CN105200014A - Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof - Google Patents
Duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain and preparation method and application thereof Download PDFInfo
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- CN105200014A CN105200014A CN201410283544.3A CN201410283544A CN105200014A CN 105200014 A CN105200014 A CN 105200014A CN 201410283544 A CN201410283544 A CN 201410283544A CN 105200014 A CN105200014 A CN 105200014A
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Abstract
The invention discloses a duck tembusu virus (DTMUV) infectious clone attenuated vaccine strain. The attenuated vaccine strain is a clone strain of a parent virus DTMUV attenuated vaccine strain FX2010-180P and has a whole-genome sequence shown as SEQ ID NO.1. The infectious clone attenuated vaccine strain is obtained through a reverse genetic operation method. The invention further discloses a preparation method and application of the DTMUV infectious clone attenuated vaccine strain. The DTMUV infectious clone attenuated vaccine strain not only can be used for developing a novel DTMUV disease vaccine but also can be used as a virus vector for expression of exogenous genes, and meanwhile the strain is an important tool for DTMUV molecular biology study.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of duck tembusu virus infections clone attenuated vaccine strain and its preparation method and application.
Background technology
Duck tembusu virus (DuckTembusuvirus, 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 (Tembusuvirus).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 attenuated vaccine strain is provided, this duck tembusu virus infections clone attenuated vaccine strain is the infections clone strain of the duck tembusu virus attenuated vaccine strain FX2010-180P 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 attenuated vaccine 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 attenuated vaccine strain, this infections clone attenuated vaccine strain is the clonal strain of parent's poison duck tembusu virus attenuated vaccine strain FX2010-180P, it has the complete genome sequence shown in SEQIDNO.1, and this infections clone attenuated vaccine 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 attenuated vaccine strain FX2010-180P 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 attenuated vaccine strain FX2010-180P.
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 attenuated vaccine strain FX2010-180P whole genome sequence: 1-2050 position nucleic acid fragment in SEQIDNO.1 sequence, 2026-3056 position nucleic acid fragment, 3022-4052 position nucleic acid fragment, 3656-6353 position nucleic acid fragment, 6216-9775 position nucleic acid fragment, 9753-10991 position nucleic acid fragment, 3656-9775 position nucleic acid fragment, 3656-10991 position nucleic acid fragment, 2026-4052 position nucleic acid fragment, or 1-4052 position nucleic acid fragment.
In another aspect of this invention, additionally provide a kind of nucleic acid molecule, comprise the complete genome sequence of the duck tembusu virus attenuated vaccine strain FX2010-180P shown in SEQIDNO.1, be also equipped with transcripting promoter at this SEQIDNO.1 sequence 5 ' end.
In another aspect of this invention, additionally provide a kind of preparation method of duck tembusu virus infections clone attenuated vaccine strain, comprise the following steps:
Each DNA fragmentation of segmentation amplification duck tembusu virus attenuated vaccine strain FX2010-180P full-length genome;
Each DNA fragmentation is cloned into carrier respectively, forms multiple recombinant vectors that can cover duck tembusu virus attenuated vaccine strain FX2010-180P full-length genome;
The full-length cDNA of the duck tembusu virus attenuated vaccine strain FX2010-180P 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 attenuated vaccine strain FX2010-180P.
In another aspect of this invention, additionally provide a kind of above-mentioned duck tembusu virus infections clone attenuated vaccine 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 attenuated vaccine 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 inserts exogenous gene sequence between the genomic NS5 gene of above-mentioned duck tembusu virus infections clone attenuated vaccine strain and 3 ' non-coding sequence, and described exogenous gene sequence is internal ribosome entry site sequence and green fluorescence protein gene sequence.
Duck tembusu virus infections clone attenuated vaccine 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 FX2010-180P genome segmentation amplification schematic diagram of the embodiment of the present invention 1;
Fig. 2 is the embodiment of the present invention 1 segmented-PCR electrophoresis result figure;
Fig. 3 is that the embodiment of the present invention 1 plasmid 3-N-pSIMPLE enzyme cuts result figure;
Fig. 4 is the PCR electrophoresis result figure of the embodiment of the present invention 1 fragment T7-1-4052;
Fig. 5 is the electrophoresis result figure of the embodiment of the present invention 1 total length PCR primer;
Fig. 6 is 72 hours DF-1 cytopathy (100 ×) figure after the embodiment of the present invention 2 transfection;
Fig. 7 is the RT-PCR qualification result figure of the embodiment of the present invention 2;
Fig. 8 is that the embodiment of the present invention 2 Revive virus infects DF-1 cell IFA detected result (200 ×) figure;
Fig. 9 is the viral growth curves figure of the embodiment of the present invention 2 Revive virus and duck tembusu virus FX2010-180P strain;
Figure 10 is that the embodiment of the present invention 3 recombinant virus genomes builds schematic diagram;
Figure 11 is the PCR result figure of the embodiment of the present invention 3 fragment 9-EG-N;
Figure 12 is that the embodiment of the present invention 3 plasmid 3-EG-N-pSIMPLE enzyme cuts result figure;
Figure 13 is the PCR result figure of the embodiment of the present invention 3 total length PCR primer;
Figure 14 is restructuring duck tembusu virus detected result (100 ×) figure that the embodiment of the present invention 3 expresses EGFP.
