
By above-mentioned connection product conversion in JM109 competent cell:
In 100 μ l competent cells, add 10 μ l ligation liquid, mixing, put 30 minutes on ice, 42 DEG C of water-bath heat shock 90s, put rapidly 2min on ice, add 1ml LB nutrient solution (not containing Amp) and mix latter 37 DEG C and shake 45 minutes.Centrifugal 3 minutes of 5000rpm, abandons liquid at the bottom of supernatant 1ml pipe and hangs bacterium and be applied to containing Amp, and smoothen the LB solid medium of X-Gal, IPTG in advance, cultivates 8-20 hour for 37 DEG C.
The single bacterium colony of picking white, is inoculated in 2-3ml containing in the test tube of Amp liquid LB nutrient solution, cultivates 12-16h for 37 DEG C, with bacterium liquid for template, and PCR method qualification positive plasmid, upstream and downstream primers designed is respectively:
M13F-47:5’-CGCCAGGGTTTTCCCAGTCACGAC-3’(SEQ ID NO.22)
M13R-47:5’-GAGCGGATAACAATTTCACACAGG-3’(SEQ ID NO.23)
Reaction conditions is as follows: 1. denaturation: 95 DEG C of 5min; 2. sex change: 95 DEG C of 30s; 3. anneal: 53 DEG C of 30s; 4. extend: 72 DEG C of 1min/1000bp; 5. extend eventually: 72 DEG C of 10min; Step 2-4 is 30 circulations.
After reaction terminates, product is carried out gel nucleic acid electroresis appraisal, what have Idiotype band is positive colony, uses the little extraction reagent kit of Axygen company plasmid to extract plasmid to the bacterium liquid of positive colony:
Being divided by bacterium liquid is filled in 2ml centrifuge tube, and centrifugal 1 minute of 12000rpm, abandons supernatant.Add 250 μ l Buffer S1, make bacterial precipitation suspend evenly, should with little clump.Add 250 μ l Buffer S2 leniently to spin upside down 4-6 time, mix and make the abundant cracking of thalline until troubled liquor becomes bright solution.Add 350 μ l Buffer S3, leniently spin upside down fully mixing 6-8 time, 12000rpm, centrifugal 10 minutes.Draw centrifugal supernatant and transfer to preparation pipe (being placed in 2mL centrifuge tube) 12000rpm, centrifugal 1 minute, abandoning filtrate.Put back centrifuge tube by preparing pipe, add 500 μ l Buffer W1,12000rpm, centrifugal 1 minute, abandons filtrate.Putting back centrifuge tube by preparing pipe, adding 700 μ l Buffer W2,12000rpm, centrifugal 1 minute, and abandon filtrate, wash once with 700 μ l Buffer W2 more in the same way and abandon filtrate, put back in 2ml centrifuge tube by preparing pipe, 12000rpm, centrifugal 1 minute.Being placed in new 1.5ml centrifuge tube by preparing pipe, adding 50 μ l deionized waters in its film central authorities, room temperature leaves standstill 1 minute, centrifugal 1 minute of 12000rpm.The plasmid extracted measures its concentration through the Epoch Microplate spectrophotometer of BioTek company, and-20 DEG C save backup.
ABI company 3500 Genetic Analyser is used to check order to the positive colony plasmid extracted.First order-checking PCR is carried out.
Reaction conditions: 96 DEG C of 1min → (96 DEG C of 10sec → 50 DEG C 5sec → 60 DEG C 4min) × 25 circulations, 4 DEG C of insulations.
