CN100384990C - Method for constructing non-virulent strain of infectious bursal disease virus - Google Patents
Method for constructing non-virulent strain of infectious bursal disease virus Download PDFInfo
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Abstract
The present invention discloses a method for constructing non-virulent strains of infectious bursa disease virus (IBDV), which comprises the following steps: 1), extracting the RNA of segments A and B of genomes from IBDV, and cloning full-length cDNA of the RNA; 2), obtaining the full-length sequence of the segment A of the genomes with VP5 genes deleted, and constructing the infectious clones of the segments A and B; 3), co-transfecting the infectious clones to culture cells in order to rescue IBDV ANhe2 strains with VP5 genes deleted. Compared with the traditional method for weakening by cell passages, the method has the advantage of more stabilization and effectively retains the immunogenicity of virus. The present invention provides an IBDV ANhe2 strain with VP5 genes deleted, wherein the virus can not express VP5 proteins, retains favorable immunogenicity at the same time of losing the pathogenicity to chickens, and has no toxicity atavism. The virus has the advantages of simple preparation and convenient use. Other characteristics of the ANhe2 strain are consistent with the traditional low virulent vaccine virus strains.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method for constructing non-virulent strain of infectious bursal disease virus.
Background technology
(Infectious bursal disease is by infectious bursal disease virus (Infectious bursal disease virus, acute, the height contagious disease that IBDV) cause IBD) to infectious bursal disease.IBDV mainly encroaches on the central immune organ fabricius bursa of chick, causes immunosuppression and immune deficiency, causes the immuning failure of other vaccine simultaneously, is one of main transmissible disease that endangers at present world's aviculture.IBDV is a diplornavirus, belongs to birnavirus section (Biranviridae) Avibirnavirus (Avibirnavirus), and its genome is made up of two sections of A, B.B sections (about 2.8kb) coding VP1 albumen is the RNA polymerase of RNA dependence.A sections (about 3.3kb) has two overlapped open reading frames (ORF), and the precursor protein VP2/4/3 of the ORF1 coding in downstream can further shear and be processed as VP2, VP4, VP3.Wherein VP2 is the main host protective antigen of virus, and is relevant with the antigenic variation and the virulence of virus; VP4 is a kind of serine protease, and the oneself who is responsible for precursor protein VP2/4/3 shears; VP3 then is one of structural protein of virus, has group specific antigen.The ORF2 of upstream (438bp) the Nonstructural Protein VP5 (about 15kDa) that encodes, the non-virus replication of this albumen institute is essential, and is pathogenic relevant with virus.
Change the codon ATG of IBDV D78 low virulent strain ORF A2 into AGG (coding Arg), the defective virus IBDV/VP5-that the no VP5 of its cRNA transfection chick-embryo cell (CEC) acquisition expresses is vero cells infection normally, and prompting VP5 duplicates also inessential at IBDV.D78 low virulent strain of usefulness VP5 defectives such as Yao (rD78NS Δ) and parental virus rD78 infect respectively 3 age in week the SPF chicken, do not cause any clinical symptom and fabricius bursa tissue injury even the former strengthens dosage of inoculation yet, the latter is opposite; And it is similar that both induce the ability of IBDV neutralizing antibody.Further studies show that, VP5 is placed CMV promotor downstream transfection chicken B lymphocyte (RP9) and chick embryo fibroblast respectively, as a result the induced expression of VP5 the apoptosis of RP9 and chick embryo fibroblast, and the cytopathy that the virus that has lacked the VP5 rescue causes is lighter than wild-type, reproduction speed is slower than wild-type, the virus quantity of cell conditioned medium liquid reduces by 30 times than wild-type, the virus that has lacked VP5 greatly reduces the ability of cytolysis releasing virus, as seen VP5 has very strong cytotoxicity, discharges to born of the same parents outside in the born of the same parents with ripe virus and viral pathogenic relevant.Therefore the VP5 associated biomolecule is learned characteristic and study, will help us further to understand causing a disease and immunologic mechanism of IBDV.
Summary of the invention
The purpose of this invention is to provide a kind of method for constructing non-virulent strain of infectious bursal disease virus.
The step of method is:
1) extracting of property bursa of Fabricius virus genome A, B sections RNA and the clone of cDNA total length thereof;
2) acquisition of the genome A sections full length sequence of VP5 genetically deficient and A, B sections Construction of Infectious Molecular Clone;
3) above-mentioned infections clone cotransfection culturing cell is saved out the infectious bursal disease virus ANhe2 strain of VP5 genetically deficient;
The extracting of described infectious bursal disease virus genome A, B sections RNA and the clone of cDNA total length thereof: the HZ2 strain infectious bursal disease virus that collecting cell is cultivated, its genome of extracting A, B sections RNA, with the accurate RT-PCR amplification gene group A of long distance, B sections cDNA total length, clone easy in pGEM-T, obtain recombinant vectors pGEM-T-A/HZ2 and pGEM-T-B/HZ2, and carry out sequencing.