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 cDNA clones, the method for fusion DNA vaccine obtains duck tembusu virus FX2010-180P strain full-length cDNA containing T7 promoter sequence, successfully save out duck tembusu virus FX2010-180P 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 FX2010-180P, utilize RT-PCR method, divide 6 sections of amplifications to obtain the DNA fragmentation of the full-length genome of Corticovirus, the end sequence of adjacent segment is overlapped, each fragment is cloned into respectively on pSIMPLE18 carrier; Then the sequence on 3 plasmids cut by enzyme, connect the fragment obtaining viral genome 3656-10991, this fragment is cloned into pSIMPLE18 carrier, final acquisition 4 is containing the plasmids covering duck tembusu virus full-length gene group.Utilize the method for PCR to introduce T7 promotor at 5 ' end, obtained the total length PCR primer of virus by fusion DNA vaccine.Utilize the method for in-vitro transcription to prepare and have infective viral cell-free transcription folder, purified virus RNA transfection is to DF-1 cell, and compared with the control, transfection is after 48 hours, and pathology appears in the cell of transfected virus RNA, and 72 hour cell pathologies are more obvious.Through RT-PCR, the multiple method qualification such as indirect immunofluorescence (IFA), proves successfully to save out viral R-FX2010-180P.Through sequence alignment, the sequence of Revive virus and the sequence of duck tembusu virus FX2010-180P strain completely the same.
The method that the present invention also utilizes PCR and enzyme to cut, inserts internal ribosome primer sites (IRES) and green fluorescence protein gene (EGFP) between the genomic NS5 gene of duck tembusu virus and 3 ' non-coding sequence.PCR method is utilized to obtain 5 ' end containing T7 promotor, the FX2010-180P strain whole genome sequence comprising IRES and GFP sequence.RNA transfection in-vitro transcription obtained is to DF-1 cell, the restructuring duck tembusu virus of expressing green fluorescent protein is saved in success, virus was passed for 3 generations continuously, and every generation all can observe fluorescent protein expression, successfully duck tembusu virus be have expressed external source EGFP albumen as virus vector.
The structure of embodiment 1 duck tembusu virus low virulent strain FX2010-180P full length cDNA clone
1. material
1.1 virus and cells
Duck tembusu virus FX2010-180P strain, DF-1 cell are preserved by China Agriculture Academe Shanghai Veterinary Institute.
2. method
The extraction of 2.1 viral RNAs
The RNAisoPlus 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-culturing fluids, add 700 μ lRNAisoplusReagent, be placed in 1.5ml without RNA enzyme centrifuge tube, after piping and druming mixing, room temperature leaves standstill 5 minutes.In mixed solution, add 200 μ l chloroforms, use forced oscillation 15s, room temperature leaves standstill 3min.12000r/min4 DEG C of centrifugal 10min.Get supernatant liquor to be transferred in another clean centrifuge tube without RNA enzyme.In supernatant liquor, add isopyknic Virahol, after mixing, room temperature leaves standstill 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.12000rpm4 DEG C of centrifugal 10min, discards liquid completely, pellet dried at room temperature 5-10 minute, after 75% ethanol all volatilizees, adds the DEPC water dissolution precipitation of the RNasefree of 20 μ l.
The design of 2.2 specific reverse transcription primers and reverse transcription
2.2.1 the design of specific reverse transcription primer
According to (LiGetal, 2014 such as this laboratory Li; Xiao Yali, 2013) reference sequences (SEQIDNO.1) of the sick FX2010-180P strain of the duck tembusu virus recorded, utilize Primer5 software design can cover 7 specific reverse transcription primer of total length, synthesized by invitrogen company, list of primers is as follows:
Table 1FX2010-180P reverse transcription primer
2.2.2 reverse transcription
In table 17 reverse transcription primer respectively being got 100 μ l joins in the centrifuge tube of 1.5ml without RNA enzyme, and totally 700 μ l mix mixing, as specific reverse transcription primer, save backup.Utilize the SuperScriptIII ThermoScript II of invitrogen company, according to specification sheets requirement, with the RNA of said extracted for template carries out reverse transcription, operation steps is as follows: 9 μ lRNA templates, 3 μ l mixing Auele Specific Primers, 1 μ l10mMdNTP, add in the PCR pipe of nuclease free, to be placed in PCR instrument 70 DEG C, 10min; Put rapidly and 2min on ice.Add 5 μ l5 × firststrandbuffer again, 1 μ l0.1MDTT, 1 μ lRNA enzyme inhibitors, 1 μ lSuperScript ThermoScript II, 6 μ l, without the aqua sterilisa of RNA enzyme, to be then placed in PCR instrument 55 DEG C, 60min; 70 DEG C, 15min, 4 DEG C of preservations.
Point fragment design of primers and the clone of 2.3 full-length gene groups
2.3.1 design of primers
First, zero position need be held to add T7 promoter sequence and protectiveness base at full length sequence 5 ' in order to in-vitro transcription can be carried out smoothly.Then the mapdraw software in DNASTAR is utilized, restriction enzyme site analysis is carried out to the genomic of FX2010-180P 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 and latter half genome sequence is connected to identical carrier.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.Concrete design of primers is listed as follows:
Table 2FX2010-180PPCR primer
According to designed primer, point fragment for template, divides fragment amplification duck tembusu virus FX2010-180P full-length gene group (see Fig. 1) with viral cDNA.
2.3.2 point 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:
Use primers F XA-T7-1F, FXA-2050R amplified fragments 1-2050, be placed in PCR instrument reaction amplification, reaction conditions: 94 DEG C, 2min; (94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 2min) × 30 circulations, 68 DEG C, 10min, 4 DEG C of preservations.
Use primers F XA-2026F, FXA-3056R amplified fragments 2026-3056; Use primers F XA-3022F, FXA-4052R amplified fragments 3022-4052, be placed in PCR instrument reaction amplification, reaction conditions: 94 DEG C, 2min; (94 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 1min) × 30 circulations; 68 DEG C, 10min, 4 DEG C of preservations.