Reaction terminates rear use alcohol EDTA method and carries out purifying to order-checking PCR primer:
Reaction product is centrifugal 1min on little whizzer, is all transferred in the centrifuge tube of 1.5ml by 20 μ l products; Pipe adds 2 μ l 125mM EDTA, 2 μ l 3M NaAc; Add 50 μ l 100% alcohol, evenly, room temperature places 15min in concussion; 12000rpm, 4 DEG C of centrifugal 30min, outwell supernatant; Add 100 μ l 70% alcohol, 12000rpm, 4 DEG C of centrifugal 15min, outwell supernatant; Repeat 70% ethanol wash 2 times, outwell supernatant, make remaining alcohol volatilization dry; Add 10 μ l Hi-Di methane amides, piping and druming is even, 95 DEG C of sex change 4min, rapid ice-cold 4min; 10 μ l samples are joined 96 orifice plates, upper machine order-checking.Use seqman software splicing in DNASTAR to analyze sequencing result, choose correct plasmid clone.The correct plasmid clone designation of 3656-6353 fragment is 3-6-pSIMPLE plasmid; The correct plasmid clone designation of 6216-9775 fragment is 6-9-pSIMPLE plasmid; The correct plasmid clone designation of 9753-10991 fragment is 9-N-pSIMPLE plasmid.Then the structure of plasmid 3-N-pSIMPLE is carried out:
To 3-6-pSIMPLE plasmid, 6-9-pSIMPLE plasmid successively uses EcoRV, and AgeI restriction enzyme carries out enzyme and cuts.EcoRV enzyme cuts system: 5 μ l 10 × H buffer, 2 μ l EcoRV restriction enzymes, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, reclaim DNA fragmentation.AgeI enzyme cuts system: 5 μ l 10 × Buffer, 1 μ l AgeI restriction enzyme, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, extract reaction solution and carry out nucleic acid gel electrophoresis, and it is the fragment of 5469bp that 3-6-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims length, and it is the fragment of 3528bp that 6-9-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims length.
The two sections of products reclaimed are carried out ligation, and system is as follows: 1 μ l 10 × Buffer, 1 μ l T4 DNA Ligase ligase enzyme, and 7 μ l6-9-pSIMPLE plasmid enzyme restrictions reclaim product, and 1 μ l3-6-pSIMPLE plasmid enzyme restriction reclaims product.Be placed in 16 DEG C of metal baths and carry out ligation in 4 hours.Product conversion will be connected in DH5 α competence intestinal bacteria, after through choosing bacterium, liquid LB cultivates, qualification positive colony, extract plasmid, after order-checking, obtain correct positive colony 3-9-pSIMPLE plasmid (covering duck tembusu virus FX2010 pnca gene group nucleotide position is the sequence of 3656-9775).
To 3-9-pSIMPLE plasmid, 9-N-pSIMPLE plasmid successively uses NotI, and AgeI restriction enzyme carries out enzyme and cuts.NotI enzyme cuts system: 5 μ l 10 × H buffer, 2 μ l NotI restriction enzymes, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, reclaim DNA fragmentation.AgeI enzyme cuts system: 5 μ l 10 × Buffer, 1 μ l AgeI restriction enzyme, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, extract reaction solution and carry out nucleic acid gel electrophoresis, and it is the fragment of 8968bp that 3-9-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims length, and it is the fragment of 1228bp that 9-N-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims length.
The two sections of products reclaimed are carried out ligation, be placed in 16 DEG C of metal baths and connect 4 hours, then product conversion will be connected in DH5 α competence intestinal bacteria, through choosing bacterium, liquid LB cultivates, qualification positive colony, extracts plasmid, obtains correct positive colony 3-N-pSIMPLE plasmid (covering duck tembusu virus FX2010 pnca gene group nucleotide position is the sequence of 3656-10991) after order-checking.
1.2.6 the acquisition of FX2010 strain full-length cDNA
By the PCR primer fragment 1-1512 of purifying, 1397-2896,2643-4020 carries out fusion DNA vaccine, to obtain 1-4020 fragment, in this, as the 5 ' end merged in full-length cDNA reaction, method is with above-mentioned three small segments for template, is that upstream and downstream primer uses pfx archaeal dna polymerase to carry out fusion DNA vaccine with FX-S-T7-1F, FX-4020R.3 ' the acquisition of holding is then carry out enzyme with ApaLI restriction enzyme to 3-N-pSIMPLE plasmid to cut.
