CN106318975A - Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof - Google Patents

Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof Download PDF

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CN106318975A
CN106318975A CN201610817565.8A CN201610817565A CN106318975A CN 106318975 A CN106318975 A CN 106318975A CN 201610817565 A CN201610817565 A CN 201610817565A CN 106318975 A CN106318975 A CN 106318975A
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bvdv
pmd
cdna
swine
borne
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朱国强
陶洁
王建业
朱礼倩
张信军
夏芃芃
孟霞
羊扬
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Yangzhou University
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Yangzhou University
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    • C12N2770/24011—Flaviviridae
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Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to swine-borne BVDV-2 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof. The swine-borne BVDV-2 strain infectious cDNA clone contains swine-borne BVDV-2 seed virus full-length cDNA, T7 RNA (ribonucleic acid) polymerase promoters are inserted in 5' terminals of the swine-borne BVDV-2 seed virus full-length cDNA, and SbfI restriction enzymes are inserted in 3' terminals of the swine-borne BVDV-2 seed virus full-length cDNA. The invention further discloses a plasmid pASH28 with the swine-borne BVDV-2 strain infectious cDNA. The plasmid is linearized and then is subjected to in-vitro transcription to obtain RNA, MDBK (Madin-Darby bovine kidney) cells are transfected by the plasmid, and bovine viral diarrhea viruses can be successfully rescued. The swine-borne BVDV-2 strain infectious cDNA clone, the application and the plasmid have the advantage that the swine-born BVDV-2 strain infectious cDNA clone can be applied to research on functional difference between different animal-borne BVDV-2 proteins and also can be used for genetically modifying and preparing high-titer attenuation BVDV vaccine.

Description

The construction method of Zhu Yuan BVDV-2 virus strain infection property cDNA clone and application
Technical field
The invention belongs to veterinary biologics technical field.It is specifically related to boar source BVDV-2 virus strain infection property cDNA gram Grand construction method and application.
Background technology
The host range of BVDV is very wide, except energy infected cattle, pig, sheep, deer, camel and multiple wild animal also can be caused to send out Sick.But until 2012, this disease is just classified as one of the disease that must circulate a notice of by OIE (OIE).
Pig infects BVDV and does not show clinical symptoms, or occurs being similar to the symptom of swine fever, thus not only gives the pre-of swine fever Anti-and control brings the biggest difficulty, and the prevention and control also making BVDV are extremely difficult.At present, the Mortality rate of BVDV in domestic swinery The most serious, it has also become a problem that can not be ignored.Wei Xiuyu etc. are between 2005～2009, to domestic 20 Duo Ge provinces and cities More than 300 Large-scale pig farm has carried out the Epidemiological study of BVDV, finds that the BVDV positive rate in each year is respectively 22.2%, 43.1%, 46.4%, 66.7% and 80.8%, trend and situation in expanding year by year are serious.Deng Yu etc. utilize shell type RT-PCR carries out examination at the clinical sample to Some Domestic area in 2007～2010, and the positive rate of detection BVDV is respectively 23.1% (2007), 27.7% (2008), 33.6% (2009) and 23.6% (2010).Liang Sheng etc. to 2010～ Within 2012, some areas of Guangxi swine diseases is investigated, and result shows that the recall rate of BVDV is 26.92%.
There is cross infection widely in BVDV and CSFV, BDV, therefore, brings tired not only to the diagnosis of BVDV and anti-system Difficulty, and obscured the quarantine of CSFV and BDV.At present, the prevention and control to BVDV abroad mainly use vaccine injection, strengthen prison Survey, eliminate immunologic tolerance and the animal of persistent infection.And at home, BVDV infects the most hidden with persistent infection and immunologic tolerance etc. Sexy dye is main, and therefore the prevention and control of BVDV should be emphatically to get rid of this inapparent infection animal.
The most potential application of reverse Genetics Technique is the development of new generation vaccine strain, by cDNA level to gene Group is modified, and can develop the efficiently marker vaccine of safety.Additionally, utilize this technology also can be in viral genome Insert exogenous gene sequence, so that RNA viruses becomes the carrier of vaccine and gene therapy.Therefore, this technology is also deep for us Enter to study cattle source BVDV and the infection mechanism of pig source BVDV and prevention and control provide effective technology platform.