Use primers F XA-3656F, FXA-MCS6353R amplified fragments 3656-6353; Be placed in PCR instrument reaction amplification, reaction conditions: 94 DEG C, 2min; (4 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 XA-6216F, FXA-9775R amplified fragments 6216-9775, be placed in PCR instrument reaction amplification, 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 XA-9753F, FXA-NotI10991R, amplified fragments 9753-10991, be placed in PCR instrument reaction amplification, 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.
Prepare 0.8% sepharose, fill a prescription as follows: 0.8g agarose+100ml0.5 × TAE damping fluid+5 μ lgoldview, mixing, microwave boils, and glue is poured into the glue groove being plugged comb in advance.10 × loadingbuffer mixing of 5 μ l is added respectively in above-mentioned 6 pipe PCR reaction solutions, being added by all liquid 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, calculate the weight of blob of viscose, this weight is as a gel volume V (wherein swap mode is 100mg=100 μ l volume).Add the BufferDE-A of 3 volumes, 75 DEG C of water-baths, melt completely to colloid, about 10min, and liquid becomes red.Add the BufferDE-B of 0.5 BufferDE-A volume, and mix liquid yellowing; Mixed solution is transferred to DNA preparation pipe (Filter column) inner, be placed in two milliliters of centrifuge tubes, on normal temperature whizzer, centrifugal 1 minute that carries out 12000rpm, abandon filtrate.Preparation pipe (Filter column) is put back in two milliliters of centrifuge tubes, adds 500 μ lBufferW1 washingss, 12000rpm, centrifugal 30 seconds, abandon filtrate.Preparation pipe (Filter column) is put back in 2ml centrifuge tube, add 700 μ lBufferW2 washingss, centrifugal 30 seconds of 12000rpm, abandons filtrate, wash a 12000rpm with 700 μ lBufferW2 again with same method, within centrifugal 1 minute, carry out centrifugal on normal temperature whizzer.2ml centrifuge tube is put back by preparing pipe, empty from 1 minute with the rotating speed of 12000rpm.To prepare pipe and be placed in the 1.5ml centrifuge tube of brand-new cleaning, preparing film central authorities and add the deionized water of 30 μ l, room temperature leaves standstill 1 minute, and 12000rpm centrifugal 1 minute eluted dna, can repeat above-mentioned final step, thus improves elution efficiency.
2.3.3 cDNA clones
The PCR primer of the 6 sections of FX2010-180P reclaimed is carried out phosphatizing treatment, operate according to the specification sheets of the T4PNK enzyme of NEB company, system is as follows: 2 μ l10 × reactionbuffer, 0.5 μ l100mMATP, 15.5 μ lDNA, 2 μ lT4PNK phosphorylating kinases; Temperature control 37 DEG C in PCR instrument, 30min.Then often 4 μ l reaction solutions got by pipe, 1 μ l blunt vector pSIMPLE18EorV/BAPVECTOR, 5 μ lTakaraSloutionI reaction solutions; Be placed in 16 DEG C of water-baths to spend the night, carry out ligation.
6 above-mentioned pipes are connected product, transforms in JM109 competent cell.In 100 μ l competent cells, add 10 μ l ligation liquid, mixing, puts ice 30 minutes.42 DEG C of water-bath heat shock 90s, put rapidly ice 2min, add 1ml liquid LB and cultivate (base not containing Amp) and mix latter 37 DEG C and shake 45 minutes.Centrifugal 3 minutes of 5000rpm, abandon liquid at the bottom of supernatant 1ML pipe to hang bacterium and be applied to containing Amp, and the X-Gal, the 10 μ lIPTG that smoothen 40 μ l in advance (put into 37 DEG C of incubators more than 30 minutes after completing, a small amount of liquid LB can be mixed into) in IPTG and X-Gal, LB solid medium, 37 DEG C cultivate 8-20 hour.The single bacterium colony of picking white, is inoculated in and contains in the test tube of Amp liquid LB nutrient solution containing 4ml, within the shaking table of 37 DEG C, cultivate 16h.Afterwards with bacterium liquid for template, identify positive plasmid by PCR method, upstream and downstream primer is respectively:
M13F-47:5’-CGCCAGGGAGGTTTGTCCCAGTCACGAC-3’(SEQIDNO.22)，
M13R-47:5’-GAGCCGGGATACACAATTTCACACAGGA-3’(SEQIDNO.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 1000bp/min; 5. extend eventually: 72 DEG C of 10min, step 2-4 are 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, first carries out 12000rpm, 1 minute centrifugal, outwells supernatant fluid.Add 300 μ lBufferS1 washingss, suspended bacterial precipitates.Also it spins upside down 8 times fully to add 300 μ l washings BufferS2 gentlenesses, mixes and makes the abundant cracking of thalline.Add the buffer B ufferS3 of 400 μ l, gentle and spin upside down mixing fully 10 times, centrifuge tube symmetry is put into whizzer, under the rotating speed of 12000rpm centrifugal 15 minutes.Draw centrifugal supernatant and transfer to preparation pipe (being placed in 2mL centrifuge tube) 12000rpm, centrifugal 1 minute, abandoning filtrate.Putting back centrifuge tube by preparing pipe, adding 500 μ lBufferW1,12000rpm, centrifugal 1 minute, abandon filtrate.Putting back centrifuge tube by preparing pipe, adding 700 μ lBufferW2,12000rpm, centrifugal 1 minute, and abandon filtrate, washing once with 700 μ lBufferW2 more in the same way and abandon filtrate, putting 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 the central authorities of film, room temperature leaves standstill 1 minute, centrifugal 1 minute of 12000rpm.
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.