37 DEG C of water-baths 2 hours, reclaim the fragment (comprising the fragment on duck tembusu virus FX2010 pnca gene group 3739-10991 fragment and part pSIMPLE carrier) that length is 7968bp.
With the two halves end DNA fragmentation of above-mentioned recovery for template uses pfu archaeal dna polymerase to carry out polyreaction,
10×pfu Buffer 5μl
2.5mMdNTP 5μl
PfuDNA polysaccharase 1 μ l
The 1-4020 fragment of equimolar ratio and 3-N-pSIMPLE plasmid enzyme restriction reclaim fragment totally 39 μ l
Reaction conditions is as follows: 92 DEG C, 2min; (92 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 6min) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.
With above-mentioned reaction solution for template, pfuDNA polysaccharase is used to carry out the reaction of total length PCR.
Reaction tubes is placed in PCR instrument, and reaction conditions is as follows: 92 DEG C, 2min; (92 DEG C, 30s; 68 DEG C, 16min) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.After carrying out gel nucleic acid electrophoresis, reclaim the object fragment that length is about 11000bp.
Check order to the duck tembusu virus FX2010 strain full-length cDNA (product called after 1-N) obtained by fusion DNA vaccine, sequencing primer is in table 3.
Table 3 FX2010 strain full-length gene group sequencing primer
Use ABI3500 Genetic Analyser to measure virus full length DNA sequence dna, method is with aforementioned.Measurement result uses DNASTAR software to analyze, and the FX2010 reference sequences recorded with laboratory compares, and guarantees that the full length cDNA sequence obtained does not change completely.
2. result
2.1FX2010 strain divides fragment amplification electrophoresis result
Be that 6 fragments carry out pcr amplification by virus full length gene element, reaction solution carried out nucleic acid gel electrophoresis, the fragment meeting object clip size of gained is reclaimed.Amplified fragments 1-1512,1397-2896,2643-4020,3656-6353,6216-9775,9753-10991 length is respectively 1538bp, 1499bp, 1377bp, 2730bp, 3559bp, 1252bp, and acquired results as shown in Figure 2.In Fig. 2,1:1-1512; 2:1397-2896; 3:2643-4020; 4:3656-6353; 5:6216-9775; 6:9753-10991.
2.2FX2010 total length two halves end electrophoresis result is merged in strain
5 ' half end merging total length is the fragment 1-4020 (4046bp) obtained that increases, and 3 ' half end is the recovery fragment (7968bp) that plasmid 3-N-pSIMPLE cuts through ApalI enzyme, and electrophoresis result as shown in Figure 3.In Fig. 3, A is the 7968bp fragment of 3 ' half end, and B is the 4046bp fragment of 5 ' half end.
2.3FX2010 strain full-length cDNA amplification electrophorogram
Obtain the full-length cDNA fragment of FX2010 strain by obtain 5 ' half end and 3 ' half end by fusion DNA vaccine amplification, result as shown in Figure 4.
The rescue of embodiment 2 duck tembusu virus FX2010 strain and qualification
1. in-vitro transcription
With above-mentioned viral cDNA for template, utilize
t7Kit in-vitro transcription test kit carries out in-vitro transcription, obtains and has infective RNA.
2. in-vitro transcription product purification
Use the lithium chloride precipitator method (Lithium chloride precipitation) to carry out the purifying of transcription product, lithium chloride (LiCl) carries for in-vitro transcription test kit.Concrete grammar is as follows:
Add 30ul Nuclease-free Water and 30ul LiCl in Transcription Termination product, mix gently ,-20 DEG C freezing at least 30 minutes, 14000xg, 4 DEG C are centrifugal 15 minutes, carefully remove supernatant, then 1ml70% ethanol (with the preparation of DEPC water) is added, 14000xg, 4 DEG C are centrifugal 15 minutes, inhale and abandon supernatant, ethanol is made to volatilize completely, add 20ul Nuclease-free Water and dissolve RNA precipitation, save backup in-70 DEG C, portioning section is used for concentration determination.