The acquisition of the genome A sections full length sequence of VP5 genetically deficient and A, B sections Construction of Infectious Molecular Clone: reclaim the long segment of pGEM-T-A/HZ2 after the NdeI enzyme is cut and again from successively win pGEM-T-AN; Carry out the rite-directed mutagenesis of PCR mediation with upstream and downstream mutant primer The2S:AGATCAGACAAACGATCGCAG and TheA:GcTaGcAATGATAGCGTTATAGAAG, TaKaRa MutanBEST Kit, the linearized vector that obtains 3603bp is after also direct self connection, acquisition contains the mutant pGEM-T-The2 of point mutation and fragment deletion, and order-checking confirms.After the EcoR I/Nde I endonuclease bamhi of pGEM-T-The2 623bp is connected with the Nde I/Kpn I fragment of the 2615bp that comes from pGEM-T-A/HZ2 is linear, directly insert the pC I carrier of cutting through EcoR I/Kpn I enzyme, obtain finally to be used for the eukaryon expression plasmid pC I-ANhe2 of transfection, its preserving number is CGMCC No.1507, with recombinant plasmid pGEM-T-B/HZ2 is skeleton, with EV5:CGCCCTTAAGACCGGTCGaTAtcGGAACGAAG and B3:ATTTCTAGAGGGGGCCCCCGCAGGCGAA is mutant primer, use the Pfu polymeric enzymatic amplification to go out the sudden change fragment of 573bp, simultaneously 573bp fragment and pGEM-T-B/HZ2 are carried out Afl II/Xba I double digestion, replace the corresponding part of pGEM-T-B/HZ2 with the 573bp fragment, obtain containing the B sections recon pGEM-T-mB/HZ2 of EcoRV mark, after order-checking confirms, cut pGEM-T-mB/HZ2 with EcoRI/XbaI/Pvu I enzyme, reclaim the 2833bp fragment, be connected with the pCI carrier of cutting through the EcoRI/XbaI enzyme, acquisition is used for the eukaryon expression plasmid pCI-mB/HZ2 of transfection, and its preserving number is CGMCC No.1506.
With above-mentioned infections clone cotransfection culturing cell, save out the infectious bursal disease virus ANhe2 strain of VP5 genetically deficient: under liposome Lipofectamine 2000 mediations, chick embryo fibroblast with pCI-ANhe2/pCI-mB cotransfection 80% degrees of fusion, pass through cytopathy, simulated virus goes down to posterity, be an anti-indirect immunofluorescence assay with anti-IBDV serum of rabbit and the anti-IBDV VP5 of rabbit serum respectively, the electron microscopic observation virus particle, RT-PCR and enzyme are cut experiment confirms such as evaluation and are saved out the infectious bursal disease virus of VP5 genetically deficient, are the ANhe2 strain with this viral nomenclature.
Description of drawings
Fig. 1 is infectious bursal disease virus provided by the invention (IBDV) genome A, B sections cDNA amplification and clone's process and collection of illustrative plates.
Fig. 2 is that infectious bursal disease virus provided by the invention (IBDV) genome A sections RT-PCR identifies collection of illustrative plates.
Fig. 3 is that infectious bursal disease virus provided by the invention (IBDV) genome B sections RT-PCR identifies collection of illustrative plates.
Fig. 4 is the genome A sections mutant of infectious bursal disease virus provided by the invention (IBDV) and the building process and the collection of illustrative plates of eukaryon expression plasmid thereof.
Fig. 5 is the order-checking situation of the genome A sections mutant of infectious bursal disease virus provided by the invention (IBDV).
Fig. 6 is that the enzyme of the eukaryon expression plasmid pCI-ANhe2 of the genome A sections mutant that contains infectious bursal disease virus (IBDV) provided by the invention is cut the evaluation collection of illustrative plates.
Fig. 7 is the building process and the collection of illustrative plates of the genome B sections mutant of infectious bursal disease virus provided by the invention (IBDV).
Fig. 8 is the order-checking situation of the genome B sections mutant of infectious bursal disease virus provided by the invention (IBDV).
Fig. 9 is that the enzyme of the eukaryon expression plasmid pCI-mB of the genome B sections mutant that contains infectious bursal disease virus (IBDV) provided by the invention is cut the evaluation collection of illustrative plates.
Figure 10 is the cytopathic effect observation (100 *) of infectious bursal disease virus provided by the invention (IBDV) gene-deleted strain ANhe2 strain.
Figure 11 is the expression (100 *) that the indirect immunofluorescence assay of infectious bursal disease virus provided by the invention (IBDV) gene-deleted strain ANhe2 strain cell culture is observed virus structural protein and Nonstructural Protein.
Embodiment
Be preserved in the colon bacillus (Escherichia coli) at China Committee for Culture Collection of Microorganisms common micro-organisms center on October 25th, 2005, preserving number is CGMCCNO1506 and CGMCCNO1507.
Embodiment 1:
Present embodiment has been described the preparation method of infectious bursal disease virus provided by the invention (IBDV) genome A, B sections full length sequence, and the whole experiment of long distance R T-PCR amplification and clone IBDV binodal fragment gene group as shown in Figure 1.
(1) RT-PCR amplification infectious bursal disease virus (IBDV) HZ2 pnca gene group A, B sections full length sequence
Chick embryo fibroblast (chick embryo fibroblast) the cell culture fluid multigelation of collection infectious bursal disease virus (IBDV) HZ2 strain 3 times; 5000rpm, 4 ℃, 5min is centrifugal, gets supernatant; 14000rpm, 4 ℃, 5min is centrifugal, gets supernatant; Supernatant adds in the dialysis tubing with funnel, places 50% polyoxyethylene glycol (PEG), puts 4 ℃ and spends the night, and concentrates 100 times; Viral liquid after concentrating with the pipettor sucking-off ,-20 ℃ of preservations are standby.