At present, the domestic research to BVDV relatively lags behind, on the one hand inadequate to the attention degree of this disease due to people, the opposing party Face is owing to furtheing investigate the technology platform of this virus very little.And the success of infection clones builds to we providing a facility And effective instrument, viral genome can be modified from molecular level and be analyzed by let us, thus further deeply Solve its gene function and disease mechanisms.Although, the most successfully build many strains BVDV and separate the infection clones of strain, but these are all Separate strain for cattle source BVDV, the most also there is no the structure of pig source BVDV-2 infection clones.Therefore, this research is first The secondary pig source BVDV-2 that constructs separates the infection clones of strain SH-28, and the correlational study for follow-up pig source BVDV establishes one Platform.
Summary of the invention
The primary and foremost purpose of the present invention is to provide a boar source BVDV-2 infectious CDNA, modifies RNA, pig source for subsequent in vitro The protein function Research on differences of BVDV-2 infection mechanism, different animals source and genotype BVDV provides an effective technology to put down Platform.
Pig source of the present invention BVDV-2 infectious CDNA, contains pig source BVDV-2 kind poison total length, at this cDNA 5 ' End inserts t7 rna polymerase promoter, inserts SbfI restricted enzyme at cDNA 3 ' end.
The invention also discloses and comprise Zhu Yuan BVDV-2 virus strain infection's property cDNA clone and the 5 ' ends at this genome cDNA End inserts t7 rna polymerase I promoter sequence, inserts plasmid pASH28 and the structure thereof of SbfI restricted enzyme at 3 ' ends Build.
The construction method of described pASH28 is: extracts pig source BVDV-2 and separates the total serum IgE of strain SH-28, with SEQ ID No.1-18 be primer by RT-PCR method expand 9 cDNA fragment: SHA of its genome, SHB, SHC, SHD, SHE, SHF, SHG, SHH, SHI, described 9 fragments be cloned into respectively in pMD 19T Simple carrier obtain pMD-A, pMD-B, pMD-C, PMD-D, pMD-E, pMD-F, pMD-G, pMD-H and pMD-I；
PMD-D (SphI+BglI), pMD-E (BglI+EcoRV) and pBR322 (SphI+EcoRV) are carried out three fragments even Connect, it is thus achieved that pBR-DE；Then pMD-C (PshAI+SphI) is connected into pBR-DE (PshAI+SphI), it is thus achieved that pBR-CDE；Simultaneously PMD-A (EagI+SphI), pMD-B (SphI+PshAI) and pBR322 (EagI+PshAI) are carried out three fragment connections, it is thus achieved that pBR-AB；Finally pBR-AB (EagI+PshAI) is connected into pBR-CDE (EagI+PshAI), builds 5 ' large fragments pBR-AE；
Then pMD-F (EcoRV+SacI) and pMD-G (SacI+XbaI) is connected into pACYC184 (EcoRV+XbaI), structure Build pAC-FG；PMD-H (XbaI+NheI) and pMD-I (NheI+ClaI) is connected into pACYC184 (XbaI+ClaI) jointly simultaneously, Obtain pAC-HI；PAC-FG (EcoRV+XbaI) and pAC-HI (EcoRV+XbaI) is connected with each other, it is thus achieved that 3 ' large fragments pAC- FI；
PBR-AE (EagI+EcoRV) and pAC-FI (EagI+EcoRV) is connected with each other the most at last, it is thus achieved that full-length cDNA infects Sex clone plasmid pASH28.
For ensureing the stability of virus full length infection clones, we select low copy carrier pACYC184 to carry out structure Build, and utilize round pcr 5 ' ends of SH-28 genome and 3 ' ends introduce T7 promoter sequence respectively and one single SbfI restriction enzyme site.Based on total length transcript 5 ' end and the 3 ' changes held, infectivity is had a certain impact, when design by T7 Promoter sequence is accurately placed in 5 ' ends；Additionally, the sequence of SbfI restriction enzyme site is CCTGCA/GG, with SH-28 genome 3 ' end Having common " C ", on the one hand decrease the introducing of redundancy base, on the other hand make plasmid linearization, being transcribed into 3 ' ends can Correct termination.For ensureing virus genomic accuracy, during reverse transcription and PCR, select SuperScript First-respectively Strand Synthesis System for RT-PCR (Invitrogen) and Expand long template PCR system(Roche).But there is a coding mutation at genome 3978bp in this full-length cDNA infection clones pASH28, It is all consistent through many time clonings and the result of order-checking.The sudden change in this site result in going out of a new restriction enzyme site AseI Existing, we do not correct this sudden change, but as distinguishing Revive virus and a genetic marker of male parent virus.Through indirectly Immunofluorescence test and RT-PCR identify, find along with the increase of passage number, and the virus titer of Revive virus vASH gradually increases Add, and reach steady statue after the 5th generation.Revive virus genome is not lost in continuous succeeding generations AseI enzyme Cut site, illustrate that its inherited character is stable.Data above confirm we with pig source BVDV-2 separate strain SH-28 as template, success Establish the full-length cDNA infection clones platform of SH-28.
