CN110129286B

CN110129286B - Recombinant infectious bursal disease virus strain and preparation method thereof

Info

Publication number: CN110129286B
Application number: CN201910496396.6A
Authority: CN
Inventors: 韦平; 陈果; 何秀苗; 姬忠华; 赵增志; 张誉文; 王威威; 黄宇; 磨美兰; 韦天超; 黄腾
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2019-06-10
Filing date: 2019-06-10
Publication date: 2021-04-13
Anticipated expiration: 2039-06-10
Also published as: CN110129286A

Abstract

The invention discloses a recombinant infectious bursal disease virus strain and a preparation method thereof. The invention provides a recombinant infectious bursal disease virus, which is obtained by replacing RNA capable of being translated to obtain a VP1 protein N-terminal structural domain in chicken infectious bursal disease virus with RNA shown in the 112 th to 612 th sites from the 5' end of a sequence 7 in a sequence table. The recombinant infectious bursal disease virus strain does not contain any exogenous gene, has the same size with the infectious bursal disease virus genome reported in the prior art, and has biological safety. The recombinant infectious bursal disease virus strain provided by the invention can be used as a vaccine candidate strain for vaccine research and development.

Description

Recombinant infectious bursal disease virus strain and preparation method thereof

Technical Field

The invention relates to a recombinant infectious bursal disease virus strain and a preparation method thereof.

Background

Infectious Bursal Disease (IBD) is an acute, highly contagious Infectious Disease that harms young chickens caused by IBDV. IBDV mainly attacks lymphoid tissues of chickens, in particular the bursa of Fabricius, the central immune organ, where the proliferation of the virus causes depletion of B-lymphocytes, thus causing immunosuppression of the chickens. Especially early infections in chicks can cause very severe and long-term immunosuppression. Immunosuppression by IBD can cause necrotic dermatitis, inclusion body hepatitis-anemia syndrome, escherichia coli infection and immune failure in chickens. IBDV has two serotypes, serogroup I and serogroup II. Of these, only serous type I IBDV is pathogenic to chickens. The epidemic strains in the early stage of IBD are mainly classical strains, and because of the extremely strong IBDV variation characteristics, the epidemics of very virulent strains and variant strains of IBDV have appeared all over the world since the eighties of the last century. IBDV has been mutated to change its antigenic and virulence properties, and IBD continues to be prevalent despite the extensive use of traditional live and inactivated vaccines.

IBDV belongs to the genus Avian Birnaviridae (Avian Birnaviridae) of the Birnaviridae family (Birnaviridae), whose genome consists of A, B two-segment double-stranded RNA (dsRNA). Segment A contains two Open Reading Frames (ORFs), wherein ORF1 encodes three viral proteins, VP2, VP3 and VP 4; ORF2 encodes the viral protein VP 5. The B-segment contains only one ORF, encoding the viral protein VP 1. The VP2 protein is the main structural protein and protective antigen of IBDV and is capable of inducing the production of neutralizing antibodies. The VP1 protein exists in two forms, one exists in the form of RdRp during virus replication; another VP1, which binds to the genome in mature virions, connects the A-segment and the B-segment of the genome, exists as structural proteins, and is covalently linked to the viral genome is called VPg. Studies have shown that the VP1 protein can also influence the virulence of IBDV.

Conventional researches on viruses and other living matters are started from ' traits ' -genes ' and used for researching the characteristics of genetic materials and the occurrence and development rules of the living matters. This is classical (forward) genetics. Classical genetics has originated in the pea test of mendelian, the father of modern genetics. The reverse genetic engineering starts with "gene" - "character", and is the direct research on the genetic material of life material. In 1996, Mundt E et al successfully rescued IBDV, which was the first report on the rescue of serotype I IBDV. Since the first time that Mundt E and the like used reverse genetic manipulation technology to research IBDV in 1996, the research on various aspects related to gene function, pathogenic mechanism, immunosuppressive mechanism and the like of IBDV was carried out by constructing IBDV genome full-length cDNA and carrying out mutation, deletion or substitution on IBDV genome. The study of IBDV by reverse genetic manipulation has become a choice and focus of more and more researchers.

Disclosure of Invention

The invention aims to provide a recombinant infectious bursal disease virus strain and a preparation method thereof.