Get the viral liquid after the 200 μ l dialysis, add in the new 0.5ml eppendorf pipe of handling with DEPC; Add SDS and Proteinase K to final concentration respectively and be 0.5% and 1mg/ml, 50 ℃, digestion 2hrs; Use isopyknic phenol/chloroform/primary isoamyl alcohol (25/24/1), chloroform/primary isoamyl alcohol (24/1) extracting respectively once, the centrifuging and taking supernatant, add 3M NaAc to final concentration be 0.3M, add the ice-cold dehydrated alcohol of 650ml again, the mixing that turns upside down ,-20 ℃ are spent the night precipitated rna; 12000rpm, 4 ℃, centrifugal 10min, precipitation 75% ethanol rinsing of precooling; Room temperature volatilization ethanol is looked precipitation capacity and is added an amount of DEPC treating water, blows and beats mixing gently; After 1% agarose gel electrophoresis was identified ,-20 ℃ of preservations were standby.
Design primer respectively at genome A, B sections full length sequence, see Table 1, the A5/A3 A sections full length sequence that is used to increase, the B5/B3 B sections full length sequence that is used to increase.All primers all are diluted to 25 μ M with the DEPC treating water, and-20 ℃ of preservations are standby.
Table 1 infectious bursal disease virus genome A, B sections cDNA amplification primers sequence
Following various composition mixings and of short duration centrifugal before using: 5 μ l viral RNA genome suspensions, 2 μ l reverse transcription primers (2.5 μ M), 2 μ l, 10 * PCR buffer, 2 μ l MgCl
2(25mM), 1 μ l dNTP (10mM), 2 μ l DTT (0.1M); 98 ℃, 5min ices cancellation 1min immediately; 50 ℃, 5min; Add 1 μ l (200U) MMLV ThermoScript II to pipe, mixing, 50 ℃, 50min; 70 ℃, 15min termination reaction, ice cancellation; Carry out the PCR reaction immediately; Or freezing preservation.Operational guidance according to the Expand HighFidelity PCR System of Roche company carries out the PCR reaction, with reverse transcription synthetic first chain is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 15sec, 60 ℃ of annealing 30sec, 68 ℃ are extended 5min; Circulate 30 times, 72 ℃ are extended 10min.Get 1～21% agarose gel electrophoresis after reaction finishes and detect, the A sections cDNA that the result obtains about 3.3kb (sees Fig. 2.1, DNA maker; 2 ﹠amp; 3, A sections cDNA) and the B sections cDNA of 2.8kb (see Fig. 3.M, DNA maker; 1, B sections cDNA).
(2) clone of IBDV HZ2 pnca gene group A, B sections full length sequence
Rubber tapping is reclaimed the PCR product and is directly connected on the pGEM-T easy carrier (Promega company).Specifically carry out to specifications, in the 0.5ml centrifuge tube, add following reagent: 2 * Buffer 5 μ l successively, pGEM-T easy 1 μ l, the PCR product 4 μ l that reclaim, T4DNA liagase 1 μ l, room temperature connects 1h, transformed into escherichia coli HD5 α, coating contains the LB flat board of penbritin, and picking list bacterium colony is with the LB substratum enlarged culturing of spending the night, the extracting plasmid DNA is carried out PCR and enzyme and is cut evaluation.Be further to confirm the exactness of sequence, each sections respectively select 3 independently positive colony check order.The primer step-by-step method is used in order-checking, carries out on the ABI3730 sequenator.To clone difference called after pGEM-T-A/HZ2 and the pGEM-T-B/HZ2 that checks order correct.Sequencing result shows that the A sections of HZ2 strain has 3259bp, and sequence is as follows:
1 ggatacgatc?ggtctgaccc?cgggggagtc?acccggggac?aggtcgccaa?ggccttgttc
61 caggatggaa?ctcctccttc?tataacgcta?tcattgatgg?tcagtagaga?tcagacaaac
121 gatcgcagcg?atgacaaacc?tgcaagatca?aacccaacag?attgttccgt?tcatacggag