In our current research, in order to improve the virus titer of Revive virus, the method that we select band poison to pass on is saved The cultivation of virus.But indirect immunofluorescence qualification result shows, infect green the most a small amount of in the cell hole of 1st generation vASH glimmering Light, and infect the green fluorescence in the cell hole of the 3rd generation vASH and the most just reach half, illustrate that virus multiplication effect is not to manage very much Thinking, although after vASH passed to for the 5th generation, its virus titer has reached stable, and the virus titer growth trend of vASH is also and parent The virus titer growth trend of this virus is consistent.Lead it is presumed that be possibly due to carry out viral passages in 24 porocyte plates Causing, owing to the hole area of 24 porocyte plates is less, and the quantity every time passing on hole when inner cell paving is too many, causes cell short I.e. growing up to monolayer in time, growth is restricted, so that virus can not be in intracellular good propagation.Therefore, in virus During rescue, select correct viral passages method the most critically important.
After this plasmid linearization, in vitro transcription becomes RNA, and transfected into MDBK cells, can successfully save out bovine viral diarrhoea Virus.This boar source BVDV-2 infectious CDNA clones can be used for studying the function difference between the BVDV albumen of different animals source, Can also be used for genetic modification and prepare high-titer attenuation BVDV vaccine.
In sum, the present invention has been successfully established pig source BVDV-2 and has separated the infection clones platform of strain SH-28, is not only The gene functional research of follow-up pig source BVDV is laid a good foundation, and is also the gene function difference between pig source BVDV and cattle source BVDV Technology platform is provided Deng research.
Accompanying drawing explanation
The scattergram of restriction enzyme site in Fig. 1 .SH-28 pnca gene group
The integrated connection strategy of Fig. 2 .SH-28 strain full length cDNA clone
Fig. 3 .SH-28 separates the PCR amplification of strain each cDNA fragment
Fig. 4. each cDNA recombiant plasmid upgrading grain qualification result
Fig. 5. each cDNA recombiant plasmid enzyme action qualification result
Fig. 6. the enzyme action of full-length genome cDNA clone plasmid pASH28 is identified
Fig. 7. the indirect immunofluorescene assay of Revive virus vASH
Fig. 8. the RT-PCR detection of different generation Revive virus vASH
The specific fragment of Fig. 9 .RT-PCR detection Revive virus vASH
Figure 10. the virus titer growth trend of Revive virus and parental virus compares
Detailed description of the invention
1. material
1.1 viruses, cell, thalline and carrier
Pig source BVDV-2 strain SH-28 is separated by this experiment and identifies that [Tao Jie, etc. pig source bovine viral diarrhea virus SH- 28 whole genome sequences separating strain and heredity fractional analysis. China's veterinary's journal, 2013,33 (3): 321-325.].Genetic engineering Bacterium E.Coli HB101 and middle copy cloning vehicle pBR322 is purchased from TAKARA company, and low copy cloning vehicle pACYC184 is purchased from NEB company.MDBK cell line is given by Univ Pennsylvania USA professor Bello.
1.2 reagent and antibody
DNA Ligation Kit LONG and pMD 19T Simple carrier are purchased from TaKaRa company；Various restricted cores Acid restriction endonuclease is purchased from NEB company；Expand long template PCR system is purchased from Roche company；Pancreatin, DMEM train Support base and Opti-MEM culture medium purchased from Gibco company；QIAprep zero R Spin Miniprep Kit is purchased from QIAGEN company； MEGAscript T7 is purchased from Ambion company；Liposome Lipofectamine LTX and PlusTMReagent and SuperScript First-Strand Synthesis System for RT-PCR is purchased from Invitrogen company；MAb Bz-53 (BVDV-2) and D89 (BVDV-1) is given by Univ Pennsylvania USA professor Bello；HRP labelling sheep anti-mouse igg, FITC labelling sheep anti-mouse igg is purchased from doctor moral company；Other conventional reagent is domestic analytical pure level product.