3. transfection
Transfectional cell selects DF-1, adopts liposome mediated-method, according to LTX transfection reagent specification sheets, by above-mentioned RNA transfection to DF-1 cell, proceeds as follows:
1) complete cell the day before yesterday, make cell density reach 70%-80% when transfection in second day;
2) get 100ul Opti-MEM respectively in the 1.5ml EP pipe of 2 RNase free, 12ul LTX is dissolved in a wherein pipe, effect 5min;
3) get 5ug RNA to be added in another pipe 100ul Opti-MEM, add the Plus Reagent of 5ul simultaneously, mix gently;
4) by above-mentioned 2) and 3) in liquid mixing, the contrast and blank that only add LTX are set simultaneously, mix gently, room temperature effect 20min;
5) time of 20min can rinse cell, uses Opti-MEM, washes twice, finally add 800ul Opti-MEM on cell;
6) be added drop-wise on cell liquid level by the transfection cocktail doing to make good use of, then light rolling makes it be evenly distributed;
7) after 6h, supernatant is abandoned in suction, and the nutrient solution used instead containing 2%FBS continues to cultivate, and notes observation of cell change of state.
8) observe 96h after transfection the latest, results transfection hole supernatant, clear to be stored in-70 DEG C of cryogenic refrigerators for subsequent use for packing mark.
4. the qualification of Revive virus
4.1 cytopathies (CPE) are identified
After getting transfection, the transfection hole supernatant inoculation DF-1 cell of 48h carries out Secondary Culture:
(1) get and complete the DF-1 cell that density reaches 80% the day before yesterday, discard cell growth medium, clean 3 times with PBS;
(2) get 500ul transfection hole supernatant to be inoculated in T-25 cell bottle, the even uniform liquid that makes that shakes gently covers whole cell bottle bottom surface, puts 37 DEG C of CO
2adsorb 1.5h in incubator, period makes it adsorb evenly every 15min slight wobble;
(3) discard liquid after having adsorbed, clean cell 3 times with PBS, the cell maintenance medium then added containing 2%FBS is about 6ml (guaranteeing to cover bottom surface completely), puts 37 DEG C containing 5%CO
2cultivate in incubator;
(4) observe 2 every day, see if there is cytopathy and produce.The most late 96h, the packing of results supernatant liquor is stored in-70 DEG C.
Result: after transfection, 48 h transfection holes and blank and transfection reagent control wells have JND, 72 hour cell pathologies are obvious, cell rounding shrinkage, partial exfoliation levitating.Within 96 hours, pathology is serious.The cellular change of 72 hours, as Fig. 5, can find by observing, now having occurred obvious cytopathy (CPE).In Fig. 5, A: Revive virus hole; B control wells.
4.2RT-PCR qualification
Get the above-mentioned supernatant liquor that goes down to posterity of 300ul and extract viral RNA, the cDNA that reverse transcription obtains dilutes 4 gradients successively and carries out PCR qualification, and use specificity duck tembusu virus primers designed (H face big prosperous et al., 2011H), primer sequence is as follows:
FX-826F5’-CATAGGCTGGAATCTGGGAAC-3’(SEQ ID NO.42)；
FX-1126R：5’-TCTGGATTCTGTCGTCACGTC-3’(SEQ ID NO.43)。
Reaction conditions: 95 DEG C, 5min; (95 DEG C, 30s53,30s, 72 DEG C, 30s) × 30 circulations, 72 DEG C, 10min.Then identified whether have specific band by agarose gel electrophoresis.
Result: pass a generation after transfection 48h, go down to posterity and get supernatant after 72h and extract viral RNA, the cDNA that reverse transcription obtains dilutes 4 gradients successively and carries out PCR qualification, result amplification obtains the specific band of FX2010 strain virus, clip size is 300bp, the existence of viral nucleic acid can be described, electrophoresis result is shown in Fig. 6.In Fig. 6,1: blank; 2:10000 × dilution cDNA; 3:1000 × dilution cDNA; 4:100 × dilution cDNA; 5:10 × dilution cDNA; 6: former times cDNA.