Carrying out order-checking after reaction terminates uses alcohol EDTA method to carry 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; Often pipe adds 2 μ l125MEDTA, 2 μ l3MNaAc; Add the alcohol of 50 μ l dehydrated alcohols or 98%, evenly, room temperature places 15min in concussion fully; 12000rpm, 4 DEG C of centrifugal 30min, outwell supernatant; Add 100 μ l70% 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 lHi-Di methane amides, evenly, 10 μ l samples are joined 96 orifice plates by 95 DEG C of sex change 4min, rapid ice-cold 4min in piping and druming, upper machine order-checking.Use seqman software splicing in DNASTAR to analyze sequencing result, choose and obtain the correct plasmid clone that 6 sections cover duck tembusu virus FX2010-180P strain full-length genome.The correct plasmid clone designation of 1-2050 fragment is T-2-pSIMPLE plasmid; The correct plasmid clone designation of 2026-3056 fragment is 2-3-pSIMPLE plasmid; The correct plasmid clone designation of 3022-4052 fragment is 3-4-pSIMPLE plasmid; 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.
2.4 the structure of plasmid 3-N-pSIMPLE
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 l10 × Hbuffer, 2 μ lEcorV 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 l10 × Buffer, 1 μ lAgeI 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 l10 × Buffer, 1 μ lT4DNALigse 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.Be converted 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 FX2010-180P 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 l10 × Hbuffer, 2 μ lNotI 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 l10 × Buffer, 1 μ lAgeI 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 9-N-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims the fragment of length greatly 1228bp.
The two sections of products reclaimed are carried out ligation, and the metal bath being placed in 16 DEG C carries out the ligation of 4 hours.Be converted 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-N-pSIMPLE plasmid (covering duck tembusu virus FX2010-180P pnca gene group nucleotide position is the sequence of 3656-10991).
2.5 fusion DNA vaccine methods obtain the full-length cDNA of duck tembusu virus FX2010-180P strain
Respectively with T-2-pSimpel, 2-3pSimpel, 3-4-pSIMPLE plasmid for template, respectively with FXA-T7-1F, FXA-2050R; FX2026F, FXA-3056R; FXA-3022F, FXA-4052R are primer, carry out pcr amplified fragment with pfxDNA polysaccharase, use nucleic acid gel to reclaim kits PCR primer, obtain T7-1-2050 fragment respectively, 2026-3056 fragment, 3022-4052 fragment.And then with fragment 2026-3056, fragment 3022-4052 is template, is that upstream and downstream primer uses pfxDNA polysaccharase to carry out fusion DNA vaccine with FXA-2026F, FXA-4052R, obtains 2026-4052 fragment.
With fragment T7-1-2050, fragment 2026-4052 is template, is that upstream and downstream primer uses pfxDNA polysaccharase to carry out fusion DNA vaccine, obtains T7-1-4052 fragment with FXA-T7-1F, FXA-4052R.
Use ApaLI restriction enzyme to carry out enzyme to 3-N-pSIMPLE plasmid to cut, reaction system is as follows: 5 μ l10 × Lbuffer, 2 μ lApaLI restriction enzymes, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, reclaim the fragment (comprising the fragment on duck tembusu virus FX2010-180P genome 3656-10991 fragment and part pSIMPLE carrier) that length is 7968bp.
With the DNA fragmentation of above-mentioned recovery two sections for template uses pfuDNA polysaccharase to carry out polyreaction, reaction system is as follows: 5 μ l10 × pfuBuffer, 5 μ l2.5mMdNTP, 1 μ lpfuDNA polysaccharase, the T7-1-4052 fragment of equimolar ratio and 3-N plasmid enzyme restriction reclaim fragment totally 39 μ l.Be placed in PCR instrument, 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 system is as follows: 5 μ l10 × pfuBuffer, 5 μ l2.5mMdNTP, 1 μ lpfuDNA polysaccharase, 2 μ l polymerisate reaction solutions, 2 μ l upstream primer FXA-T7-1F, 2 μ l downstream primer FXA-10991R, 33 μ lH2O.Be placed in PCR instrument, reaction conditions is as follows: 2 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.
The duck tembusu virus FX2010-180P full-length cDNA obtained for fusion DNA vaccine checks order, and sequencing primer is listed as follows:
Table 3FX2010-180P full-length gene group sequencing primer
Use on ABI3500 Genetic Analyser and virus full length DNA sequence dna is measured, measurement result uses DNASTAR software to analyze, FX2010-180P reference sequences (the LiGetal recorded with this seminar, 2013) compare, do not change completely through sequence alignment gained full length cDNA sequence.
3. result
The result of 3.1 segmented-PCRs
Be that 6 fragments carry out pcr amplification by virus full length gene element, carry out nucleic acid gel electrophoresis after amplification, acquired results as shown in Figure 2, reclaims the fragment meeting object clip size of gained.In Fig. 2, A:5000marker; B: fragment T7-1-2050 (2080bp); C: fragment 2026-3056 (1030bp); D: fragment 3022-4052bp (1030bp); E: fragment 3656-6353 (2697bp); F: fragment 6216-9775 (3559bp); G: fragment 9753-10991 (1228bp).
3.2 plasmid 3-N-pSIMPLE enzymes cut result
Use ApaLI restriction enzyme to carry out enzyme to 3-N-pSIMPLE plasmid to cut, the enzyme that the fragment that recovery length is 7968bp obtains cuts result as shown in Figure 3.
The PCR result of 3.3 fragment T7-1-4052
Utilize fusion DNA vaccine method to obtain T7-1-4052 fragment, reaction solution is carried out nucleic acid gel electrophoresis, electrophoresis result is as shown in figure .4, and it is the fragment of 4084bp that length is reclaimed in cutting.