The invention firstly provides a recombinant infectious bursal disease virus, which is obtained by replacing RNA capable of being translated to obtain a VP1 protein N-terminal structural domain in the chicken infectious bursal disease virus with RNA shown in the 112 th to 612 th sites from the 5' end of a sequence 7 in a sequence table.

The chicken infectious bursal disease virus can be specifically an infectious bursal disease virus NN1172 strain.

The RNA sequence of the recombinant infectious bursal disease virus genome segment A is shown as a sequence 6 in a sequence table.

The RNA sequence of the recombinant infectious bursal disease virus genome B segment is shown as a sequence 7 in a sequence table.

The invention also protects a recombinant infectious bursal disease virus, which is obtained by cotransfecting the vector A containing the specific segment A and the vector B containing the specific segment B into chick embryo fibroblast and rescuing the chick embryo fibroblast by using a reverse genetics technology;

the specific fragment A comprises cDNA corresponding to a chicken infectious bursal disease virus genome A segment;

the specific fragment B comprises cDNA corresponding to a chicken infectious bursal disease virus genome B segment;

the B segment of the virus genome comprises a cDNA segment which codes for the N-terminal structural domain of infectious bursal disease virus VP1 protein, and the segment is shown as the sequence 5 from the 170 th to the 670 th position of the 5' end.

The cDNA corresponding to the chicken infectious bursal disease virus genome B segment is shown as 59-2885 bits from the 5' end of the sequence 5 in the sequence table.

The cDNA corresponding to the genome A segment of the infectious bursal disease virus is shown as the 59 th-3318 th site from the 5' end of the sequence 3 of the sequence table.

The specific fragment A sequentially has a coding sequence of hammerhead nuclease HamRz, cDNA corresponding to the chicken infectious bursal disease virus genome A segment and a coding sequence of hepatitis delta virus ribozyme HdvRz from 5 'end to 3' end.

The specific fragment B is sequentially provided with a coding sequence of hammerhead nuclease HamRz, cDNA corresponding to the chicken infectious bursal disease virus genome B segment and a coding sequence of hepatitis delta virus ribozyme HdvRz from 5 'end to 3' end.

The coding sequence of hammerhead nuclease Hamrz is shown as 1st to 58 th from 5 'end of a sequence 3 in a sequence table or is shown as 1st to 58 th from 5' end of a sequence 5 in the sequence table.

The coding sequence of the hepatitis delta virus ribozyme Hdvrz is shown as 3319 to 3406 th site from 5 'end of a sequence 3 of a sequence table or is shown as 2886 to 2973 th site from 5' end of the sequence 5 of the sequence table.

The specific fragment A is shown as a sequence 3 in a sequence table.

The specific fragment B is shown as a sequence 5 in a sequence table.

The vector A is a recombinant expression vector obtained by inserting DNA molecules shown in a sequence 3 in a sequence table between EcoR V enzyme cutting sites of a eukaryotic expression vector pVAX 1.

The vector B is a recombinant expression vector obtained by inserting DNA molecules shown in a sequence 5 in a sequence table between EcoR V enzyme cutting sites of a eukaryotic expression vector pVAX 1.

In the method, when the co-transfection is carried out, the mass ratio of the vector A to the vector B is 1: 1.

in the method, cells are harvested 24h after cotransfection, and the recombinant virus solution is obtained after repeated freeze thawing for 3 times.

The recombinant virus liquid can be inoculated to chick embryos for passage to obtain the recombinant virus capable of being stably passed.

The recombinant virus can be inoculated to chick embryos for passage through a chick embryo chorioallantoic membrane approach.

The generation method comprises the following steps: selecting 9-day-old SPF (specific pathogen free) chick embryos, illuminating the eggs with an egg illuminating lamp, drawing a circle at a position without blood vessels on the side surface of an air chamber of the chick embryos by using a pencil to serve as a mark of a punching position, disinfecting the positions of the air hole mark and the air chamber of the chick embryos by using iodine, and then disinfecting by using 75% alcohol. The chick embryo is placed on a superclean workbench, small holes are respectively punched at the marked punching position and the upper part of the chick embryo air chamber by using ophthalmological forceps, the small holes at the upper part of the chick embryo air chamber are sucked by using an ear sucking ball, and an artificial air chamber is formed on the side surface of the chick embryo. 0.2mL of virus liquid is inoculated in the artificial air chamber at the side of the chick embryo. Culturing chick embryo in a 37 deg.C incubator for 96h, harvesting chick embryo chorioallantoic membrane, grinding to obtain virus liquid, and continuously passaging for 3 times to obtain virus liquid.