1.3 key instrument
2720Thermal Cycler PCR instrument (Applied Biosystems), digital Labworks image acquisition and analysis software JD- 801 (Jetta companies), DYY-604 type voltage stabilization and current stabilization electrophresis apparatus (Nanjing New Campus Biological Technology Institute), liquid-transfering gun (Eppendorf), trace desk centrifuge (Thermol), CO2 gas incubator (Thermol), constant-temperature metal bath (win by Hangzhou Day Science and Technology Ltd.), MP120-2 electronic scale (Shanghai Ken Qiang Instrument Ltd.), ultra cold storage freezer (Haier), IMT2 is glimmering Light microscope (Olympus).
2. method
The structure of 2.1 SH-28 strain full-length cDNA infection clones
The PCR amplification of the most each cDNA fragment and clone
Extract pig source BVDV-2 and separate the total serum IgE of strain SH-28, expanded 9 cDNA of its genome by RT-PCR method Fragment, respectively SHA, SHB, SHC, SHD, SHE, SHF, SHG, SHH, SHI, the primer expanding each fragment is shown in Table 1.In order to ensure Amplified production and the fidelity of former sequence, select hi-fi, highly active SuperScript First-Strand Synthesis System for RT-PCR carries out reverse transcription, also selects the Roche Expand of hi-fi during PCR reaction long template PCR system。
Reclaim each pcr amplified fragment of purification, utilize " TA " cloning that each fragment is cloned into pMD 19T Simple respectively In carrier, connect product and convert HB101 competent cell, screened the sun of each cDNA fragment by the ammonia benzyl resistance marker on carrier Property recon pMD-A, pMD-B, pMD-C, pMD-D, pMD-E, pMD-F, pMD-G, pMD-H and pMD-I.
The each fragment primer of table 1.SH-28 infection clones
2.1.2 the screening of cDNA positive colony and qualification
In the ammonia benzyl resistant panel of above-mentioned conversion, the single colony inoculation of picking is in the LB liquid of ammonia benzyl resistance, and 37 DEG C are shaken Swing cultivation 16h；Then use alkaline lysis method of extracting plasmid, and carry out electroresis appraisal with the agarose gel of 0.8%.Further Carry out double digestion qualification with corresponding restricted enzyme, finally each cDNA positive colony filtered out is served marine growth engineering Technology Co., Ltd. checks order, and each sample repeats to check order 3 times.
2.1.3 the connection of full-length cDNA and clone
Owing to BVDV full-length genome is about 12.3kb, the single restriction enzyme site wherein comprised is not typically the most conventional Restricted enzyme, therefore we select full-length genome cDNA is divided into the strategy that 2 large fragments connect.With in full-length genome Between position single restriction enzyme site EcoRV for boundary, be divided into 5 ' large fragments and 3 ' large fragments, thus avoid conventional restriction enzyme site Between interfere (Fig. 1), integrated connection strategy is as shown in Figure 2.
In view of restriction enzyme site unicity on each cDNA fragment and carrier and the problem of order, 5 ' large fragments are connected into In pBR322 carrier, 3 ' large fragments are then connected in pACYC184 carrier, and two large fragments are connected the most at last, are cloned into together In pACYC184 carrier, complete the connection of full-length cDNA infection clones.The concrete Connection Step of 5 ' large fragments is: first will PMD-D (SphI+BglI), pMD-E (BglI+EcoRV) and pBR322 (SphI+EcoRV) carry out three fragment connections, it is thus achieved that pBR- DE；Then pMD-C (PshAI+SphI) is connected into pBR-DE (PshAI+SphI), it is thus achieved that pBR-CDE；Simultaneously by pMD-A (EagI + SphI), pMD-B (SphI+PshAI) and pBR322 (EagI+PshAI) carry out three fragment connections, it is thus achieved that pBR-AB；Finally will PBR-AB (EagI+PshAI) is connected into pBR-CDE (EagI+PshAI), builds 5 ' large fragments pBR-AE.
The concrete Connection Step of 3 ' big segments is: first by pMD-F (EcoRV+SacI) and pMD-G (SacI+XbaI) even Enter pACYC184 (EcoRV+XbaI), build pAC-FG；Altogether by pMD-H (XbaI+NheI) and pMD-I (NheI+ClaI) simultaneously With being connected into pACYC184 (XbaI+ClaI), it is thus achieved that pAC-HI；Finally by pAC-FG (EcoRV+XbaI) and pAC-HI (EcoRV+ XbaI) it is connected with each other, it is thus achieved that 3 ' large fragments pAC-FI.
Finally carry out the connection of two large fragments, pBR-AE (EagI+EcoRV) and pAC-FI (EagI+EcoRV) is mutual Connect, it is thus achieved that full-length cDNA infective cloned plasmids pASH28.Above enzyme action, reclaim, connection etc. belongs to routine operation, according to " Molecular Cloning: A Laboratory guide " operates.