181 ccttctgatg?ccaacaaccg?gaccggcgtc?cattccggac?gacaccctgg?agaagcacac
241 tctcaggtca?gagacctcga?cctacaattt?gactgtgggg?gacacagggt?cagggctaat
301 tgtctttttc?cctggattcc?ctggctcaat?tgtgggtgct?cactacacac?tgcagagcaa
361 tgggaactac?aagttcgatc?agatgctcct?gactgcccag?aacctaccgg?ccagttacaa
421 ctactgcagg?ctagtgagtc?ggagtctcac?agtgaggtca?agcacacttc?ctggtggcgt
481 ttatgcacta?aacggcacca?taaacgccgt?gaccttccaa?ggaagcctga?gtgaactgac
541 agatgttagc?tacaatgggt?tgatgtctgc?aacagccaac?atcaacgaca?aaattgggaa
601 cgtcctagta?ggggaagggg?tcaccgtcct?cagcttaccc?acatcatatg?atcttgggta
661 tgtgaggctt?ggtgacccca?ttcccgcaat?agggcttgac?ccaaaaatgg?tagccacatg
721 tgacagcagt?aacaggccca?gagtctacac?cataactgca?gccgatgatt?accaattctc
781 atcacagtac?caaccaggtg?gggtaacaat?cacactgttc?tcagccaaca?ttgatgccat
841 cacaagcctc?agcgttgggg?gagagctcgt?gtttcaaaca?agcgtccatg?gccttgtact
901 gggcgccacc?atctacctca?taggcttcga?tgggacagcg?gtaatcacca?gggctgtggc
961 cgcaaacaat?gggctgacga?ccggcaccga?caaccttttg?ccattcaatc?ttgtgattcc
1021?aacaaacgag?ataacccagc?caatcacatc?catcaaactg?gagatagtga?cctccaaaag
1081?tggtggtcag?gcaggggatc?agatgtcatg?gtccgcaaga?gggagcctag?cagtgacgat
1141?ccatggtggc?aactatccag?gggccctccg?tcccgtcacg?ctagtggcct?acgaaagagt
1201?ggcaacagga?tccgtcgtta?cggtcgctgg?ggtgagcaac?ttcgagctga?tcccaaatcc
1261?tgaactagca?aagaacctgg?ttacagaata?cggccgattt?gacccaggag?ccatgaacta
1321?cacaaaattg?atactgagtg?agaggggccg?tcttggcatc?aagaccgtct?ggccaacaag
1381?ggagtacact?gactttcgtg?aatacttcat?ggaggtggcc?gacctcaact?ctcccctgaa
1441?gattgcagga?gcattcggct?tcaaagacat?aatccgggcc?ataaggagga?tagctgtgcc
1501?ggtggtctcc?acattgttcc?cacctgccgc?tcccctagcc?catgcaattg?gggaaggtgt
1561?agactacctg?ctgggcgatg?aggcacaggc?tgcttcagga?actgctcgag?ccgcgtcagg
1621?aaaagcaaga?gctgcctcag?gccgcataag?gcagctgact?ctcgccgccg?acaaggggta
1681?cgaggtagtc?gcgaatctat?tccaggtgcc?ccagaatccc?gtagtcgacg?ggattcttgc
1741?ttcacctggg?gtactccgcg?gtgcacacaa?cctcgactgc?gtgttaagag?agggtgccac
1801?gctattccct?gtggttatta?cgacagtgga?agacgccatg?acacccaaag?cattgaacag
1861?caaaatgttt?gctgtcattg?aaggcgtgcg?agaagacctc?caacctcctt?ctcaaagagg
1921?atccttcata?cgaactctct?ctggacacag?agtctatgga?tatgctccag?gtggggtact
1981?tccactggag?actgggagag?actacaccgt?tgtcccaata?gatgatgtct?gggacgacag
2041?cattatgctg?tccaaagatc?ccatacctcc?tattgtggga?aacagtggaa?atctagccat
2101?agcttacatg?gatgtgtttc?gacccaaagt?cccaatccat?gtggctatga?cgggagccct
2161?caatgcttgt?ggcgagattg?agaaagtaag?ctttagaagc?accaagctcg?ccactgcaca
2221?ccgacttggc?cttaagttgg?ctggtcccgg?agcattcgat?gtaaacaccg?ggcccaactg
2281?ggcaacgttc?atcaaacgtt?tccctcacaa?tccacgcgac?tgggacaggc?ccccctacct
2341?caaccaacca?taccttccac?ccaatgcagg?acgccagtac?caccttgcca?tggctgcatc
2401?agagttcaaa?gagacccccg?aactcgagag?tgccgtcaga?gcaatggaag?cagcagccaa
2461?cgtggaccca?ctattccaac?ctgcactcag?tgtgttcatg?tggctggaag?agaatgggat
2521?tgtgactgac?atggccaact?tcgcactcag?cgacccgaac?gcccatcggg?tgcgaaattt
2581?tcttgcaaac?gcaccacaag?caggcagcaa?gtcgcaaagg?gccaagtacg?ggacagcagg
2641?ctacggagtg?gaggctcggg?gccccacacc?agaggaagca?cagagggaaa?aagacacacg
2701?gatctcaaag?aagatggaga?ccatgggcat?ctactttgca?acaccagaat?gggtagcact
2761?caatgggcac?agagggccaa?gccccggcca?gctaaagtac?tggcagaaca?cacgagaaat
2821?accggaccca?aacgaggact?atctagacta?cgtgcatgca?gagaagagcc?ggttggcatc
2881?agaagaacaa?atccaaaggg?cagctacgtc?gatctacggg?gctccaggac?aggcagagcc
2941?accccaagct?ttcatagacg?aagttgccaa?agtctatgaa?atcaaccatg?gacgtggccc
3001?aaaccaagaa?cagatgaaag?atctgctctt?gactgcgatg?gagatgaagc?atcgcaatcc
3061?caggcgggct?ctaccaaagc?ccaagccaaa?acccaatgct?ccaacacaga?gaccccctgg
3121?tcggctgggc?cgctggatca?ggaccgtctc?tgatgaggac?cttgagtgag?gctcctggga
3181?gtctcccgac?accacccgcg?caggtgtgga?caccaattcg?gccttacaac?atccaaattg
3241?gatccgttcg?cgggtcccc
The B sections has 2827bp, and sequence is as follows:
1 ggatacgatg?ggtctgaccc?tctgggagtc?acgaattaac?atggctacta?ggggcgatgc