The amplification of 3.4 total length PCR primer
Fusion DNA vaccine method is utilized to obtain the duck tembusu virus FX2010-180P strain total length PCR primer of 5 ' end containing T7 promotor, reaction solution is carried out nucleic acid gel electrophoresis, it is the fragment of 11021bp that length is reclaimed in cutting, ABI3500 Genetic Analyser is used to measure virus full length PCR primer sequence, measurement result uses DNASTAR software to analyze, the FX2010-180P reference sequences (SEQIDNO.1) recorded with this seminar compares, and does not change completely through sequence alignment gained total length PCR primer sequence.The electrophoresis result of total length PCR primer as shown in Figure 5.
The rescue of embodiment 2 duck tembusu virus low virulent strain FX2010-180P and qualification
1. in-vitro transcription
With above-mentioned virus full length PCR primer for template, utilize
in-vitro transcription test kit carries out in-vitro transcription, and preparation has infective RNA.
37 DEG C are reacted 5 hours, react and carry out in PCR instrument.
Use the lithium chloride precipitator method to carry out purifying to transcription product, add 30 μ lNuclease-freeWater and 30 μ lLiCl in Transcription Termination product, mix gently.Carefully remove supernatant, add 1ml70% ethanol (with the preparation of DEPC water), 4 DEG C, 14000g, centrifugal 15min, inhale and abandon supernatant, and ethanol is volatilized completely, adds 20 μ lNuclease-freeWater and dissolve RNA precipitation, in-70 DEG C of preservations.
2. transfection
Use LTX transfection reagent box by above-mentioned RNA transfection to DF-1 cell, the day before yesterday completes cell, makes the density of cell density cell when transfection in second day reach 80%; In the 1.5mlEP pipe of 2 RNasefree, add 100 μ lOpti-MEM nutrient solutions respectively, 10 μ lLTX are dissolved in a wherein pipe, effect 5min; Get 5ugRNA to be added among another pipe 100 μ lOpti-MEM nutrient solution, add the LTXPlusReagent of 5 μ l simultaneously, mix gently; Two above-mentioned pipe liquid are mixed, the reagent controls and blank that only add LTX is set simultaneously, mixes gently, room temperature effect 20min; Utilize the time of this 20min to rinse cell, use Opti-MEM nutrient solution to wash twice, finally add 800 μ lOpti-MEM on cell; Be added drop-wise on cell liquid level uniformly by the transfection cocktail doing to make good use of, then light rolling makes it be evenly distributed; 37 DEG C of cell culture incubators are cultivated.Inhale supernatant discarded after 8h, the DMEM nutrient solution used instead containing 2%FBS continues to cultivate, and notes observation of cell change of state.
Result: after transfection, 48 h transfection holes have contrasted significant difference with blank and transfection reagent, 72 hour cell pathologies are obvious, and cell rounding shrinkage, come off levitating.96 hours serious change.The cellular change of 72 hours, as Fig. 6, can find by observing, now having occurred obvious cytopathy (CPE).In Fig. 6, A: Revive virus hole; B: transfection reagent control wells; C: blank control wells.
3. Viral diagnosis
After transfection 96h, draw transfection hole supernatant F1 generation, the culture dish of the DF-1 cell completed before being forwarded to goes down to posterity, the FBS nutrient solution continuation cultivation 48h of 2% used instead by PBS damping fluid after washing 3 times.Draw culture suspension, packing is saved to-70 DEG C of cryogenic refrigerators.
3.1RT-PCR qualification
Get the above-mentioned F2 of 300 μ l and carry out molecular biology identification for suspension.Extract the RNA in Revive virus F2 generation by step described in embodiment 1, reverse transcription obtains cDNA, and use specificity duck tembusu virus primers designed (Yan Pixi etc., 2011), carry out PCR qualification, primer sequence is as follows:
FX-826F：CATAGGCTGGAATCTGGGAAC(SEQIDNO.40)；
FX-1126R：TCTGGATTCTGTCGTCACGTC(SEQIDNO.41)。
CDNA template concentrations used is followed successively by former times, 10 times dilutions, 100 times of dilutions and 1000 times of dilutions respectively.
Result: as shown in Figure 7, former times, 10 times dilutions, 100 times of dilutions and 1000 times of cDNA templates of diluting can amplify the object fragment that size is 300bp.In Fig. 7, A:2000Marker; B: former times; C:10 doubly dilutes; D:100 doubly dilutes; E:1000 doubly dilutes; F negative control.
3.2IFA qualification
The virus that rescue obtains is F1 generation, after the culture dish in F2 generation noted earlier is drawn suspension, carries out indirect immunofluorescence (Immunofluorescenceassay, IFA) qualification to the DF-1 cell be adsorbed on ware.In the culture dish of every hole, add 500 μ l4% paraformaldehydes carry out 4 DEG C and spend the night fixing, PBS damping fluid washes 3 times afterwards, each 5min, and little shaking table more slower speeds rocks.Every hole adds 500 μ l0.5%TritonX-100 and carries out penetrating, and little shaking table rocks 15min at a slow speed, and PBS damping fluid washes 3 times afterwards, and each 5min more slower speeds rocks on little shaking table.Add the 1%BSA of 500 μ l, little shaking table rocks at a slow speed closed 30min, PBS damping fluid and on little shaking table, rocks at a slow speed washing 3 times, each 5min.Add 300 μ l duck tembusu virus E protein monoclonal antibodies to rock at a slow speed on little shaking table and hatch 1h, add PBS damping fluid afterwards and on little shaking table, rock at a slow speed washing 3 times, each 5min.Lucifuge adds 500 μ lFITC against murine two anti-(diluting 400 times), 37 DEG C are rocked at a slow speed and hatch 1h on little shaking table, add PBST damping fluid (adding the PBS damping fluid of Tween-20) and wash 3 times, each 5min, last every hole adds 500 μ lPBS damping fluids, observes under inverted fluorescence microscope.
