CN100384990C - 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 bursal disease virus genome and the cloning of the full-length cDNA thereof; Construction of segmental infectious clones; 3) The above-mentioned infectious clones were co-transfected into cultured cells, and the infectious bursal disease virus ANhe2 strain with VP5 gene deletion was rescued; this method is more stable than traditional cell passage attenuation methods, And effectively retain 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 an avirulent strain of infectious bursal disease virus

technical field

The invention belongs to the field of biotechnology and relates to a method for constructing an avirulent strain of infectious bursal disease virus.

Background technique

Infectious bursal disease (IBD) is an acute, highly contagious infectious disease caused by infectious bursal disease virus (IBDV). IBDV mainly invades the central immune organ of chicks, the bursa of Fabricius, leading to immunosuppression and immunodeficiency, and at the same time causing immune failure of other vaccines. It is currently one of the main infectious diseases that endanger the poultry industry in the world. IBDV is a double-stranded RNA virus belonging to the genus Avibirnavirus of the family Biranviridae, and its genome consists of two segments, A and B. The B segment (about 2.8kb) encodes the VP1 protein, which is an RNA-dependent RNA polymerase. The A segment (about 3.3kb) has two overlapping open reading frames (ORFs), and the downstream ORF1 encodes the precursor protein VP2/4/3, which can be further cut and processed into VP2, VP4, and VP3. Among them, VP2 is the main host protective antigen of the virus, which is related to the antigenic variation and virulence of the virus; VP4 is a serine protease responsible for the self-cleavage of the precursor protein VP2/4/3; VP3 is one of the structural proteins of the virus One, with group-specific antigens. The upstream ORF2 (438bp) encodes a non-structural protein VP5 (about 15kDa), which is not necessary for viral replication and is related to viral pathogenicity.

The start code ATG of IBDV D78 attenuated strain ORF A2 was replaced with AGG (encoding Arg), and its cRNA was transfected into chicken embryo cells (CEC) to obtain a defective virus IBDV/VP5 without VP5 expression, which could normally infect Vero cells, suggesting that VP5 Replication in IBDV is not necessary. Yao et al. infected 3-week-old SPF chickens with VP5-deficient D78 attenuated strain (rD78NSΔ) and parental virus rD78 respectively. The ability to induce IBDV neutralizing antibodies was similar. Further studies showed that VP5 was placed downstream of the CMV promoter to transfect chicken B lymphocytes (RP9) and chicken embryo fibroblasts, respectively. As a result, the expression of VP5 induced the apoptosis of RP9 and chicken embryo fibroblasts, while the deletion of VP5 The rescued virus causes milder cytopathy than the wild type, the replication speed is slower than the wild type, and the virus amount in the cell supernatant is 30 times lower than that of the wild type. The virus lacking VP5 greatly reduces the ability of the cell to lyse and release the virus. It can be seen that VP5 It has strong cytotoxicity, which is related to the release of mature virus from intracellular to extracellular and the pathogenicity of the virus. Therefore, research on the biological characteristics of VP5 will help us further understand the pathogenic and immune mechanisms of IBDV.

Contents of the invention

The object of the present invention is to provide a method for constructing an avirulent strain of infectious bursal disease virus.

The steps of the method are:

1) Extraction of the RNA of the A and B segments of the bursal disease virus genome and the cloning of its full-length cDNA;

2) Acquisition of the full-length sequence of the A segment of the genome with VP5 gene deletion and the construction of infectious clones of the A and B segments;

3) The above-mentioned infectious clones were co-transfected into cultured cells, and the infectious bursal disease virus ANhe2 strain with VP5 gene deletion was rescued;

The extraction of the RNA of the A and B segments of the infectious bursal disease virus genome and the cloning of its full-length cDNA: collect the HZ2 strain of infectious bursal disease virus cultured in cells, and extract the A and B segments of the genome Amplify the full-length cDNA of genome A and B segments by long-distance precise RT-PCR, clone them in pGEM-T easy, obtain recombinant vectors pGEM-T-A/HZ2 and pGEM-T-B/HZ2, and perform sequence determination.