61 ccgccgctaa?ttgccatgtt?agtggctcct?cttcttgatg?attctgccac?catgagtgac
121 attttcaaca?gtccacaggc?gcgaagcaag?atctcagcag?cgttcggcat?aaagcctact
181 gctggacaag?acgtggaaga?actcttgatc?cctaaagtct?gggtgccacc?tgaggatccg
241 cttgccagcc?ctagtcgact?ggcaaagttc?ctcagagaga?acggctacaa?agttttgcag
301 ccacggtctc?tgcccgagaa?tgaggagtat?gagaccgacc?aaatactccc?agacttagca
361 tggatgcgac?agatagaagg?ggctgtttta?aaacctactc?tatctctccc?cattggaggc
421 caggagtact?tcccaaagta?ctacccaaca?catcgcccta?gcaaggagaa?gaccaatgcg
481 tacccgccag?acatcgcact?actcaagcag?atgatttacc?tgtttctcca?ggttccagag
541 gccaacgagg?gcctaaagga?tgaagtaacc?ctcctgaccc?aaaatataag?ggataaggcc
601 tatggaagtg?ggacctacat?ggggcaagca?actcgacttg?tagccatgaa?ggaggttgcc
661 actgggagaa?acccgaacaa?ggatcctcta?aaacttgggt?acacttttga?gagcatcgcg
721 cagctgcttg?acatcacact?accggtaggc?ccacccggtg?aggatgacaa?gccctgggtg
781 ccactcacaa?gagtgccgtc?gcggatgttg?gtgctgacgg?gagacgtaga?tggcgacttt
841 gaggttgaag?attaccttcc?caaaatcaac?ctcaagtcat?caagtggact?accatatgta
901 ggtcgcacca?aaggagagac?aattggcgag?atgatagcta?tctcaaacca?gtttctcaga
961 gagctatcaa?cactgttgaa?gcaaggtgca?gggacaaagg?ggtcaaacaa?gaagaagcta
1021?ctcagcatgt?taagtgacta?ttggtactta?tcatgcgggc?ttttgtttcc?aaaggctgaa
1081?aggtacgaca?aaagcacatg?gctcaccaag?acccggaaca?tatggtcagc?tccatcccca
1141?acacacctca?tgatctccat?gatcacctgg?cccgtgatgt?ccaacagccc?aaacaacgtg
1201?ttgaacattg?aagggtgtcc?atcactctac?aaattcaacc?cgttcagagg?agggttgaac
1261?aggatcgtcg?agtggatatt?ggctccggaa?gaacccaagg?ctcttgtata?tgcggacaac
1321?atatacattg?tccactcaaa?cacgtggtac?tcaattgacc?tagagaaggg?cgaggcaaac
1381?tgcacacgcc?aacacatgca?agccgcaatg?tactacatcc?tcaccagagg?gtggtcagac
1441?aacggcgacc?caatgttcaa?tcaaacatgg?gccacctttg?caatgaacat?tgcccctgct
1501?ctagtggtag?actcatcgtg?tctgataatg?aacctgcaaa?ttaagaccta?tggtcaaggc
1561?agcgggaatg?cagccacgtt?catcaataac?cacctcttga?gcacgctagt?gcttgaccag
1621?tggaacctga?tgagacagcc?cagaccagac?agcgaggagt?tcaaatcaat?tgaggacaag
1681?ctaggcatca?acttcaagat?tgagaggtcc?attgatgaca?tcaggggcaa?gctgagacag
1741?cttgtccccc?ttgcacaacc?agggtacctg?agtggggggg?ttgaaccaga?acaatccagc
1801?ccaactgttg?agcttgacct?actagggtgg?tcagctacat?acagcaaaga?tctcgggatc
1861?tatgtgccgg?tgcttgacaa?ggaacgccta?ttttgttctg?cggcgtatcc?caagggagta
1921?gagaacaaga?gtctcaagtc?taaagtcggg?atcgagcagg?catacaaggt?agtcaggtat
1981?gaggcgttga?ggttggtagg?tggttggaac?tacccactcc?tgaacaaagc?ctgcaagaat
2041?aacgcaggcg?ctgctcggcg?gcatctggag?gccaaggggt?tcccgctcga?cgagttccta
2101?gccgagtggt?ccgggctgtc?agagttcggt?gaggccttcg?aaggcttcaa?tatcaagctg
2161?accgtaacat?ctgagagcct?agccgaactg?aacaagccag?taccccccaa?gcccccaaat
2221?gtcaacagac?cagtcaacac?tgggggtctc?aaggcagtca?gcaacgccct?taagaccggt
2281?cggtacagga?acgaagccgg?actgagtggt?ctcgtccttc?tagccacagc?aaggagccgt
2341?ctgcaagacg?cagttaaggc?caaggcagaa?gccgagaaac?tccacaagtc?caagccagac
2401?gaccccgatg?cagactggtt?tgaaagatca?gaaactctgt?cagaccttct?ggagaaagcc
2461?gacatcgcca?gtaaggtcgc?ccactcagca?ctcgtggaaa?caagcgacgc?tcttgaagca
2521?gttcggtcga?cttccgtgta?cacccccaag?tacccagaag?tcaagaaccc?acagaccgcc
2581?tccaaccccg?ttgttgggct?ccacctgccc?gccaagagag?ccaccggtgt?ccaggccgct
2641?cttctcggag?caggaacgag?ccgaccaatg?gggatggagg?ccccaacacg?gtccaagaac
2701?gccgtgaaaa?tggccaaacg?gcggcaacgc?caaaaagaga?gccgccaata?gccatgatgg
2761?gaaccactca?agaagaggac?actaatccca?gaccccgtat?ccccggcctt?cgcctgcggg
2821?ggccccc
Embodiment 2:
Present embodiment has been described the preparation method of genome A sections full length sequence of infectious bursal disease virus provided by the invention (IBDV) VP5 genetically deficient and A, B sections Construction of Infectious Molecular Clone.
(1) contain the structure of eukaryon expression plasmid of the A sections of VP5 genetically deficient, whole building process as shown in Figure 4.