Result: as shown in Figure 8, F2 is for after Revive virus inoculation DF-1 monolayer cell 48h, and present visible specific fluorescence signal in cells infected kytoplasm, cell controls then has no fluorescence.A: Revive virus hole; B: negative control hole.
4. Revive virus genome sequencing
Extract RNA by after the cells and supernatant multigelation 3 times of Revive virus, utilize RT-PCR segmentation to increase the full-length gene group of Revive virus, the primer is in table 2.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 FX2010-180P full-length gene group.
Result: the two sequence is identical, there is not any sudden change in Revive virus sequence sequence compared with former strain.
5. the analysis of the growth characteristics of Revive virus
The TCID50 of 5.1 Revive virus measures
With aseptic PBS, tested seed culture of viruses is carried out serial dilution by 10 multiple proportions, get 10
-4~ 10
-74 extent of dilution are seeded in the DF-1 cell 96 porocyte culture plates growing to 80 ~ 90% respectively, and each extent of dilution inoculates 4 holes, every hole 0.1ml; 37 DEG C act on 1 ~ 2 hour, change to the DMEM containing 2% foetal calf serum, at 5%CO with PBS after cleaning 3 times
2continue cultivation 6 with under 37 DEG C of conditions, observe and record the cytopathic cell hole of appearance, calculating viral level (Reed.L.J.etal, 1938) by Reed-Muench method.
The growth in vitro curve determination of 5.2 viruses
By DF-1 passage to T25 cell bottle, in Revive virus F2 generation, is pressed virus infection plural number (Multiplicityofinfection with duck tembusu virus FX2010-180P strain virus, MOI) be 0.1 amount inoculation well-grown grow up to the DF-1 cell of individual layer, adsorb 1.5 hours, discard virus liquid, wash 3 times with PBS, add 2% maintenance medium, at 5%CO
2cultivate 120h, every 12h with continuation under 37 DEG C of conditions and gather in the crops a cell conditioned medium, each 200 μ l carry out virus titer mensuration.Draw the growth curve of virus on DF-1 cell.
Result: with DF-1 raji cell assay Raji Revive virus R-FX2010-180P strain F2 for the growth in vitro curve with duck tembusu virus FX2010-180P strain virus as shown in Figure 9, Revive virus and the growth characteristics of parental virus on DF-1 cell are without significant difference.
Embodiment 3 with duck tembusu virus for vector expression foreign protein
1. material
1.1 virus and cells
The sick FX2010-180P strain of duck tembusu virus is preserved by this laboratory; DF-1 cell is preserved by this laboratory.
2. method
The insertion point of 2.1 foreign genes is selected and design of primers
2.1.1 the constructing plan of recombinant virus genomes
As shown in Figure 10, between the NS5 gene and 3 ' non-coding sequence of FX2010-180P pnca gene group, once insert sequence and the green fluorescence protein gene (EGFP) of internal ribosome entry site (IRES).
2.1.2 design of primers
In order to IRES+EGFP sequence being inserted duck tembusu virus genome, utilize PCR method, at 5 ' end of IRES sequence, and 3 ' end of EGFP sequence introduces one section of particular sequence of duck tembusu virus FX2010-180P respectively.NS5 gene is the genomic 3 ' end of duck tembusu virus, is the encoding gene of the least significant end of full-length genome, is close to 3 ' end non-coding sequence.And the genomic open reading frame of duck tembusu virus FX2010-180P ends at 10373 place's Nucleotide, thus be exactly be equivalent to IRES+EGFP sequence to be inserted into duck tembusu virus 10373 and 10374 place's Nucleotide between.Utilize fusion DNA vaccine method to be incorporated in the total length PCR primer of FX2010-180P by IRES and EGFP, design of primers is as shown in table 4, and the 5 ' end of primers F XA-NotI10991R with the addition of protectiveness base and NotI restriction enzyme site.
Table 4 recombinant virus primer
The structure of 2.2 plasmid 3-EGFP-N-pSIMPLE
With pIRES2-EGFP plasmid for template, use primer NS5-IRESF, EGFP-3UTRR is that upstream and downstream primer carries out PCR, obtain the base of 5 ' end containing duck tembusu virus genome 10355-10373 place, the DNA fragmentation of the 3 ' IRES-EGFP of end containing duck tembusu virus genome 10374-10392 place base.
With 9-N-pSIMPLE plasmid for template, use FXA-9753F, NS5-IRESR to be that upstream and downstream primer carries out PCR, obtain 3 ' end holds the duck tembusu virus genome 9753-10373 place of front 18 bases gene fragment containing 5 ' of IRES.
With 9-N-pSIMPLE plasmid for template, use EGFP-3UTRF, FXA-NotI-10991R to be that upstream and downstream primer carries out PCR, obtain the gene fragment at the duck tembusu virus genome 10374-10991 place of 3 ' end 18 bases of 5 ' end containing EGFP.
The DNA fragmentation obtained with above-mentioned 3 kinds of PCR primer is for template, with FXA-9753F, FXA-NotI10991R is that upstream and downstream primer carries out fusion DNA vaccine, obtains the inner gene fragment containing IRES+EGFP sequence duck tembusu virus genome 9753-10991 place, fragment called after 9-EG-N.