Obtaining the full-length sequence of segment A of the genome with VP5 gene deletion and the construction of infectious clones of segments A and B: recovering the long fragment of pGEM-T-A/HZ2 digested with NdeI and re-self-ligating to obtain pGEM-T-AN; The above downstream mutation primers The2S: AGATCAGACAAACGATCGCAG and TheA: GcTaGcAATGATAGCGTTATAGAAG, TaKaRa MutanBEST Kit were used for PCR-mediated site-directed mutagenesis to obtain a 3603bp linearized vector and directly self-ligated to obtain the mutant pGEM-T-The2 containing point mutations and fragment deletions , and confirmed by sequencing. After the 623bp EcoR I/Nde I fragment of pGEM-T-The2 was linearly ligated with the 2615bp Nde I/Kpn I fragment from pGEM-T-A/HZ2, it was directly inserted into pC I digested by EcoR I/Kpn I Vector, obtain the final eukaryotic expression plasmid pC I-ANhe2 for transfection, its preservation number is CGMCC No.1507, with the recombinant plasmid pGEM-T-B/HZ2 as the backbone, with EV5: CGCCCTTAAGACCGGTCGaTAtcGGAACGAAG and B3: ATTTCTAGAGGGGGCCCCCGCAGGCGAA as mutation primers, Use Pfu polymerase to amplify a 573bp mutant fragment, perform Afl II/Xba I double enzyme digestion on the 573bp fragment and pGEM-T-B/HZ2 at the same time, replace the corresponding part of pGEM-T-B/HZ2 with the 573bp fragment, and obtain the EcoRV-labeled fragment Segment B recombinant pGEM-T-mB/HZ2, after sequencing confirmation, pGEM-T-mB/HZ2 was digested with EcoRI/XbaI/Pvu I, the 2833bp fragment was recovered, and ligated with the pCI vector digested with EcoRI/XbaI, The eukaryotic expression plasmid pCI-mB/HZ2 used for transfection was obtained, and its preservation number is CGMCC No.1506.

Co-transfect the cultured cells with the above-mentioned infectious clones, and rescue the infectious bursal disease virus ANhe2 strain with VP5 gene deletion: under the mediation of liposome Lipofectamine 2000, co-transfect 80% of the cells with pCI-ANhe2/pCI-mB Confluent chicken embryo fibroblasts were identified through cytopathic, simulated virus passage, indirect immunofluorescence test with rabbit anti-IBDV serum and rabbit anti-IBDV VP5 serum as primary antibodies, electron microscope observation of virus particles, RT-PCR and enzyme digestion and other experiments confirmed that the infectious bursal disease virus with VP5 gene deletion was rescued, and the virus was named ANhe2 strain.

Description of drawings

Fig. 1 is the infectious bursal disease virus (IBDV) genome A, B segment cDNA amplification and cloning process and map provided by the present invention.

Fig. 2 is a RT-PCR identification map of the infectious bursal disease virus (IBDV) genome segment A provided by the present invention.

Fig. 3 is the RT-PCR identification map of the infectious bursal disease virus (IBDV) genome segment B provided by the present invention.

Fig. 4 is the construction process and map of the genome segment A mutant of infectious bursal disease virus (IBDV) and its eukaryotic expression plasmid provided by the present invention.

Fig. 5 is the sequencing situation of the mutant of the genome segment A of infectious bursal disease virus (IBDV) provided by the present invention.

Fig. 6 is an enzyme digestion identification map of the eukaryotic expression plasmid pCI-ANhe2 containing the genome A segment mutant of infectious bursal disease virus (IBDV) provided by the present invention.

Fig. 7 is the construction process and map of the genome B segment mutant of infectious bursal disease virus (IBDV) provided by the present invention.

Fig. 8 is the sequencing situation of the genome B segment mutant of infectious bursal disease virus (IBDV) provided by the present invention.

Fig. 9 is an enzyme digestion identification map of the eukaryotic expression plasmid pCI-mB containing the genome segment B mutant of infectious bursal disease virus (IBDV) provided by the present invention.

Figure 10 is the observation of the cytopathic effect of the infectious bursal disease virus (IBDV) gene deletion strain ANhe2 strain provided by the present invention (100×).