The invention also protects the application of any recombinant infectious bursal disease virus in the preparation of infectious bursal disease virus vaccines.

The invention also protects the recombinant infectious bursal disease virus in the preparation of a product for preventing and/or treating the infectious bursal disease virus.

The invention also protects an infectious bursal disease virus vaccine, and the active component of the infectious bursal disease virus vaccine is the recombinant infectious bursal disease virus.

The invention also protects a product, the active component of which is any one of the recombinant infectious bursal disease virus; the product is used for preventing and/or treating infectious bursal disease virus.

The invention provides a recombinant infectious bursal disease virus strain, which is obtained by carrying out gene recombination on an infectious bursal disease virus strain obtained by domestic separation by utilizing a reverse genetics operation technology. The genome of the recombinant infectious bursal disease virus strain provided by the invention is mainly derived from an infectious bursal disease virus strain separated from domestic chicken flocks, and part of genes are derived from an infectious bursal disease virus vaccine strain (B87), so that the recombinant infectious bursal disease virus strain serving as a vaccine candidate strain is more suitable for preventing domestic infectious bursal disease epidemic chicken flocks. The recombinant infectious bursal disease virus strain does not contain any exogenous gene, has the same size with the infectious bursal disease virus genome reported in the prior art, and has biological safety. The recombinant infectious bursal disease virus strain provided by the invention can be used as a vaccine candidate strain for vaccine research and development.

Drawings

FIG. 1 is a schematic diagram of a method for amplifying the A-segment and the B-segment of the genome of strain NN 1172.

FIG. 2 shows the results of the electrophoretic identification of the steps in example 1.

FIG. 3 is a schematic diagram of a method for constructing a vector for expressing the A-segment and the B-segment of the genome of strain NN 1172.

FIG. 4 is a schematic diagram showing an alternative method of gene sequence of the B segment part of NN1172 genome.

FIG. 5 is a graph of the in vivo replication kinetics of the virus.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Infectious bursal disease virus strain NN 1172: reference documents: jizhouhua, Jiangwei, Jiapeng billow, old fruit, Zhao Zeo Zhi, zhuyanwen, He xiu Miao, Weiping, immune protection test of different IBD vaccines against infection of IBDV virulent strain NN1172 [ J ]. Heilongjiang animal husbandry and veterinary, 2017(18):157 + 159+298 ]; the public is available from Guangxi university.

Eukaryotic expression vector pVAX 1: sammer Feishale science and technology (China) Co., Ltd., Cat number: and V26020.

Infectious bursal disease live vaccine (strain B87): china veterinary microbial strain preservation management center, strain preservation number CVCCAV 140.

9-day-old SPF chick embryos: beijing Meiliya Viton laboratory animal technology Co.

21-day-old three-yellow chicken: guangxi Zhu's farming-grazing company, Inc.

Example 1 acquisition of A-segment and B-segment of genome of infectious bursal disease Virus NN1172 Strain

A schematic diagram of the method for amplifying the A-segment and the B-segment of the genome of strain NN1172 is shown in FIG. 1.

The primers used in this example are shown in Table 1.

TABLE 1

1. Total RNA of the infectious bursal disease virus NN1172 strain is extracted and is reversely transcribed into cDNA.

2. The cDNA obtained in step 1 was used as a template, and PCR amplification was carried out using a primer pair consisting of primer A1-F and primer A1-R to obtain a genomic fragment A1 of 1441bp in size (lane 1 of FIG. 2).

3. Using the cDNA obtained in step 1 as a template, PCR amplification was performed using a primer pair consisting of primer A2-F and primer A2-R to obtain a genomic fragment A2 of 1840bp in size (lane 2 of FIG. 2).

4. The cDNA obtained in step 1 was used as a template, and PCR amplification was performed using a primer pair consisting of a primer B1-F and a primer B1-R to obtain a genomic fragment B1 (lane 4 of FIG. 2) of 1364bp in size.

5. The cDNA obtained in step 1 was used as a template, and PCR amplification was carried out using a primer pair consisting of the primer B2-F and the primer B2-R to obtain a 1484bp genomic fragment B2 (lane 5 of FIG. 2).

