CN1804029A - Method for constructing non-virulent strain of infectious bursal disease virus - Google Patents

Abstract

The invention discloses a method for constructing an avirulent strain of infectious bursal disease virus. The steps of the method are as follows: 1) extraction of the RNA of the A and B segments of the infectious bursal disease virus genome and the cloning of the full-length cDNA thereof; 2) obtaining the full-length sequence of the genome A segment of the VP5 gene deletion and Construction of infectious clones in section B; 3) co-transfection of cultured cells with the above-mentioned infectious clones to rescue the infectious bursal disease virus ANhe2 strain with VP5 gene deletion; this method is more stable than the traditional method of cell passage attenuation , and effectively retained the immunogenicity of the virus. The invention provides a VP5 gene-deleted infectious bursal disease virus (IBDV) ANhe2 strain, the virus does not express VP5 protein, loses pathogenicity to chickens, and retains good immunogenicity, and Virulence does not return to the ancestors. The virus is simple to prepare and convenient to use. Other characteristics of the ANhe2 strain were consistent with traditional attenuated vaccine strains.

Description

Method for constructing non-virulent strain of infectious bursal disease virus

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 Ndel 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 EcoRI/Nde I endonuclease bamhi of pGEM-T-The2623bp is connected with the Nde I/Kpn I fragment of the 2615bp that comes from pGEM-T-A/HZ2 is linear, directly insert the pCI carrier of cutting through EcoR I/Kpn I 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 Af1 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 CGMCCN01506 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

Primer	Sequence #	Binding site *	Purposes
				A5	GGATACGATCGGTCTGACCCCGG	A：1～23	The amplification of genome A sections
A3	AT GGTACCGGGGACCCGCGAACGGAT	A：3243～3260
			B5	AT GAATTCGGATACGATGGGTCTGACCCT	B：1～21	The amplification of genome B sections
B3	ATT TCTAGAGGGGGCCCCCGCAGGCGAA	B：2809～2827

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), 2ul 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 GaTAtcGGAACGAAG (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, DNAmaker, 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

Group	Dosage (TCID 50)	Fabricius bursa pathology after the observation immunity changes			Untoward reaction
						Head exempted from back 3 days	Two exempted from back 3 days	Two exempted from back 10 days
		ANhe2	5×10 2	0 */5					0/5	0/5	Do not have
5×10 3	0/5				0/5	0/5	Do not have
			5×10 4	0/5				0/5	0/5	Do not have
5×10 5	0/5				0/5	0/5	Do not have
			HZ2	5×10 3				3/5	3/5	0/5	Do not have
Normally	/	0/5			0/5	0/5	Do not have

Claims (4)

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;

2. a kind of method for constructing non-virulent strain of infectious bursal disease virus according to claim 1, it is characterized in that, 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 in pGEM-Teasy, obtain recombinant vectors pGEM-T-A/HZ2 and pGEM-T-B/HZ2, and carry out sequencing.

3. a kind of method for constructing non-virulent strain of infectious bursal disease virus according to claim 1, it is characterized in that 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 NbeI 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 EcoRI/NdeI endonuclease bamhi of pGEM-T-The2 623bp 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:ATTTCTMGAGGGGGCCCCCGCAGGCGAA 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 AflII/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.

4. 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 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.

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