4.3 indirect immunofluorescences (IFA) are identified
DF-1 cell is laid on six orifice plates by the day before yesterday, treats that its density reaches 80%, and get the above-mentioned supernatant liquor 300ul that goes down to posterity and inoculate as stated above, in cell culture incubator, cultivate the laggard row indirect immunofluorescene assay of 48h, method is as follows:
(1) fixing: discard cell culture fluid, clean 3 times with PBS, each 5min, in the culture dish of every hole, add 500ul4% paraformaldehyde room temperature fix 15min, PBS damping fluid washes 3 times afterwards, each 5min, and little shaking table more slower speeds rocks;
(2) penetrating: every hole adds 500ul0.5%TritonX-100 and carries out penetrating, and on little shaking table, room temperature rocks 15min at a slow speed, and PBS damping fluid washes 3 times afterwards, each 5min;
(3) close: add 500ul1%BSA, room temperature closes 30min, PBS buffer solution 3 times, each 5min;
(4) one anti-bindings: add 300ul duck tembusu virus E protein monoclonal antibody and hatch 1h at 37 DEG C, use PBST (adding the PBS damping fluid of Tween-20) to wash 3 times, each 5min afterwards;
(5) two anti-bindings: lucifuge adds 500ul FITC against murine two anti-(diluting 400 times), hatches 1h for 37 DEG C, washs 3 times with PBST, each 5min;
(6) observe: last every hole adds 500ul PBS damping fluid, observes under inverted fluorescence microscope (200 ×).
Result: make IFA experimental identification Revive virus by mouse-anti duck tembusu virus E protein monoclonal antibody.Result shows, and after Revive virus inoculation DF-1 monolayer cell 48h, present visible specific fluorescence signal in cells infected kytoplasm, cell controls then has no fluorescence, can illustrate that the special albumen of virus is expressed (see Fig. 7).In Fig. 7, A: Revive virus hole; B: negative control hole.
4.4 chicken embryo pathogenicities
By the cell conditioned medium of transfection 48h with dose inoculation 7 age in days of 100ul/ piece healthy SPF chicken embryo.Chicken embryo is observed with egg lamp before inoculation, dead germ, unfertilized embryo and unhealthy chicken embryo are abandoned, delimit air chamber, aseptically through allantoic cavity inoculation, after paraffin sealing, cultivate in 37 DEG C of incubators, every day at least discards according to chicken embryo dead in embryo twice, 24h, continues to cultivate 2-7 days, chicken embryo dead is during this period put 4 DEG C of refrigerator cold-storages and is spent the night, and makes idiosome vasoconstriction.Whether after sterile collection 24h, the idiosome glass homogenizer grinding of dead chicken embryo evenly, is placed in 2ml centrifuge tube, 12000rpm, 4 DEG C of centrifugal 10min, and it is for subsequent use to get supernatant fluid packing, and extract viral nucleic acid identifying virus and exist.
Result: observe after the cell conditioned medium of transfection 48h being inoculated 7 ages in days healthy SPF chicken embryo, chicken embryo was died off in one week, dead chicken embryo is visible, and significantly oedema is hemorrhage, meet the Symptoms of duck tembusu virus infected chicken embryo, and confirm in chicken embryo, be separated to the nucleic acid of Revive virus through nucleic acid extraction qualification.In 84h, dead chicken embryo symptom as shown in Figure 8.In Fig. 8, A: inoculation transfectional cell supernatant chicken embryo; B: normal chicken embryo.
4.5 Sequence Identification
The passage cell culture supernatant of Revive virus is extracted RNA, and utilize RT-PCR to divide the full-length cDNA fragment of 9 sections of Revive virus that increase, the primer is in table 3.Use 3500 Genetic Analyser of ABI company to carry out the sequencing of full-length gene group, splicing obtains the whole genome sequence of Revive virus, compares with FX2010 strain full-length gene order (SEQ ID NO.1).
Both result displays sequence is identical.Not only prove virus rescue success, and there is not any sudden change in Revive virus sequence compared with former strain.