Figure 11 shows the expression of virus structural and non-structural proteins observed by indirect immunofluorescence test of the infectious bursal disease virus (IBDV) gene deletion strain ANhe2 strain cell culture provided by the present invention (100×).

Detailed ways

On October 25, 2005, the Escherichia coli (Escherichia coli) was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation numbers are CGMCCNO1506 and CGMCCNO1507.

Example 1:

This example describes the method for obtaining the full-length sequence of the A and B segments of the infectious bursal disease virus (IBDV) genome provided by the present invention, and the entire experimental process of long-distance RT-PCR amplification and cloning of the IBDV double-segment genome As shown in Figure 1.

(1) RT-PCR amplification of the full-length sequences of the genome segments A and B of the infectious bursal disease virus (IBDV) HZ2 strain

Collect the chicken embryo fibroblast (chicken embryo fibroblast) cell culture medium of infectious bursal disease virus (IBDV) HZ2 strain repeatedly freeze-thawed 3 times; 5000rpm, 4 ℃, 5min centrifugation, take the supernatant; Centrifuge for 5 minutes to take the supernatant; add the supernatant to the dialysis bag with a funnel, place it in 50% polyethylene glycol (PEG), put it at 4°C overnight, and concentrate it 100 times; suck out the concentrated virus solution with a pipette, Store at -20°C for later use.

Take 200μl of the dialyzed virus solution and add it to a new 0.5ml eppendorf tube treated with DEPC; add SDS and proteinase K to the final concentration of 0.5% and 1mg/ml respectively, digest at 50°C for 2hrs; phenol/chloroform/isoamyl alcohol (25/24/1) and chloroform/isoamyl alcohol (24/1) to extract once, centrifuge to get the supernatant, add 3M NaAc to a final concentration of 0.3M, then add 650ml ice-cold anhydrous Water and ethanol, mix upside down, -20°C overnight, precipitate RNA; 12000rpm, 4°C, centrifuge for 10min, rinse the precipitate with pre-cooled 75% ethanol; evaporate ethanol at room temperature, add appropriate amount of DEPC-treated water depending on the amount of precipitation, and gently pipette Mix well; after identification by 1% agarose gel electrophoresis, store at -20°C for later use.

Primers were designed for the full-length sequences of the A and B segments of the genome, as shown in Table 1. A5/A3 was used to amplify the full-length sequence of the A segment, and B5/B3 was used to amplify the full-length sequence of the B segment. All primers were diluted to 25 μM with DEPC-treated water and stored at -20°C for future use.

Table 1 Infectious Bursal Disease Virus Genome A, B Segment cDNA Amplification Primer Sequence

Mix and briefly centrifuge the following components before use: 5μl viral RNA genome suspension, 2μl reverse transcription primer (2.5μM), 2μl 10×PCR buffer, 2μl MgCl ₂ (25mM), 1μl dNTP (10mM), 2μl DTT (0.1M); 98°C, 5min, immediately quenched on ice for 1min; 50°C, 5min; add 1μl (200U) MMLV reverse transcriptase to the tube, mix well, 50°C, 50min; 70°C, 15min to stop the reaction, ice Quench; run PCR reaction immediately; or freeze. The PCR reaction was carried out according to the operation guide of the Expand High Fidelity PCR System of Roche Company, using the first strand synthesized by reverse transcription as a template, and the PCR reaction parameters were pre-denaturation at 94°C for 3min, denaturation at 94°C for 15sec, annealing at 60°C for 30sec, and extension at 68°C for 5min ; Cycle 30 times, extend at 72°C for 10 min. After the reaction was completed, 1-21% agarose gel electrophoresis was used for detection, and the result was about 3.3 kb segment A cDNA (see Figure 2. 1, DNA maker; 2 & 3, A segment cDNA) and 2.8 kb segment B segment cDNA (see Figure 3. M, DNA maker; 1, segment B cDNA).