2.1.4 the qualification of full-length cDNA infective cloned plasmids
According toSpin Miniprep Kit description extracting full-length cDNA infective cloned plasmids pASH28, Concrete operation step is: take 4mL bacterium solution, and 12,000g are centrifuged 2min collects thalline, goes the most residual night；Add 250 μ L Buffer P1, Resuspended thalline；Add 250 μ L Buffer P2, overturn 4～6 times gently；Add 350 μ L Buffer N3, overturn 4～6 the most gently Secondary, room temperature places 5min；4 DEG C, 12,000g are centrifuged 10min, carefully draw supernatant, are placed in QIAprep adsorption column, and room temperature is quiet Put 2min, then 12,000g is centrifuged 30s；Liquid in collecting pipe is inhaled again and puts in QIAprep adsorption column, repeat above-mentioned behaviour Make 1 time；Then adding 750 μ L Buffer PE to wash 1 time, 12,000g are centrifuged 30s；Discarding liquid in collecting pipe, 12,000g are centrifuged 1min, abandons most residual liquid；Then QIAprep adsorption column is placed on a new 1.5mL dactylethrae, 37 DEG C of dry 5min；Add 30 μ L Ultra-pure water is in adsorption column, and room temperature stands 2min；12,000g are centrifuged 30s, and centrifugal gained liquid is the pASH28 matter of purification Grain.
Use PCR amplification and the method for digestion with restriction enzyme, full length cDNA clone plasmid pASH28 is carried out dual Identify.PCR identifies and selects wherein 4 pairs of cDNA amplimers (SHC～SHF)；In order to identify full length cDNA clone plasmid further Accuracy, select three groups of restricted enzyme to carry out enzyme action qualification, select EcoRV and PshAI double digestion for first group, second group With XhoI and XbaI double digestion, the 3rd group is SacI single endonuclease digestion.Enzyme action system is 1 μ L plasmid, 0.3 μ L restricted enzyme, X μ L Buffer, moisturizing to 20 μ L (the corresponding different Buffer of X: different restricted enzyme), 37 DEG C of water-bath 1h, then use 1% agarose gel electrophoresis identifies enzyme action result.
2.2 in vitro transcription
First with SbfI restricted enzyme single endonuclease digestion infective cloned plasmids, it is allowed to linearisation；Then enzyme action is terminated anti- Should, in above-mentioned enzyme action system, i.e. add 1/20 volume 0.5M EDTA, 1/10 volume 3M NaAc and 2 times of volume dehydrated alcohol also Put-20 DEG C of precipitation 30min；13,000g are centrifuged 15min, abandon supernatant, drying precipitated, then with appropriate ultra-pure water dissolution precipitation, extremely Final concentration of 0.5～1 μ g/ μ L.Transcribe to eliminate the RNase that may produce or introduce during upgrading grain and enzyme action or other Inhibitor, carries out following process: add final concentration of 100～200 μ g/mL E.C. 3.4.21.64 and 0.5%SDS, 50 DEG C of water-bath 30min, Then with phenol/chloroform, the method purified product of alcohol precipitation, finally with 8 μ L RNase-free H2O dissolution precipitation.
According to MEGAscript T7 test kit description, the linearization plasmid through above-mentioned process is carried out in vitro transcription, tool Precursor reactant system is: the 8 linearizing infective cloned plasmids of μ L, each 2 μ L of ATP, CTP, GTP, UTP, 2 μ L 10 × Reaction After Buffer, 2 μ L Enzyme Mix, fully mixing, hatch 4h for 37 DEG C；Add 1 μ L TURBO DNase, 37 DEG C of effects 15min；It is subsequently adding 30 μ L RNase-free H2O and 30 μ L LiCl ,-20 DEG C of precipitation more than 30min；4 DEG C of 13,000g from Heart 15min；Abandoning supernatant, 70% ethanol washes one time；It is dried, finally with 20 μ L RNase-free H2O dissolution precipitation is standby.