Cut pGEM-T-A/HZ2 with the NdeI enzyme, reclaim long segment (2645bp), and, obtain to contain the reorganization T carrier pGEM-T-AN of IBDV A sections 5 ' end 647bp again from connecting.With upstream and downstream mutant primer (The2S:AGATCAGACAAACGATCGCAG, 129-149nt; TheA:GcTaGcAATGATAGCGTTATAGAAG, 77-101nt, NheI), TaKaRa MutanBEST Kit carries out the rite-directed mutagenesis of PCR mediation, obtain linearized vector and direct self connection of 3603bp, acquisition contains the mutant pGEM-T-The2 of point mutation and fragment deletion, and order-checking confirms (to see Fig. 5, for the sequence GCTAGC after the sudden change, be the NheI restriction enzyme site in the square frame; In the arrow indication bracket is wild virus gene order GATGGTCAGTAG.)。With the EcoRI/NdeI endonuclease bamhi (623bp) of pGEM-T-The2 and the NdeI/KpnI fragment (2615bp) that comes from pGEM-T-A/HZ2 are linear be connected after, directly insert the pCI carrier of cutting through the EcoRI/KpnI enzyme, obtain finally to be used for the eukaryon expression plasmid pCI-ANhe2 of transfection, and enzyme is cut evaluation (Fig. 6.Swimming lane M, DNA maker, 1, the XhoI enzyme cuts, 2, the NdeI enzyme cuts; Result and expection in full accord.)。
(2) contain the structure of B sections eukaryon expression plasmid, whole building process as shown in Figure 7.
With recombinant plasmid pGEM-T-B/HZ2 is skeleton, introduces new restriction enzyme site EcoRV (GATATC) as molecule marker on B sections cDNA by the rite-directed mutagenesis of PCR mediation.Mutant primer EV5:CGCC
CTTAAGACCGGTC
GGAACGAAG (2265～2296nt), wherein: "
CTTAAG" be Afl II site unique on the B sections; The sequence of small letter black matrix mark (a, t, c) is a metathetical point mutation Nucleotide.These sudden changes do not change the amino acid coding of VP1 gene on the B sections.With recombinant plasmid pGEM-T-B/HZ2 is template, and EV5 and B3 are primer, uses the Pfu polymeric enzymatic amplification to go out the fragment of 573bp.Simultaneously 573bp fragment and pGEM-T-B/HZ2 are carried out Afl II/Xba I double digestion, replace the corresponding part of pGEM-T-B/HZ2 with the 573bp fragment, obtain containing the B sections recon pGEM-T-mB/HZ2 of EcoRV mark, and order-checking confirms that (Fig. 8, pGEM-T-B/HZ2 are depicted as the gene order of wild virus B sections corresponding position; PT-mB is depicted as the corresponding gene order of sudden change back B sections, inserts the EcoRV restriction enzyme site).Cut pGEM-T-mB/HZ2 with EcoRI/XbaI/Pvu I enzyme, reclaim long segment (2833bp), be connected, obtain pCI-mB/HZ2, and enzyme is cut evaluation (Fig. 9 with the pCI carrier of cutting through the EcoRI/XbaI enzyme.M, DNA maker, 1﹠amp; 2, the EcoRV/XhoI enzyme is cut; Result and expection in full accord.)。
Embodiment 3:
Present embodiment has been described the preparation method of infectious bursal disease virus provided by the invention (IBDV) VP5 genetically deficient strain ANhe2 strain.
Press the test kit specification sheets, with Concert
TMHigh purity plasmid purification system kit extracting pCI-ANhe2 and two kinds of ultrapure plasmids of transfection level of pCI-mB.Under liposome Lipofectamine 2000 mediations,, establish the only control group of transfection liposome simultaneously with the chick embryo fibroblast of pCI-ANhe2/pCI-mB cotransfection 80% degrees of fusion.The chick embryo fibroblast that is used for transfection and correlation test is cultivated on 6 orifice plates.Behind the transfection 72h, with each the group cell conditioned medium respectively simulated virus infect new fresh cell, " go down to posterity " successively 5 times.
Chick embryo fibroblast 24h after transfection begins to occur pathology, and aggravation gradually.Continue to be cultured to 72h after the transfection, the new fresh cell of harvested cell culture supernatants direct infection constantly carries out later on cell culture supernatant " going down to posterity " by this way similarly.Two plasmids are combined in and have all occurred cytopathy (CPE, Figure 10 A) at every turn when " going down to posterity ", and this CPE is very similar to the normal IBDV infection CPE that culturing cell produced, the main cell circle that shows as contracts, reunite, break, refractivity strengthens, and particulate material increases; And go down to posterity since the 2nd time, similar CPE (Figure 10 B) does not appear in transfection liposome control group.More than IBDV is saved out in prompting, and the IBDV that is saved constantly duplicates on chick embryo fibroblast and goes down to posterity.
Adopt indirect immunofluorescence to detect the viral protein expression situation, each test group is one anti-with anti-IBDV serum of rabbit and the anti-IBDV VP5 of rabbit serum respectively, and the goat-anti chicken IgG of FITC mark is two anti-, detects the expression of virus structural protein and Nonstructural Protein.Chick embryo fibroblast behind the simulated infection 36h, when being anti-a detection with the anti-IBDV antiserum(antisera) of rabbit, can " going down to posterity " cell in observe specific yellow-green fluorescence (Figure 11 B); And when detecting, then can not observe specificity fluorescent (Figure 11 D) with the anti-IBDV VP5 of rabbit antiserum(antisera).No matter be that anti-IBDV serum of rabbit or the anti-IBDV VP5 of rabbit serum all can detect specific yellow-green fluorescence (Figure 11 A, C) from the viral HZ2 strain of female parent cells infected.With any serum is an anti-specificity fluorescent (Figure 11 E, F) of all can not observing from control group.The The above results prompting, VP5 albumen is not expressed after the new transfection combination pCI-ANhe2/pCI-mB transfection that makes up.Behind the collecting cell nutrient solution ultracentrifugation, abandoning supernatant liquor suspends with the TEN solution weight, the phospho-wolframic acid negative staining can observe under Electronic Speculum that diameter is arranged in the culture supernatants is that the virus particle of 55～60nm, no cyst membrane exists, and this is consistent with the distinctive morphological specificity of birnavirus coe virus.