Embodiment 3 E gene replaces the rescue of restructuring duck tembusu virus
1. materials and methods
1.1 material
1.1.1 virus and cell
Plasmid, the DF-1 cell of duck tembusu virus FX-2010 and FX2010-180P segmentation amplified fragments and segmentation structure are preserved by China Agriculture Academe Shanghai Veterinary Institute.
1.2 method
1.2.1 the structure of recombinant virus infection cDNA
1.2.1.1 recombinant virus genomes construction strategy
Weakly obtain FX2010-180P low virulent strain by FX-2010 strain people is exquisite, the recombinant virus in this research is the E gene E gene of FX-2010 strain being replaced with FX2010-180P low virulent strain, and retains other sector strategy (Fig. 9) of strong virus gene group.By the E gene order (table 4) of comparison two strains, find different sites, then according to each constant gene segment C distribution (table 5) design primer (table 6), utilize the plasmid SmaI-2092-1994-10 built, 24M7-3,1-2-Psimple, 2-3-pSIMPLE are template, segmentation is increased, and finally merges the full-length cDNA obtaining recombinant virus.
Table 4 FX2010 strain and FX2010-180P virus different amino acids site, E gene coding region
Table 5 duck tembusu virus FX2010 pnca gene stack features
Primer needed for table 6 construction of recombinant virus full-length cDNA
1.2.1.2 the acquisition of recombinant virus full-length cDNA
Fragment amplification is all use Pfx archaeal dna polymerase to carry out, and the polymerization of last full length DNA uses pfuDNA polysaccharase with fusion, and system program is with aforementioned.
(1) with plasmid SmaI-2092-1994-10 for template, use primers F X-S-T7-1F and CEF-232-1032R, amplified fragments 1-1032;
(2) with plasmid 1-2-pSIMPLE for template, use primer CEF-232-1011F and CEF-232-2050R amplified fragments 1011-2050; With plasmid 2-3-pSIMPLE for template, use primer CEF-232-2026F, FX-2896R, amplified fragments 2026-2896; Then the two sections of fragments obtained are done fusion DNA vaccine, do primer with CEF-232-1011F, FX-2896R, two fragments are template, merge and obtain fragment 1011-2896 (this section comprises the sequence site of the low virulent strain FX2010-180P that there are differences);
(3) with plasmid 24M7-3 for template, use primer 2 876F and FX-4020R, amplified fragments 2876-4020;
(4) with above-mentioned three fragments obtained for template, merge with primers F X-S-T7-1F and FX-4020R and obtain 1-4020 recombinant fragment, in this, as 5 ' end of last fusion reaction;
(5) 3 ' acquisitions of holding are then carry out enzyme with the 3-N-pSIMPLE plasmid that ApaLI restriction enzyme is obtained to embodiment 1 to cut, and reaction system is as follows: 5ul10 × Lbuffer, 2ul ApaLI restriction enzyme, 2ug plasmid DNA, make up water is to 50ul.37 DEG C of water-baths 2 hours, reclaim the fragment (comprising the fragment on duck tembusu virus FX2010 pnca gene group 3656-10991 fragment and part pSIMPLE carrier) that length is 7968bp;
(6) then merge according to method described in embodiment 1 full-length cDNA obtaining recombinant virus, and sequence verification, sequencing primer is in table 3.
1.2.2 the rescue of recombinant virus and qualification
1.2.2.1 in-vitro transcription and purifying
Method is with embodiment 1.
1.2.2.2 transfection
Method is with embodiment 1.
1.2.2.3 the qualification of recombinant virus
Mainly through cytopathy (CPE) and order-checking qualification, method is with embodiment 1.
2. the rescue result of restructuring duck tembusu virus
2.1 electrophoresis result
Divide fragment amplification each small segment according to the reorganization scheme designed, then obtain full length DNA with the method for fusion DNA vaccine, each fragment electrophoretic result as shown in Figure 10.In Figure 10, A: point fragment amplification figure; B: the two halves end electrophorogram merging total length; C: recombinant virus full-length cDNA electrophorogram; Wherein, 1: fragment 1-1032 (1058bp); 2: fragment 1011-2896 (1885bp); 3: fragment 2876-4020 (1144bp); 4:5 ' half end 1-4020 (4060bp); 5:3 ' half end (7968bp); 6: full-length cDNA (11017bp).