2.3 cell transfecting
24h before transfection, takes the MDBK cell that growth conditions is good, with trypsinization and adjust cell concentration, inoculates in right amount In 24 porocyte culture plates, put in CO2 gas incubator and cultivate.Within second day, when cell monolayer degrees of fusion reaches about 80%, incite somebody to action 24 porocyte plate inner cell supernatants discard, and PBS washs 2 times, add Opti-MEM culture medium.Referring next to Lipofectamine LTX and PlusTMReagent description transfects: first preparation DNA-liposome turbid liquor, takes 2 1.5mL finger-types Pipe, adds 100 μ L Opti-MEM, 1 μ g RNA and 1 μ L Plus Reagent, another dactylethrae in one of them dactylethrae Middle addition 100 μ L Opti-MEM and 3 μ L lipofectamine LTX, rear chamber is gentle and quiet puts 5min in mixing gently；Then by two Suspension in dactylethrae is added together, and rear chamber is gentle and quiet puts 25min in soft mixing；24 porocyte plate inner cell supernatants are discarded, Add DNA-liposome turbid liquor, then 24 porocyte plates are placed in CO2 gas incubator；After quiescent culture 6h, by cell Supernatant changes the DMEM complete medium containing 10%HS into, then cell is placed in 37 DEG C of CO2 gas incubator continuation and cultivates.
The qualification of 2.4 infectious virus particles
2.4.1 the enrichment culture of Revive virus
In order to improve the titre of Revive virus, the method selecting band poison cell to pass poison carries out the enrichment culture of virus.Transfection After in vitro transcription thing RNA 48～72h, collect and preserve 1st generation cell culture supernatant, PBS cell one time, then use 0.02% trypsinization also carries out Secondary Culture in the ratio of 1:3；When cell degrees of fusion reaches 100%, repeat aforesaid operations, Carry out the passing on of Revive virus vASH, enrichment culture.
2.4.2 indirect immunofluorescene assay
VASH and SH-28 is inoculated in respectively the MDBK cell in 24 porocyte plates, carries out indirect immunofluorescene assay.Training After supporting 48h, discarding cell conditioned medium, PBST washs 3 times, each 5min；200 μ L 70% ethanol are added fixing thin after drying at room temperature Born of the same parents ,-20 DEG C of fixing 15min；Discard fixative, ibid wash；The most each hole add 150 μ L monoclonal antibodies Bz-53 (1: 3000), it is placed in wet box 37 DEG C and hatches 1h；Discard one to resist, ibid wash；Add 150 μ L FITC labellings sheep anti-mouse igg (1: 400), in wet box 37 DEG C hatch 30min；Discarding two to resist, ibid wash, the glycerol being eventually adding 1 10% finishes filming, and then places Observed result under fluorescence microscope.Ghost comparison is set simultaneously.
2.4.3 RT-PCR detection
Total serum IgE the reverse transcription of extracting the 3rd, 5,10 generation Revive virus vASH become cDNA, then draw with the public detection of BVDV Thing FBVDVI-II (5 '-CATGCCCATAGTAGGAC-3 ' SEQ ID No.19)/RBVDVI-II (5 '- CCATGTGCCATGTACAG-3 ' SEQ ID No.20) carry out PCR amplification, the viral level of the different generation Revive virus of detection becomes Change.Additionally, with the RNA of the tenth generation Revive virus as template, choose the wherein 4 cDNA amplimer to building infection clones (SHC～SHF) carries out RT-PCR qualification, and will comprise the fragment of AseI restriction enzyme site (with the sheet of SHD primer pair amplifies gained Section) serve marine growth engineering finite technology company and check order and analyze.
2.4.4 the drafting of virus titer growth curve
Revive virus vASH and parental virus SH-28 is infected monolayer MDBK cell respectively, and infective dose is 1MOI.Absorption 1h After, discard cell conditioned medium, be subsequently adding the DMEM containing 2%HS and maintain liquid to continue to cultivate.After infection 12h, 24h, 48h, 60h, 72h collect cell ,-20 DEG C of preservations respectively, and measure its TCID respectively50.Measure virus TCID50Concrete grammar be first Virus liquid being made continuous print 10 times dilution, is inoculated in the most respectively in 96 porocyte plates, 100 μ L/ holes, each dilution factor repeats to connect Plant 8 holes；After absorption 1h, cell conditioned medium is changed into the DMEM containing 2%HS and maintains liquid；After cultivating about 72h, with reference in this chapter 2.7.2 Operating procedure carry out indirect immunofluorescene assay, then according to Reed-Muench method calculate each time point virus TCID50.Last with the time as abscissa, TCID50For vertical coordinate, draw virus titer growth curve.