(2) Cloning of the full-length sequence of the A and B segments of the genome of the IBDV HZ2 strain

The PCR product recovered from rubber tapping was directly connected to the pGEM-T easy vector (Promega). Specifically, follow the instructions. In a 0.5ml centrifuge tube, add the following reagents in sequence: 2×Buffer 5μl, pGEM-T easy 1μl, recovered PCR product 4μl, T4DNA liagase 1μl, connect at room temperature for 1h, transform Escherichia coli HD5α, and coat with Ampicillin LB plate, pick a single colony, expand culture overnight in LB medium, extract plasmid DNA, and carry out PCR and enzyme digestion identification. In order to further confirm the correctness of the sequence, three independent positive clones were selected for each segment to be sequenced. Sequencing was performed on an ABI3730 sequencer using the primer-walking method. The correctly sequenced clones were named pGEM-T-A/HZ2 and pGEM-T-B/HZ2, respectively. Sequencing results showed that the A segment of the HZ2 strain had a total of 3259 bp, and the sequence was 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 actggagag 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 gagaccccccg 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 segment has a total of 2827bp, and the 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 atgattacc 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 ccaccccggtg 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 acaacacctca 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 taccactcc 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

Example 2:

This example describes the method for obtaining the full-length sequence of segment A of the genome of infectious bursal disease virus (IBDV) VP5 gene deletion provided by the present invention and the construction of infectious clones of segments A and B.

(1) Construction of eukaryotic expression plasmid containing segment A of VP5 gene deletion, the whole construction process is shown in FIG. 4 .

Digest pGEM-T-A/HZ2 with NdeI, recover the long fragment (2645bp), and re-self-ligate to obtain the recombinant T vector pGEM-T-AN containing 647bp of the 5' end of the IBDV A segment. The above downstream mutation primers (The2S: AGATCAGACAAACGATCGCAG, 129-149nt; TheA: GcTaGcAATGATAGCGTTATAGAAG, 77-101nt, NheI) and TaKaRa MutanBEST Kit were used for PCR-mediated site-directed mutagenesis to obtain a 3603bp linearized vector and directly self-ligated to obtain a vector containing a point mutation and the mutant pGEM-T-The2 with fragment deletion, and confirmed by sequencing (see Figure 5, the mutated sequence GCTAGC in the box, which is the NheI restriction site; the wild virus gene sequence GATGGTCAGTAG in the square brackets indicated by the arrow. ). The EcoRI/NdeI fragment (623bp) of pGEM-T-The2 was linearly ligated with the NdeI/KpnI fragment (2615bp) from pGEM-T-A/HZ2, and then directly inserted into the pCI vector digested with EcoRI/KpnI to obtain the final The eukaryotic expression plasmid pCI-ANhe2 used for transfection was digested and identified (Figure 6. Lane M, DNA maker, 1, XhoI digestion, 2, NdeI digestion; the results were exactly as expected.).

(2) Construction of a eukaryotic expression plasmid containing segment B, the entire construction process is shown in FIG. 7 .

GGAACGAAG(2265～2296nt)，其中：“CTTAAG”为B节段上唯一的Afl II位点；小写黑体标记的序列(a、t、c)为置换的点突变核苷酸。这些突变没有改变B节段上VP1基因的氨基酸编码。以重组质粒pGEM-T-B/HZ2为模板，EV5与B3为引物，使用Pfu聚合酶扩增出573bp的片段。同时对573bp片段和pGEM-T-B/HZ2进行Afl II/Xba I双酶切，用573bp片段替换pGEM-T-B/HZ2的对应部分，得到含有EcoRV标记的B节段重组子pGEM-T-mB/HZ2，并测序证实(图8，pGEM-T-B/HZ2所示为野生病毒B节段相应位置的基因序列；pT-mB所示为突变后B节段相应的基因序列，插入EcoRV酶切位点)。以EcoRI/XbaI/Pvu I酶切pGEM-T-mB/HZ2，回收长片段(2833bp)，与经过EcoRI/XbaI酶切的pCI载体连接，获得pCI-mB/HZ2，并酶切鉴定(图9。M、DNA maker，1&2、EcoRV/XhoI酶切；结果与预期的完全一致。)。Using the recombinant plasmid pGEM-TB/HZ2 as the backbone, a new restriction site EcoRV (GATATC) was introduced into the B segment cDNA by PCR-mediated site-directed mutagenesis as a molecular marker. Mutation Primer EV5: CGCC CTTAAG ACCGGTC