Although cotransfection with repeatedly do not contact other IBDV in the passage process, but be to determine that the genome of this rIBDV is really from pCI-ANhe2 and pCI-mB, for this reason with the A5/A3 primer to amplifying the A sections cDNA of rIBDV again, cut the molecule marker that PCR product checking rite-directed mutagenesis is introduced with the NheI enzyme, the band that can detect the expection molecular weight exists, and order-checking confirms.
Being derived from pCI-ANhe2/pCI-mB, and the rIBDV called after ANhe2 strain of in chick embryo fibroblast, being rescued.
Embodiment 4:
Present embodiment has been described the production method of infectious bursal disease virus provided by the invention (IBDV) VP5 genetically deficient strain ANhe2 strain.
IBDV ANhe2 strain cell toxicant is inoculated 11 embryo SPF in age chicken embryos with the amount of 100 μ l/ embryos, collects allantoic fluid after 4 days, and the centrifugal 10min of 8000rpm collects supernatant.Supernatant is inoculated the chick embryo fibroblast that grows up to individual layer with 10 times of gradient dilutions, begins observation of cell pathology situation from inoculating back 24h, up to 120h; Calculate cell cultures by the Reed-Muench method and organize median infective dose (TCID
50).After the result showed that the ANhe2 strain is by SPF chicken embryo propagation, virus titer can reach 10
5.5TCID
50
All allantoic fluid supernatants if be directly used in inoculation, then are diluted to 5 * 10 with stroke-physiological saline solution by after the bacterial filter filtration sterilization in 0.22 μ m aperture
4TCID
50/ ml concentration.The immunizing dose of every plumage chicken is by 5 * 10
3TCID
50Virus quantity calculate.Last dosage of inoculation is 100 μ l/ plumages.
If the virus-virus (allantoic fluid supernatant) after the degerming is used for preserving, can directly allantoic fluid be sealed in-20 ℃ of freezing preservations in the vaccine bottle; As the need prolonged preservation, can carry out lyophilize to allantoic fluid after, be sealed in-20 ℃ or-70 ℃ of freezing preservations in the vaccine bottle.
Embodiment 5:
Present embodiment has been described infectious bursal disease virus provided by the invention (IBDV) VP5 genetically deficient strain ANhe2 strain to the security of chick and pathogenic.
(1) security of genetically deficient virus and pathogenic
Test is divided into 6 groups, i.e. four dosage groups (5 * 10 of ANhe2 strain virus
2, 5 * 10
3, 5 * 10
4With 10
5TCID
50/ plumage), dosage group (5 * 10 of HZ2 strain virus
3TCID
50/ plumage), the normal control group, every group of 15 chickens, each test group isolated rearing, personal management.Two kinds of viruses are all by the inoculation of drinking-water approach, and 10 age in days head exempt from, 28 age in days booster immunizations.Should cut off the water 2 hours before the inoculation, so that the chicken group produces thirsty sense; In inoculating preceding 72 hours and inoculating back 24 hours, in tap water, must not add any medicine and sterilizing agent.Every day is observed the clinical response of chicken in immunity back, and slaughters 5 respectively in 10 days behind 3 days, booster immunization after head exempts from back 3 days, booster immunization, after whole body, internal organs are checked, cuts open and gets the fabricius bursa, uses 10% formaldehyde fixed, carries out histopathological examination.
The results are shown in Table 2 (
*Expression has the chicken quantity of fabricius bursa damage): after four dosage groups of ANhe2 strain virus and the immunity of maternal viral HZ2 strain virus, observed for 3 weeks continuously, any untoward reaction does not all appear in chick clinically.Slaughtered in 3 days after the first immunisation, on inspection, the fabricius bursa, spleen etc. all do not have the variation of observing, and histopathology is observed and shown: all fabricius bursa of four dosage immune group of ANhe2 strain virus are all normal, and the HZ2 immune group has the fabricius bursa (3/5) of 3 chickens slight damage to occur.Slaughtered in 3 days behind the booster immunization, the result shows that the fabricius bursa of all immune group does not all have the variation of observing, and histopathology is observed and shown: four dosage immune group of ANhe2 strain virus are all normal, and the HZ2 immune group has 3 chickens (3/5) slight damage to occur.Slaughtered in 10 days behind the booster immunization, all test group chicken bursas do not have pathology to be changed.The The above results explanation, the ANhe2 strain virus has still lost pathogenic to chick, be safe to chick, and the weak malicious seedling of HZ2 also has certain virulence to chick.