3. result
The structure of 3.1 SH-28 strain full-length cDNA infection clones and qualification
3.1.1 SH-28 pnca gene group cDNA fragment amplification
Separate the whole genome sequence of strain SH-28 according to pig source BVDV-2, design 9, to cDNA primer (table 1), extracts virus Total serum IgE, utilizes RT-PCR method Successful amplification to go out to cover 9 cDNA fragments of SH-28 full-length genome, swimming lane 1-9 be SHA, SHB, SHC, SHD, SHE, SHF, SHG, SHH and SHI pcr amplified fragment, each fragments specific is all preferable, and length is respectively 344bp, 2194bp, 1179bp, 3205bp, 1281bp, 2243bp, 2611bp, 2539bp and 680bp, be all consistent (figure with expection 3)。
The qualification of the most each cDNA fragment cloned plasmids
PCR expanding 9 cDNA fragments of gained and cuts after glue reclaims, " TA " is cloned in pMD 19T Simple carrier, Plasmid identification result such as Fig. 4, swimming lane 1-9 are each cDNA recombiant plasmid, size be respectively 3036bp, 4884bp, 3871bp, 5897bp, 3973bp, 4935bp, 5303bp, 5228bp and 3372bp, be all consistent with expection.Corresponding with each cDNA fragment both sides Restricted enzyme carry out double digestion qualification, enzyme action result such as Fig. 5, swimming lane 1-9 enzyme action successively be 278bp+2758bp, 2024bp+2862bp、857bp+3014bp、2695bp+3202bp、750bp+3223bp、1750bp+3185bp、2260bp+ 3043bp, 1159bp+4072bp, 544bp+2828bp two band, size is the most correct.The doubtful sun of correct each fragment will be identified The order-checking of marine growth engineering finite technology company is served in sex clone, after sequence alignment is correct, it is thus achieved that the positive colony of each fragment PMD-A, pMD-B, pMD-C, pMD-D, pMD-E, pMD-F, pMD-G, pMD-H and pMD-I.It should be noted that pMD-D fragment In have individual base to there occurs sudden change, thus resulted in an AseI restriction enzyme site, the result through repeatedly checking order is all consistent 's.
3.1.3 the qualification of full-length genome cDNA infective cloned plasmids
According to routine operation, will identify that correct each cDNA fragment carries out enzyme action, connection according to suitable connection strategy, Rear clone enters pACYC184, it is thus achieved that pig source BVDV-2 separates the full-length genome cDNA infective cloned plasmids pASH28 of strain SH-28. Selecting three groups of restricted enzyme to carry out enzyme action qualification (Fig. 6), swimming lane 1 is pASH28 plasmid；Swimming lane 2 is for using EcoRV and PshAI The result of double digestion plasmid, 3170bp, 4303bp and 8173bp tri-band of attaining the Way；Swimming lane 3 is for using XhoI and XbaI double digestion Result, it is thus achieved that 1796bp, 3488bp and 10362bp tri-band；Swimming lane 4 is SacI single endonuclease digestion result, it is thus achieved that 659bp, 6982bp With 8005bp tri-band, three groups of enzyme action results are all correct, are consistent with expection.Above qualification result explanation pASH28 be intended, Stable pig source BVDV-2 full-length genome cDNA infection clones.
The rescue of 3.2 vASH viruses and qualification
3.2.1 indirect immunofluorescene assay
With MAb Bz-53, the 1st, 3,5 generation Revive virus vASH are carried out indirect immunofluorescene assay, result such as Fig. 7, MDBK Redgreen fluorescence (Fig. 7 A) in cell controls, a green fluorescence only tuftlet (Fig. 7 B) in 1st generation vASH infection cell hole, the 3rd Account for for the amount of fluorescence in vASH infection cell hole a greater part of (Fig. 7 C) in the visual field, and the visual field in the 5th generation vASH infection cell hole It is specificity green fluorescence (Fig. 7 D) the most entirely.Increase along with passage number is described, infectious virus particle gets more and more, and arrives During 5 generation, the virus titer of Revive virus vASH reaches stable.
3.2.2 RT-PCR detection
Extract the total serum IgE of the 1st, 3,5 generation Revive virus vASH, with BVDV public detection primer (FBVDVI-II/RBVDVI- II) carrying out RT-PCR amplification, as Fig. 8, swimming lane 1-3 represent the 1st, 3,5 generation viruses respectively, result shows the increasing along with passage number Adding, the viral level of Revive virus vASH is also gradually increased.
With the 10th generation vASH total serum IgE as template, choosing 4 pairs of primers and carry out RT-PCR detection, such as Fig. 9, swimming lane 1～4 is respectively It is the result utilizing amplimer SHC～SHF to carry out RT-PCR detection, has all amplified the specific band of expection size.For The hereditary stability of further qualification vASH, clones its D fragment (using SHD primer pair amplifies gained fragment) and checks order point Analysis, finds to remain an AseI restriction enzyme site sequence in the gene order of this fragment, illustrates that Revive virus is at continuous print In succeeding generations, its inherited character still remains stable.