GGAACGAAG (2265-2296nt), wherein: " CTTAAG " is the only Afl II site on the B segment; the sequence marked in lowercase boldface (a, t, c) is the substituted point mutation nucleotide. These mutations did not alter the amino acid code of the VP1 gene on the B segment. Using the recombinant plasmid pGEM-TB/HZ2 as a template and EV5 and B3 as primers, a 573bp fragment was amplified using Pfu polymerase. Simultaneously perform Afl II/Xba I double enzyme digestion on the 573bp fragment and pGEM-TB/HZ2, replace the corresponding part of pGEM-TB/HZ2 with the 573bp fragment, and obtain the B segment recombinant pGEM-T-mB/HZ2 containing the EcoRV marker , and confirmed by sequencing (Figure 8, pGEM-TB/HZ2 shows the gene sequence of the corresponding position of the wild virus B segment; pT-mB shows the gene sequence corresponding to the B segment after the mutation, inserted into the EcoRV restriction site) . pGEM-T-mB/HZ2 was digested with EcoRI/XbaI/Pvu I, and the long fragment (2833bp) was recovered, ligated with the pCI vector digested with EcoRI/XbaI to obtain pCI-mB/HZ2, and identified by enzyme digestion (Figure 9 .M, DNA maker, 1&2, EcoRV/XhoI digestion; the results are exactly as expected.).

Example 3:

This example describes the method for obtaining the infectious bursal disease virus (IBDV) VP5 gene-deleted strain ANhe2 provided by the present invention.

According to the kit instructions, two transfection-grade ultrapure plasmids, pCI-ANhe2 and pCI-mB, were extracted with Concert ^TM high purity plasmid purification system kit. Under the mediation of liposome Lipofectamine 2000, pCI-ANhe2/pCI-mB co-transfected chicken embryo fibroblasts with 80% confluence, and set up a control group only transfected with liposome. Chicken embryo fibroblasts used for transfection and related experiments were cultured on 6-well plates. After 72 hours of transfection, the supernatants of the cells in each group were respectively simulated to infect the fresh cells with the virus, and were "passaged" 5 times in sequence.

Chicken embryo fibroblasts began to appear lesions 24 hours after transfection, and gradually intensified. The culture was continued until 72 hours after transfection, and the cell culture supernatant was harvested to directly infect fresh cells. In this way, the cell culture supernatant was continuously "passaged" in a similar manner. Cytopathic changes (CPE, Figure 10A) appeared in each "passage" of the two plasmid combinations. This CPE is very similar to the CPE produced by normal IBDV-infected cultured cells. The main manifestations are cell shrinkage, agglomeration, rupture, increased refraction, and increased granular matter; and from the second passage, similar CPE did not appear in the transfected liposome control group (Fig. 10B). The above tips have rescued IBDV, and the rescued IBDV has been continuously replicated and passaged on chicken embryo fibroblasts.

The expression of viral proteins was detected by indirect immunofluorescence method. In each test group, rabbit anti-IBDV serum and rabbit anti-IBDV VP5 serum were used as primary antibodies, and FITC-labeled goat anti-chicken IgG was used as secondary antibody to detect viral structural and non-structural proteins. expression. Chicken embryo fibroblasts after 36 hours of simulated infection were tested with rabbit anti-IBDV antiserum as the primary antibody, and specific yellow-green fluorescence was observed in the "passage" cells (Fig. 11B); while rabbit anti-IBDV VP5 antibody When serum was detected, specific fluorescence could not be observed (Fig. 11D). Both the rabbit anti-IBDV serum and the rabbit anti-IBDV VP5 serum could detect specific yellow-green fluorescence from cells infected with the parental virus HZ2 strain (Fig. 11A, C). No specific fluorescence could be observed from the control group using any serum as the primary antibody (Fig. 11E, F). The above results suggested that VP5 protein was not expressed after transfection with the newly constructed transfection combination pCI-ANhe2/pCI-mB. Collect the cell culture medium after ultracentrifugation, discard the supernatant and resuspend it with TEN solution, and negatively stain with phosphotungstic acid. Under the electron microscope, it can be observed that there are virus particles with a diameter of 55-60nm and no envelope in the culture supernatant. , which is consistent with the unique morphological characteristics of the Binnaviridae virus.