To 3-N-pSIMPLE plasmid, DNA fragmentation 9-EG-N successively uses NotI, and AgeI restriction enzyme carries out enzyme and cuts.NotI enzyme cuts system: 10 μ l10 × Hbuffer, 4 μ lNotI restriction enzymes, 4 μ gDNA, make up water to 100 μ l.37 DEG C of water-baths 2 hours, reclaim DNA fragmentation.AgeI enzyme cuts system: 10 μ l10 × Buffer, 2 μ lAgeI restriction enzymes, 4 μ gDNA, make up water to 100 μ l.React 2 hours at water-bath under 37 DEG C of conditions, extract reaction solution and carry out nucleic acid gel electrophoresis, it is the fragment of 8968bp that 3-N-pSIMPLE plasmid enzyme restriction product electrophoresis result reclaims length, and 9-EG-N fragment digestion products electrophoresis result reclaims the fragment of length greatly 2533bp.
The two sections of products reclaimed are carried out ligation, is placed in 16 DEG C of metal baths and carries out ligation in 4 hours.Be converted in DH5 α competence intestinal bacteria, after through choosing bacterium, liquid LB cultivates, qualification positive colony, extract plasmid, correct positive colony 3-EG-N-pSIMPLE plasmid (covering duck tembusu virus FX2010-180P pnca gene group nucleotide position is the sequence of 3656-10991, wherein inserts the sequence of IRES and EGFP between 10373 and 10374) is obtained after ABI company 3500 Genetic Analyser order-checking.
2.3 fusion DNA vaccine method obtains the total length PCR primer of recombinant virus
Build the T-2-pSIMPLE preserved respectively with laboratory, 2-3pSIMPEL, 3-4-pSIMPLE plasmid is template, respectively with FXA-T7-1F, FXA-2050R, FXA-3056R; FXA-3022F, FXA-4052R are primer, carry out pcr amplified fragment with pfxDNA polysaccharase, use nucleic acid gel to reclaim kits PCR primer, obtain T7-1-2050 fragment respectively, 2026-3056 fragment, 3022-4052 fragment.And then with fragment 2026-3056, fragment 3022-4052 is template, is that upstream and downstream primer uses pfxDNA polysaccharase to carry out fusion DNA vaccine with FXA-2026F, FXA-4052R, obtains 2026-4052 fragment.
With fragment T7-1-2050, fragment 2026-4052 is template, is that upstream and downstream primer uses pfxDNA polysaccharase to carry out fusion DNA vaccine, obtains T7-1-4052 fragment with FXA-T7-1F, FXA-4052R.
Use ApaLI restriction enzyme to carry out enzyme to 3-EG-N-pSIMPLE plasmid to cut, reaction system is as follows: 5 μ l10 × Lbuffer, 2 μ lApaLI restriction enzymes, 2 μ g plasmid DNA, make up water to 50 μ l.37 DEG C of water-baths 2 hours, recovery length is that the fragment of 9273bp (comprises duck tembusu virus FX2010-180P genome 3656-10991 fragment, between 10373 and 10374, wherein insert the sequence of IRES and EGFP, and the fragment on part pSIMPLE carrier).
With the DNA fragmentation of above-mentioned recovery two sections for template uses pfuDNA polysaccharase to carry out polyreaction, reaction system is as follows: 5 μ l10 × pfuBuffer, 5 μ l2.5mMdNTP, 1 μ lpfuDNA polysaccharase, and the T7-1-4052 fragment of equimolar ratio and 3-EG-N plasmid enzyme restriction reclaim fragment totally 39 μ l.Be placed in PCR instrument, reaction conditions is as follows: 92 DEG C, 2min; (92 DEG C, 15s; 53 DEG C, 30s; 68 DEG C, 8min) × 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 system is as follows: 5 μ l10 × pfuBuffer, 5 μ l2.5mMdNTP, 1 μ lpfuDNA polysaccharase, 2 μ l polymerisate reaction solutions, 2 μ l upstream primer FXA-T7-1F, 2 μ l downstream primer FXA-10991R, 33 μ lH2O.Be placed in PCR instrument, reaction conditions is as follows: 2 DEG C, 2min; (2 DEG C, 30s; 68 DEG C, 18min) × 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 FX2010-180P total length PCR primer obtained by fusion DNA vaccine, sequencing primer primer listed by embodiment 1 table 3 adds the pair of primers of following table 5 again.
1 pair of primer that the order-checking of table 5 recombinant virus full-length gene group increases
Use on 3500 type Genetic Analyser of ABI company and recombinant virus total length PCR primer sequence is measured, measurement result uses DNASTAR software to analyze, compare with the recombinant virus sequence of expection, meet expection through sequence alignment gained total length PCR primer sequence.
The rescue of 2.4 recombinant viruses
2.4.1 in-vitro transcription
With above-mentioned virus full length PCR primer for template, utilize
in-vitro transcription test kit carries out in-vitro transcription, and preparation has infective RNA.Concrete steps are with embodiment 2.
2.4.2 transfection
Transfection procedure is with embodiment 2.From F1 generation, recombinant virus goes down to posterity by DF-1 cell continuously, the expression of basis of microscopic observation green fluorescent protein, after 72 hours, reclaims the recombinant virus of the F1 generation of transfection gained.
Going down to posterity of 2.5 rescue recombinant viruses
From F1 generation, recombinant virus goes down to posterity by DF-1 cell continuously, basis of microscopic observation fluorescence, reclaims virus in 72 hours, and continuous passage, to F5 generation, observes the expression of green fluorescent protein.
2.6 Revive virus genome sequencings
Will F2 generation of rescue recombinant virus, extract RNA after the cell culture supernatant multigelation 3 times in F5 generation, utilize RT-PCR segmentation to increase the full-length gene group of Revive virus, the primer is shown in embodiment 1 table 1,2 and 3.Amplified fragments and sequencing primer are delivered to Shanghai Mei Ji biology to check order.