2.2 cytopathy
After transfection 60 hours start to show cytopathy, and after 72 hours, pathology is obvious, cell rounding shrinkage, partial exfoliation levitating.Within 96 hours, pathology is serious.The cellular change of 80 hours, as Figure 11, can find by observing, now having occurred obvious cytopathy (CPE).In Figure 11, A: Revive virus hole; B: control wells.
2.3 order-checking comparison results
The passage cell culture supernatant of Revive virus is extracted RNA, RT-PCR is utilized to divide the full length DNA fragment of 9 sections of Revive virus that increase, full-length gene group is checked order, sequencing result is compared with the sequence of experimental design after the splicing of seqman software, both result displays sequence is identical, for substituted for the E constant gene segment C of low virulent strain FX2010-180P under virulent strain FX2010 genome background.Not only prove virus rescue success, and there is not any sudden change in Revive virus sequence compared with expection recombinant strain.
The above embodiment only have expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a duck tembusu virus infections clone strain, it is characterized in that, this infections clone strain is the clonal strain of parent's poison duck tembusu virus virulent strain FX2010, it has the complete genome sequence shown in SEQ ID NO.1, and this infections clone strain is obtained by reverse genetic manipulation method.
2. duck tembusu virus infections clone strain according to claim 1, it is characterized in that, described reverse genetic manipulation method comprises the following steps:
By segmentation amplification, cDNA clones and fusion DNA vaccine, obtain the full-length cDNA of the duck tembusu virus virulent strain FX2010 containing transcriptional promoter sequence;
Described cDNA is obtained RNA through in-vitro transcription, then this RNA transfection cell is rescued from cell the infections clone strain obtaining duck tembusu virus virulent strain FX2010.
3. duck tembusu virus infections clone strain according to claim 2, it is characterized in that, described transcripting promoter is T7 promotor.
4. a recombinant vectors, it is characterized in that, comprise the partial nucleic acid fragment in following duck tembusu virus virulent strain FX2010 whole genome sequence: 3656-6353 position nucleic acid fragment, 6216-9775 position nucleic acid fragment, 9753-10991 position nucleic acid fragment, 3656-9775 position nucleic acid fragment or 3656-10991 position nucleic acid fragment in SEQ ID NO.1 sequence.
5. a nucleic acid molecule, it is characterized in that, comprise the complete genome sequence that the 1-1512 position nucleic acid fragment of the FX2010 of duck tembusu virus virulent strain shown in SEQ ID NO.1 whole genome sequence, 1397-2896 position nucleic acid fragment, 2643-4020 position nucleic acid fragment or SEQ ID NO.1 sequence 5 ' end is also equipped with transcripting promoter.
6. a preparation method for duck tembusu virus infections clone strain, is characterized in that, comprise the following steps:
Each DNA fragmentation of segmentation amplification duck tembusu virus full-length genome;
Part DNA fragmentation being cloned into carrier respectively, then obtaining the recombinant vectors containing covering duck tembusu virus genome 3656-10991 position nucleotide sequence through subclone;
The full-length cDNA of the duck tembusu virus containing transcriptional promoter sequence is obtained by fusion DNA vaccine;
Described cDNA is obtained RNA through in-vitro transcription, then this RNA transfection cell is rescued from cell the infections clone strain obtaining duck tembusu virus.
7. duck tembusu virus infections clone strain according to claim 1 prevents in preparation or treats the application in the vaccine of duck tembusu virus disease.
8. duck tembusu virus infections clone strain according to claim 1 is as the application of virus vector.
9. to recombinate a duck tembusu virus, it is characterized in that, this recombinant virus the E gene of duck tembusu virus infections clone strain described in claim 1 is replaced with the E gene of duck tembusu virus low virulent strain and the restructuring duck tembusu virus that is built into.
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