Although there was no contact with other IBDV during co-transfection and multiple cell passages, in order to confirm that the genome of this rIBDV was indeed derived from pCI-ANhe2 and pCI-mB, the A5/A3 primer pair was used to re-amplify the A of rIBDV. Segmental cDNA was digested with NheI to verify the molecular markers introduced by site-directed mutagenesis. Bands with expected molecular weights could be detected and confirmed by sequencing.

The rIBDV derived from pCI-ANhe2/pCI-mB and rescued in chicken embryo fibroblasts was named ANhe2 strain.

Example 4:

This example describes the production method of the infectious bursal disease virus (IBDV) VP5 gene deletion strain ANhe2 provided by the present invention.

IBDV ANhe2 strain cytotoxicity was used to inoculate 11-embryo-age SPF chicken embryos at an amount of 100 μl/embryo. After 4 days, the allantoic fluid was collected, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was inoculated into a monolayer of chicken embryo fibroblasts by 10-fold serial dilution, and the cytopathic condition was observed from 24h after inoculation until 120h; the half-infection dose (TCID ₅₀ ) of the cell culture tissue was calculated according to the Reed-Muench method. The results showed that the virus titer of ANhe2 strain could reach 10 ^5.5 TCID ₅₀ after being propagated through SPF chicken embryos.

All allantoic fluid supernatants were sterilized by filtration through a bacterial filter with a pore size of 0.22 μm, and if used directly for inoculation, they were diluted with sterile normal saline to a concentration of 5×10 ⁴ TCID ₅₀ /ml. The immunization dose of each chicken is calculated according to the virus load of 5×10 ³ TCID ₅₀ . The final inoculation dose was 100 μl/feather.

If the sterilized virus (supernatant of allantoic fluid) is used for preservation, the allantoic fluid can be directly sealed in a vaccine bottle and stored at -20°C; if long-term storage is required, the allantoic fluid can be freeze-dried, Store in a sealed vaccine bottle at -20°C or -70°C.

Example 5:

This example describes the safety and pathogenicity of the infectious bursal disease virus (IBDV) VP5 gene deletion strain ANhe2 strain on chicks provided by the present invention.

(1) Safety and pathogenicity of gene deletion virus

The test was divided into 6 groups, four dose groups of ANhe2 strain virus (5×10 ² , 5×10 ³ , 5×10 ⁴ and 10 ⁵ TCID ₅₀ /feather), one dose group of HZ2 strain virus (5×10 ³ TCID ₅₀ /feather), normal control group, every group of 15 chickens, each test group is kept in isolation and managed by special personnel. The two viruses were inoculated through drinking water, the first immunization at the age of 10 days, and the booster immunization at the age of 28 days. Water should be stopped for 2 hours before inoculation to make the chickens feel thirsty; within 72 hours before inoculation and within 24 hours after inoculation, no drugs and disinfectants should be added to drinking water. Observe the clinical response of the chickens every day after immunization, and kill 5 chickens respectively 3 days after the first immunization, 3 days after the booster immunization, and 10 days after the booster immunization. Formaldehyde fixed, for histopathological examination.

The results are shown in Table 2 ( ^* indicates the number of chickens with bursal damage): after immunization of four dose groups of ANhe2 strain virus and HZ2 strain virus of the mother virus, the chicks were observed continuously for 3 weeks, and no adverse reactions occurred in the chicks clinically. Three days after the first immunization, the bursa and spleen had no visual changes. Histopathological observations showed that all bursa of the ANhe2 virus strain immunized with four doses were normal, and the HZ2 immunized group had 3 The bursa of Fabricius (3/5) of all birds showed slight damage. 3 days after the booster immunization, the results showed that the bursa of Fabricius in all immunized groups had no visual changes, and histopathological observation showed that: the four doses of ANhe2 strain virus immunized groups were all normal, while the HZ2 immunized group had 3 chickens (3 /5) Slight damage occurs. After culling 10 days after the booster immunization, there was no pathological change in the bursa of Fabricius in all test groups. The above results show that the ANhe2 strain virus has lost its pathogenicity to chickens and is safe to chickens, while the attenuated HZ2 vaccine has certain toxicity to chickens.