6. The genomic fragment A1 obtained in step 2 and the genomic fragment A2 obtained in step 3 were subjected to fusion PCR using a primer pair consisting of primer A1-F and primer A2-R to obtain a genomic A segment of 3260bp in size (lane 3 of FIG. 2).

7. The genomic fragment B1 obtained in step 4 and the genomic fragment B2 obtained in step 5 were subjected to fusion PCR using a primer pair consisting of primers B1-F and B2-R to obtain a 2827bp genomic B segment (lane 6 of FIG. 2).

The genome A segment is shown as a sequence 1 in a sequence table after sequencing (wherein, the 1st to 1441 th positions from the 5' end are genome segment A1, the 1421 st to 3260 th positions are genome segment A2, and the 1421 st to 1441 th positions are consensus segments of A1 and A2).

And sequencing to obtain the genome B segment shown as a sequence 2 in the sequence table (wherein, the 1st to the 1364 th positions from the 5' end are genome segment B1, the 1344 th to the 2827 th positions are genome segment B2, and the 1344 th to the 1364 th positions are consensus segments of B1 and B2).

Example 2 construction of vectors for expressing A-segment and B-segment of genome of NN1172 Strain

A schematic diagram of the construction of vectors for expressing the A-segment and B-segment of the genome of strain NN1172 is shown in FIG. 3.

Inserting DNA molecules shown in a sequence 3 in a sequence table among EcoR V enzyme cutting sites of a eukaryotic expression vector pVAX1 to obtain a recombinant expression vector pVAX1-NN 1172A.

In the sequence 3 of the sequence table, the 1st to 58 th positions from the 5' end are coding sequences of hammerhead nuclease HamRz, the 59 th to 3318 th positions are genome A segments of NN1172 strains, and the 3319 th to 3406 th positions are coding sequences of hepatitis delta virus ribozyme HdvRz.

Inserting DNA molecules shown in a sequence 4 in a sequence table between EcoR V enzyme cutting sites of a eukaryotic expression vector pVAX1 to obtain a recombinant expression vector pVAX1-NN 1172B.

In the sequence 4 of the sequence table, the 1st to 58 th positions from the 5' end are coding sequences of hammerhead nuclease HamRz, the 59 th to 2885 th positions are genome B segments of NN1172 strains, and the 2886 th to 2973 th positions are coding sequences of hepatitis delta virus ribozyme HdvRz.

Example 3 replacement of partial Gene sequences of the B segment of the NN1172 genome

A schematic representation of an alternative gene sequence to the B segment of the NN1172 genome is shown in FIG. 4.

1. A B87 genome B segment BI gene fragment is amplified from an infectious bursal disease live vaccine (B87 strain), and is shown as the 170 th to 670 th sites from the 5' end of a sequence 5 of a sequence table.

2. The B87 genome B segment BI gene fragment obtained in the step 1 is used for replacing a partial sequence of the NN1172 strain genome B segment in the recombinant expression vector pVAX1-NN1172B obtained in the example 2 to obtain a recombinant expression vector pVAX1-NN 1172B-delta BI.

Through sequencing verification, the recombinant expression vector pVAX1-NN 1172B-delta B I is a recombinant expression vector obtained by inserting DNA molecules shown in a sequence 5 in a sequence table among EcoR V enzyme cutting sites of a eukaryotic expression vector pVAX 1.

In the sequence 5 of the sequence table, the 1st to 58 th positions from the 5' end are the coding sequence of hammerhead nuclease Hamrz, the 59 th to 2885 th positions are the replaced infectious bursal disease virus genome B segment (wherein, the 170 th to 670 th positions are B I gene segments), and the 2886 th to 2973 th positions are the coding sequence of hepatitis D virus ribozyme Hdvrz.

The BI gene fragment encodes the N-terminal domain of the VP1 protein.

Example 4 rescue of recombinant viral strain rNN1172- Δ BI

Preparation of chicken embryo fibroblast

1. Selecting 9-day-old SPF chick embryos, and disinfecting the chick embryos by using iodine tincture and alcohol.

2. After step 1 was completed, the chick embryo air chamber was opened with tweezers, the chick embryos were gently picked up into a sterile dish, and a few drops of Phosphate Buffered Saline (PBS) were added to keep the embryo bodies moist.