Table 2 infectious bursal disease virus ANhe2 strain is to the pathogenic of chick and safety testing result
Claims (2)
1. a method for constructing non-virulent strain of infectious bursal disease virus is characterized in that, the step of method is:
1) extracting of infectious bursal disease virus genome A, B sections RNA and the clone of cDNA total length thereof;
2) acquisition of the genome A sections full length sequence of infectious bursal disease virus VP5 genetically deficient and A, B sections Construction of Infectious Molecular Clone;
3) above-mentioned infections clone cotransfection culturing cell is saved out the infectious bursal disease virus ANhe2 strain of VP5 genetically deficient;
The extracting of described infectious bursal disease virus genome A, B sections RNA and the clone of cDNA total length thereof: the HZ2 strain infectious bursal disease virus that collecting cell is cultivated, its genome of extracting A, B sections RNA, with the accurate RT-PCR amplification gene group A of long distance, B sections cDNA total length, clone easy in pGEM-T, obtain recombinant vectors pGEM-T-A/HZ2 and pGEM-T-B/HZ2, and carry out sequencing;
The acquisition of the genome A sections full length sequence of described VP5 genetically deficient and A, B sections Construction of Infectious Molecular Clone: reclaim the long segment of pGEM-T-A/HZ2 after the NdeI enzyme is cut and again from successively win pGEM-T-AN; With upstream and downstream mutant primer The2S:AGATCAGACAAACGATCGCAG and TheA:GcTaGcAATGATAGCGTTATAGAAG, TaKaRa MutanBEST Kit carries out the rite-directed mutagenesis of PCR mediation, the linearized vector that obtains 3603bp is after also direct self connection, acquisition contains the mutant pGEM-T-The2 of point mutation and fragment deletion, and order-checking confirms, after the EcoRI/NdeI endonuclease bamhi of pGEM-T-The2623bp is connected with the NdeI/KpnI fragment of the 2615bp that comes from pGEM-T-A/HZ2 is linear, directly insert the pCI carrier of cutting through the EcoRI/KpnI enzyme, obtain finally to be used for the eukaryon expression plasmid pCI-ANhe2 of transfection, its preserving number is CGMCC No.1507, with recombinant plasmid pGEM-T-B/HZ2 is skeleton, with EV5:CGCCCTTAAGACCGGTCGaTAtcGGAACGAAG and B3:ATTTCTAGAGGGGGCCCCCGCAGGCGAA is mutant primer, use the Pfu polymeric enzymatic amplification to go out the sudden change fragment of 573bp, simultaneously 573bp fragment and pGEM-T-B/HZ2 are carried out the Af1II/XbaI double digestion, replace the corresponding part of pGEM-T-B/HZ2 with the 573bp fragment, obtain containing the B sections recon pGEM-T-mB/HZ2 of EcoRV mark, after order-checking confirms, cut pGEM-T-mB/HZ2 with the EcoRI/XbaI/PvuI enzyme, reclaim the 2833bp fragment, be connected with the pCI carrier of cutting through the EcoRI/XbaI enzyme, acquisition is used for the eukaryon expression plasmid pCI-mB/HZ2 of transfection, and its preserving number is CGMCC No.1506.
2. a kind of method for constructing non-virulent strain of infectious bursal disease virus according to claim 1, it is characterized in that, described with above-mentioned infections clone cotransfection culturing cell, save out the infectious bursal disease virus ANhe2 strain of VP5 genetically deficient: under liposome Lipofectamine 2000 mediations, chick embryo fibroblast with pCI-ANhe2/pCI-mB cotransfection 80% degrees of fusion, pass through cytopathy, simulated virus goes down to posterity, be an anti-indirect immunofluorescence assay with anti-IBDV serum of rabbit and the anti-IBDV VP5 of rabbit serum respectively, the electron microscopic observation virus particle, RT-PCR and enzyme are cut identification experiment and are confirmed to save out the infectious bursal disease virus of VP5 genetically deficient, are the ANhe2 strain with this viral nomenclature.
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| CN (1) | CN100384990C (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0887412A1 (en) * | 1997-05-26 | 1998-12-30 | Akzo Nobel N.V. | Recombinant birnavirus vaccine |
| CN1366041A (en) * | 2000-07-07 | 2002-08-28 | 阿克佐诺贝尔公司 | Broad spectrum infectious chicken blader disease virus vaccine |
| CN1373807A (en) * | 1999-07-14 | 2002-10-09 | Id-莱利斯塔德动物育种及动物保健研究所公司 | Mosaic infections bursal disease virus vaccines |
| WO2004085634A2 (en) * | 2003-03-24 | 2004-10-07 | Akzo Nobel N.V. | Infectious bursal disease virus mutants and vaccines |
-
2005
- 2005-11-14 CN CNB2005100615505A patent/CN100384990C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0887412A1 (en) * | 1997-05-26 | 1998-12-30 | Akzo Nobel N.V. | Recombinant birnavirus vaccine |
| CN1373807A (en) * | 1999-07-14 | 2002-10-09 | Id-莱利斯塔德动物育种及动物保健研究所公司 | Mosaic infections bursal disease virus vaccines |
| CN1366041A (en) * | 2000-07-07 | 2002-08-28 | 阿克佐诺贝尔公司 | Broad spectrum infectious chicken blader disease virus vaccine |
| WO2004085634A2 (en) * | 2003-03-24 | 2004-10-07 | Akzo Nobel N.V. | Infectious bursal disease virus mutants and vaccines |
Non-Patent Citations (2)
| Title |
|---|
| Generation of a Mutant Infectious Bursal Disease VirusThatDoes Not Cause Bursal Lesions. KUN YAO,et al.JOURNAL OF VIROLOGY,Vol.72 No.4. 1998 |
| Generation of a Mutant Infectious Bursal Disease VirusThatDoes Not Cause Bursal Lesions. KUN YAO,et al.JOURNAL OF VIROLOGY,Vol.72 No.4. 1998 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1804029A (en) | 2006-07-19 |
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