Table 2 Pathogenicity and safety test results of infectious bursal disease virus ANhe2 strain to chicks

Claims (2)

1. A method for constructing an infectious bursal disease virus avirulent strain, characterized in that the steps of the method are: 1) Extraction of the RNA of the A and B segments of the genome of infectious bursal disease virus and the cloning of its full-length cDNA; 2) Acquisition of the full-length sequence of the genome A segment of the infectious bursal disease virus VP5 gene deletion and the construction of infectious clones of the A and B segments; 3) The above-mentioned infectious clones were co-transfected into cultured cells, and the infectious bursal disease virus ANhe2 strain with VP5 gene deletion was rescued; The extraction of the RNA of the A and B segments of the infectious bursal disease virus genome and the cloning of its full-length cDNA: collect the HZ2 strain of infectious bursal disease virus cultured in cells, and extract the A and B segments of the genome Segment RNA, using long-distance accurate RT-PCR to amplify the full-length cDNA of genome A and B segments, clone it in pGEM-T easy, obtain recombinant vectors pGEM-T-A/HZ2 and pGEM-T-B/HZ2, and perform sequence determination; Obtaining the full-length sequence of segment A of the genome with deletion of the VP5 gene and construction of infectious clones of segments A and B: recovering the long fragment of pGEM-T-A/HZ2 digested with NdeI and re-self-ligating to obtain pGEM-T -AN; The above downstream mutation primers The2S: AGATCAGACAAACGATCGCAG and TheA: GcTaGcAATGATAGCGTTATAGAAG, TaKaRa MutanBEST Kit were used for PCR-mediated site-directed mutagenesis to obtain a 3603bp linearized vector and direct self-ligation to obtain a mutant pGEM containing point mutations and fragment deletions- T-The2, and confirmed by sequencing, the EcoRI/NdeI digested fragment of pGEM-T-The2623bp was linearly ligated with the 2615bp NdeI/KpnI fragment from pGEM-T-A/HZ2, and directly inserted into pCI digested with EcoRI/KpnI Vector, obtain the final eukaryotic expression plasmid pCI-ANhe2 for transfection, its preservation number is CGMCC No.1507, with the recombinant plasmid pGEM-T-B/HZ2 as the backbone, with EV5: CGCCCTTAAGACCGGTCGaATAtcGGAACGAAG and B3: ATTTCTAGAGGGGGCCCCCGCAGGCGAA as mutation primers, using A 573bp mutant fragment was amplified by Pfu polymerase, and Af1II/XbaI double digestion was performed on the 573bp fragment and pGEM-T-B/HZ2 at the same time, and the corresponding part of pGEM-T-B/HZ2 was replaced with the 573bp fragment to obtain the B segment containing the EcoRV marker Recombinant pGEM-T-mB/HZ2, after sequencing confirmation, pGEM-T-mB/HZ2 was digested with EcoRI/XbaI/PvuI, the 2833bp fragment was recovered, ligated with the pCI vector digested with EcoRI/XbaI, and obtained for transfection The eukaryotic expression plasmid pCI-mB/HZ2 transfected, its preservation number is CGMCC No.1506. 2. a kind of infectious bursal disease virus avirulence strain construction method according to claim 1, is characterized in that, described above-mentioned infectious clone co-transfects cultured cell, rescues the VP5 gene deletion Infectious bursal disease virus ANhe2 strain: Under the mediation of liposome Lipofectamine 2000, chicken embryo fibroblasts with 80% confluence were co-transfected with pCI-ANhe2/pCI-mB. Using rabbit anti-IBDV serum and rabbit anti-IBDV VP5 serum as the primary antibody, indirect immunofluorescence test, electron microscope observation of virus particles, RT-PCR and enzyme digestion identification experiments confirmed that the infectious bursal disease virus with VP5 gene deletion was rescued. The virus was named ANhe2 strain.

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