3. result
The PCR result of 3.1 fragment 9-EG-N
Utilize fusion DNA vaccine method to obtain 9-EG-N fragment, reaction solution is carried out nucleic acid gel electrophoresis, it is the fragment of 2533bp that length is reclaimed in cutting, and electrophoresis result as shown in figure 11.
3.2 plasmid 3-EG-N-pSIMPLE enzymes cut result
Use ApaLI restriction enzyme to carry out enzyme to 3-EG-N-pSIMPLE plasmid to cut, as shown in figure 12, reclaim length is the endonuclease bamhi of 9273bp to the result that enzyme is cut.
The amplification of 3.3 recombinant virus total length PCR primer
Fusion DNA vaccine method is utilized to obtain the restructuring duck tembusu virus total length PCR primer of the expressing green fluorescent protein of 5 ' end containing T7 promotor, reaction solution is carried out nucleic acid gel electrophoresis, it is the fragment of 11021bp that length is reclaimed in cutting, use on 3500 type Genetic Analyser of ABI company and recombinant virus total length PCR primer sequence is measured, measurement result uses DNASTAR software to analyze, compare with the recombinant virus sequence of expection, meet expection through sequence alignment gained total length PCR primer sequence.Total length PCR primer electrophoresis result as shown in figure 13.
The rescue result of 3.4 recombinant viruses
From F1 generation, recombinant virus goes down to posterity by DF-1 cell continuously, basis of microscopic observation fluorescence, within 72 hours, reclaim the nutrient solution of virus, continuous passage, to F5 generation, finds the increase along with passage number, the expression amount of fluorescin successively decreases successively, until pass to F5 substantially cannot see fluorescence spot for rear.The rescue of recombinant virus and go down to posterity situation as shown in figure 14.
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 attenuated vaccine strain, it is characterized in that, this infections clone attenuated vaccine strain is the clonal strain of parent's poison duck tembusu virus attenuated vaccine strain FX2010-180P, it has the complete genome sequence shown in SEQIDNO.1, and this infections clone attenuated vaccine strain is obtained by reverse genetic manipulation method.
2. duck tembusu virus infections clone attenuated vaccine 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 attenuated vaccine strain FX2010-180P 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 attenuated vaccine strain FX2010-180P.
3. duck tembusu virus infections clone attenuated vaccine 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 attenuated vaccine strain FX2010-180P whole genome sequence: 1-2050 position nucleic acid fragment, 2026-3056 position nucleic acid fragment, 3022-4052 position nucleic acid fragment, 3656-6353 position nucleic acid fragment, 6216-9775 position nucleic acid fragment, 9753-10991 position nucleic acid fragment, 3656-9775 position nucleic acid fragment, 3656-10991 position nucleic acid fragment, 2026-4052 position nucleic acid fragment or 1-4052 position nucleic acid fragment in SEQIDNO.1 sequence.
5. a nucleic acid molecule, is characterized in that, comprises the complete genome sequence of the duck tembusu virus attenuated vaccine strain FX2010-180P shown in SEQIDNO.1, is also equipped with transcripting promoter at this SEQIDNO.1 sequence 5 ' end.
6. a preparation method for duck tembusu virus infections clone attenuated vaccine strain, is characterized in that, comprise the following steps:
Each DNA fragmentation of segmentation amplification duck tembusu virus attenuated vaccine strain FX2010-180P full-length genome;
Each DNA fragmentation is cloned into carrier respectively, forms multiple recombinant vectors that can cover duck tembusu virus attenuated vaccine strain FX2010-180P full-length genome;
The full-length cDNA of the duck tembusu virus attenuated vaccine strain FX2010-180P 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 attenuated vaccine strain FX2010-180P.
7. duck tembusu virus infections clone attenuated vaccine 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 attenuated vaccine strain according to claim 1 is as the application of virus vector.
9. a restructuring duck tembusu virus, it is characterized in that, this recombinant virus inserts exogenous gene sequence between the genomic NS5 gene of duck tembusu virus infections clone attenuated vaccine strain and 3 ' non-coding sequence described in claim 1, and described exogenous gene sequence is internal ribosome entry site sequence and green fluorescence protein gene sequence.
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| CN117448284A (en) * | 2023-11-02 | 2024-01-26 | 广东省农业科学院动物卫生研究所 | Recombinant tembusu virus expressing EGFP (enhanced green fluorescent protein) through stable passage and construction method |
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| CN107475294A (en) * | 2017-08-16 | 2017-12-15 | 四川农业大学 | Carry preparation method of duck Tan Busu reporter virus of renilla luciferase and products thereof and application |
| CN107475294B (en) * | 2017-08-16 | 2020-10-09 | 四川农业大学 | Preparation method of duck tembusu report virus carrying renilla luciferase, product and application thereof |
| CN111676198A (en) * | 2020-05-19 | 2020-09-18 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Method for quickly constructing duck tembusu virus reverse genetic strain |
| CN113234693A (en) * | 2021-05-17 | 2021-08-10 | 四川农业大学 | Duck tembusu virus low virulent strain and preparation method and application thereof |
| CN113234693B (en) * | 2021-05-17 | 2022-09-06 | 四川农业大学 | Duck tembusu virus low virulent strain and preparation method and application thereof |
| CN114015723A (en) * | 2021-11-05 | 2022-02-08 | 四川农业大学 | Duck tembusu virus plasmid vector, low virulent strain, preparation method and application thereof |
| CN117448284A (en) * | 2023-11-02 | 2024-01-26 | 广东省农业科学院动物卫生研究所 | Recombinant tembusu virus expressing EGFP (enhanced green fluorescent protein) through stable passage and construction method |
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