3. And (3) after the step 2 is finished, removing internal organs, limbs and heads of the chick embryos, washing the chick embryos for 2 times by PBS, sufficiently shearing the chick embryos by sharp-pointed scissors to a crushed mud sample, and washing the chick embryos for 2-3 times by PBS to remove red blood cells.

4. After completion of step 3, the washed tissue fragments were aspirated into a cell vial, digested for 5min at 37 ℃ in 2mL of PBS containing 0.25% pancreatin, and the supernatant carefully discarded.

5. And (4) after the step 4 is finished, adding 5mL of DMEM medium containing 5% newborn calf serum, slightly blowing and beating the digested tissue blocks to fully release the cells, sucking cell supernatants of the cells, filtering the cell supernatants by 6 layers of medical gauze, and centrifuging the mixed liquid of the filtered cells and the culture medium for 5min at the speed of 800 r/min.

6. After completion of step 5, the centrifuged supernatant was discarded, and an appropriate amount of 5% newborn bovine serum DMEM medium was added to resuspend the cells at 1 × 10⁶Cell counts/mL 6 well cell culture plates were plated with 2mL of cell suspension per well.

7. The cells are cultured in a cell culture box with the temperature of 37 ℃ and the CO2 content of 5 percent, and the cells grow into a monolayer after being cultured for 24 hours.

Second, rescue of recombinant viruses

1. By using

2000 transfection reagent 500. mu.g of the recombinant expression vector pVAX1-NN1172A prepared in example 2 and 500. mu.g of the recombinant expression vector pVAX1-NN1172B- Δ BI prepared in example 3 were co-transfected into the primary chicken embryo fibroblasts prepared in step one, and after transfection, the cells were harvested 24h, and after repeated freeze-thawing for 3 times, a virus solution was obtained.

2. Taking 0.2mL of virus solution prepared in the step 1, and inoculating 9-day-old SPF chick embryos through a chick embryo chorioallantoic membrane approach, wherein the specific method comprises the following steps: selecting 9-day-old SPF (specific pathogen free) chick embryos, illuminating the eggs with an egg illuminating lamp, drawing a circle at a position without blood vessels on the side surface of an air chamber of the chick embryos by using a pencil to serve as a mark of a punching position, disinfecting the positions of the air hole mark and the air chamber of the chick embryos by using iodine, and then disinfecting by using 75% alcohol. The chick embryo is placed on a superclean workbench, small holes are respectively punched at the marked punching position and the upper part of the chick embryo air chamber by using ophthalmological forceps, the small holes at the upper part of the chick embryo air chamber are sucked by using an ear sucking ball, and an artificial air chamber is formed on the side surface of the chick embryo. 0.2mL of virus liquid is inoculated in the artificial air chamber at the side of the chick embryo.

3. After the step 2 is completed, the chick embryos are placed in a 37 ℃ incubator for 96h, chick embryo chorioallantoic membranes are harvested, virus liquid is obtained by grinding and is continuously passaged for 3 times, and the recombinant virus rNN 1172-delta B I is obtained.

4. Total RNA from recombinant virus rNN 1172-. DELTA.BII was extracted and reverse transcribed to cDNA.

5. And (3) sequencing the genome A segment and the genome B segment in the cDNA obtained in the step (4), wherein the sequencing result shows that the genome A segment is shown as 59 th to 3318 th from the 5 'end of the sequence 3 in the sequence table, and the genome B segment is shown as 59 th to 2885 th from the 5' end of the sequence 5 in the sequence table.

In recombinant viruses, the corresponding segment of the genomic B segment has been replaced with a segment of the BI gene.

The RNA sequence of the recombinant virus genome segment A is shown as a sequence 6 in a sequence table.

The RNA sequence of the recombinant virus genome B segment is shown as a sequence 7 in a sequence table.

Example 4 biological Properties of recombinant Virus rNN1172- Δ BI

One, rNN 1172-Delta B I of recombinant virus and ELD of wild strain NN1172₅₀Measurement of

And (3) virus to be detected: recombinant virus rNN1172- Δ BI and wild strain NN 1172.

1. Diluting virus solution to be tested by 10 times with PBS, selecting 10 times^-2～10^-76 dilutions were used to inoculate 9-day-old SPF chick embryos via the chorioallantoic membrane route (see method described in example 3), 5 embryos per dilution, and 0.2mL virus solution per embryo. Meanwhile, 2 SPF embryos of 9 days old were inoculated with 0.2mL of PBS, respectively, and used as a control group.