The comparison of 3.3 virus titer growth trends
Draw the virus titer growth curve of vASH and parental virus vASH, find that three has and preferably grow spy Property, and vASH and parental virus SH-28 has similar virus titer growth trend (Figure 10).

Claims (5)

1. the plasmid containing Zhu Yuan BVDV-2 virus strain infection property cDNA, it is characterised in that this plasmid is pASH28, contains Zhu Yuan BVDV-2 virus strain infection property cDNA clone, the 5 ' ends at this genome cDNA insert T7 rna plymerase i promoter sequences Row, insert SbfI restricted enzyme at 3 ' ends.
2. described in claim 1, contain the construction method of the plasmid pASH28 of Zhu Yuan BVDV-2 virus strain infection property cDNA, its feature It is that its step is as follows:
Extract pig source BVDV-2 and separate the total serum IgE of strain SH-28, expanded by RT-PCR method for primer with SEQ ID No.1-18 9 cDNA fragments of its genome: SHA, SHB, SHC, SHD, SHE, SHF, SHG, SHH, SHI, described 9 fragments are cloned respectively Obtain to pMD 19 T Simple carrier pMD-A, pMD-B, pMD-C, pMD-D, pMD-E, pMD-F, pMD-G, pMD-H and pMD-I；
PMD-D, pMD-E and pBR322 are carried out three fragment connections, it is thus achieved that pBR-DE；Then pMD-C is connected into pBR-DE, it is thus achieved that pBR-CDE；PMD-A, pMD-B and pBR322 are carried out three fragment connections simultaneously, it is thus achieved that pBR-AB；Finally pBR-AB is connected into PBR-CDE, builds 5 ' large fragments pBR-AE；
Then pMD-F and pMD-G is connected into pACYC184, builds pAC-FG；PMD-H and pMD-I is connected into jointly simultaneously PACYC184, it is thus achieved that pAC-HI；PAC-FG with pAC-HI is connected with each other, it is thus achieved that 3 ' large fragments pAC-FI；
PBR-AE and pAC-FI is connected with each other the most at last, it is thus achieved that full-length cDNA infective cloned plasmids pASH28.
3. the plasmid containing Zhu Yuan BVDV-2 virus strain infection property cDNA described in claim 1 is at research different animals source BVDV egg The purposes in function difference between Bai.
4. the plasmid containing Zhu Yuan BVDV-2 virus strain infection property cDNA described in claim 1 is preparing high-titer attenuation BVDV epidemic disease Application in Seedling.
5. the plasmid containing Zhu Yuan BVDV-2 virus strain infection property cDNA described in claim 1 is at rescue bovine viral diarrhea virus In application.
CN201610817565.8A 2016-09-12 2016-09-12 Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof Pending CN106318975A (en)

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CN106893732A (en) * 2017-04-18 2017-06-27 扬州大学 A kind of rescue method of Goose Parvovirus clone
CN107881260A (en) * 2017-11-17 2018-04-06 扬州大学 Bovine viral diarrhea virus carries out the method for separating identification in a kind of cow's serum
CN108611329A (en) * 2018-05-11 2018-10-02 上海市农业科学院 Express pig source BVDV recombinant viruses and the application of Porcine epidemic diarrhea virus S genes
CN114395568A (en) * 2021-10-29 2022-04-26 扬州大学 Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

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* Cited by examiner, † Cited by third party
Title
蒙明璐: "猪源牛病毒性腹泻病毒RT-PCR检测方法的建立即表达BVDV保护性抗原花丛中猪痘病毒的哦古剑", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 *
陶洁: "猪源BVDV-2感染性克隆的构建及非结构蛋白Npro的相关功能探析", 《中国博士学位论文全文数据库（农业科技辑）》 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106893732A (en) * 2017-04-18 2017-06-27 扬州大学 A kind of rescue method of Goose Parvovirus clone
CN107881260A (en) * 2017-11-17 2018-04-06 扬州大学 Bovine viral diarrhea virus carries out the method for separating identification in a kind of cow's serum
CN108611329A (en) * 2018-05-11 2018-10-02 上海市农业科学院 Express pig source BVDV recombinant viruses and the application of Porcine epidemic diarrhea virus S genes
CN114395568A (en) * 2021-10-29 2022-04-26 扬州大学 Porcine epidemic diarrhea virus infectious cDNA clone and construction method and application thereof

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