2. After the step 1 is finished, the inoculated chick embryos are placed in a 37 ℃ incubator for culture, the chick embryos are observed for 2 times every day and continuously observed for 7 days, the chick embryos which die within 24 hours are removed, and the chick embryo median lethal dose (ELD) is carried out according to the Reed-Muench method₅₀) Determination of log ELD₅₀The logarithm of the virus dilution + distance ratio x logarithm of the dilution factor higher than 50%.

The results show that ELD of recombinant virus rNN 1172-. DELTA.BI₅₀Is 10^-5.16ELD₅₀0.2mL of ELD of wild strain NN1172₅₀Is 10^-6.25/0.2mL。

SPF chick embryos inoculated with recombinant strain rNN 1172-delta B I show typical symptoms of infectious bursal disease virus infection, severe embryo hemorrhage and ELD₅₀Is 10^-5.16ELD₅₀0.2mL, less than wild strain NN1172ELD₅₀。

Second, the replication capacity of recombinant virus rNN 1172-delta B I in chicken

1. The 21-day-old three-yellow chickens were randomly divided into 3 groups of 15 chickens each, and each group was kept in isolation.

2. The experiments were carried out in three groups:

experimental group (recombinant virus rNN1172- Δ bi): the virus is attacked by a dropping route, and the virus attacking dose of each chicken is 0.2mL of recombinant virus rNN 1172-delta B I virus liquid (10)⁴ELD₅₀)。

Experimental group (wild strain NN 1172): the oral administration is used for counteracting toxic substance, and each chicken counteracts toxic substanceThe dosage is 0.2mL wild strain NN1172 virus liquid (10)⁴ELD₅₀)。

Control group: each chicken had a 0.2mL aliquot of sterile PBS.

After the challenge, the mental state of the chicken flocks is observed and recorded every day until 7 days after the challenge. 5 chickens were killed at each

group

3, 5, 7 days after infection, and bursal tissue was collected. Placing the bursa of Fabricius collected 3, 5 and 7 days after the toxicity attack in 2mL of EP tubes, adding 2 sterile steel balls into each EP tube, shaking the EP tubes in an electric shaker until the bursa of Fabricius becomes homogenate, centrifuging at 12000r/min for 5min, taking out the supernatant, and extracting total RNA.

3. And (3) taking the total RNA obtained in the step (2), and detecting the virus load by adopting a real-time fluorescent quantitative PCR method.

(1) The PrimeScript 1st Strand cDNA Synthesis kit (Takara, Japan) was used to reverse transcribe RNA into cDNA using IBDV-R (5'-CCCTGCCTGACCACCACTT-3') as primer, the detailed procedure was performed according to the instructions.

(2) And (2) carrying out qRT-PCR by using the cDNA obtained in the step (1) as a template.

The reaction system was 20. mu.l, and included 10. mu.l of 2 XSSYBR Premix Ex Taq (Takara, Japan), 2. mu.l of template cDNA (20-100ng), 0.4. mu.l (10. mu.M) of each of the forward primer (IBDV-F:5'-ACCGGCACCGACAACCTTA-3') and the reverse primer (IBDV-R), and 7.2. mu.l of water (nuclease-free).

The qRT-PCR reaction was carried out at iQ ^TM5 Multi color Real-Time PCR Detection System (Bio-Rad, Hercules, Calif., USA), the specific procedure was: 30s at 1.95 ℃; (ii) 39 cycles of 2.94 ℃ for 30s, 56 ℃ for 15s, 72 ℃ for 30s, fluorescence being detected at the end of each cycle; 3. the dissolution curve analysis was from 55 ℃ to 95 ℃ at 0.2 ℃/s.

And averaging the obtained results, calculating the virus load, and drawing an in vivo replication kinetic curve of the virus.

The results are shown in FIG. 5 and Table 2.

The results show that recombinant strain rNN1172- Δ BI is less replication competent in chickens than wild strain NN 1172.

TABLE 2

Thirdly, the pathogenicity of the recombinant virus rNN 1172-delta B I to chicken

1. The 21-day-old three-yellow chickens were randomly divided into 3 groups of 10 chickens each, and each group was separately raised.

2. The experiments were carried out in three groups:

Experimental group (wild strain NN 1172): the virus is attacked by a dropping way, and the virus attacking dose of each chicken is 0.2mL wild strain NN1172 virus liquid (10)⁴ELD₅₀)。

Control group: each chicken had a 0.2mL aliquot of sterile PBS.

And observing and recording the mental state of the chicken flocks every day after the challenge, and counting the mortality rate after 7 days after the challenge.

The results are shown in Table 3.

The results showed that recombinant strain rNN1172- Δ BI did not cause death of experimental chicks, while wild strain NN1172 caused death of 30% of experimental chicks. The recombinant strain rNN 1172-delta B I has obviously lower toxicity to chickens than the wild strain NN 1172.

TABLE 3

Sequence listing

<110> Guangxi university

<120> recombinant infectious bursal disease virus strain and preparation method thereof

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<170> SIPOSequenceListing 1.0

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gatcgcagcg atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag 180

ccttctgatg ccaacaaccg gaccggcgtc cattccggac gacaccctag agaagcacac 240

tctcaggtca gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat 300

tgtctttttc cctggtttcc ctggctcaat tgtgggtgct cactacacac tgcagagcaa 360

tgggaactac aagttcgatc agatgctcct gactgcccag aacctaccgg ccagctacaa 420

ctactgcagg ctagtgagtc ggagtctcac agtgaggtca agcacactcc ctggtggcgt 480

ttatgcatta aacggaacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac 540

agatgttagc tacaatgggt tgatgtctgc aacggccaac atcaacgaca aaattgggaa 600

cgtcctagta ggggaagggg taactgtcct aagcttgccc acatcatatg atctggggta 660

tgtgagactc ggtgacccca ttcccgctat agggcttgac ccaaagatgg tagcaacatg 720

tgacagcagt gacaggccca gagtctacac cataactgca gccaatgatt accaattctc 780

atcacagtac caagcaggtg gagtgacaat tacactgttc tcagcaaaca tcgatgccat 840

tacaagcctc agcatcgggg gggaactcgt gtttcaaaca agcgtccaag gccttatact 900

gggcgctacc atctacctta taggcttcga tgggactgcg gtaatcacca gagctgtggc 960

cgcaaacaat gggctaacgg ccggcactga caaccttatg ccattcaata ttgtgatacc 1020

gaccagcgag ataacccagc caatcacatc catcaaactg gagatagtta cctccaaaag 1080

tggtggtcag gcgggggatc agatgtcatg gtcagcaagt gggagcctag cggtgacgat 1140

ccacggcggc aactatccag gggccctccg tcccgtcaca ctagtagcct acgaaagagt 1200

ggctacagga tctgtcgtta cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc 1260

tgaactagca aagaacctga tcacagaata cggccgattt gacccaggag ccatgaacta 1320

cacaaaattg atactgagtg agagggaccg tcttggcatc aagaccgtat ggccaacaag 1380

ggagtacact gactttcgcg agtacttcat ggaggtggcc gacctcaact ctcccctgaa 1440

gattgcagga gcatttggct tcaaagacat aattcgggcc ttaaggagga tagctgtgcc 1500

ggtggtctcc acactgttcc cacccgccgc tcctctggcc catgcaattg gggaaggtgt 1560

agactatctt ctgggcgacg aggcacaggc tgcttcagga actgctcgag ccgcgtcagg 1620

aaaagcaaga gctgcctcag gccgcataag gcagctaacc ctcgccgccg acaaggggta 1680

cgaggtagtc gcgaatctgt ttcaggtgcc ccagaatcct gtagtcgacg ggattctcgc 1740

ttcacctggg atactccgcg gcgcacacaa cctcgactgc gtgctgagag agggtgccac 1800

gctattccct gtggtcatca ccacagtgga agatgccatg acacccaaag cactgaacag 1860

caaaatgttt gctgtcattg aaggcgtgcg agaagacctc caacctccat ctcaaagagg 1920

atccttcata cgaactctct ccggacatag agtctatgga tatgctccag atggggtact 1980

tccactggag actgggagag attacaccgt ggtcccaata gatgatgtct gggacgacag 2040

cattatgctg tccaaagacc ccatacctcc tattgtggga aacagcggga acctcgccat 2100

agcttacatg gatgtgtttc gacccaaagt ccccatccat gtagccatga cgggagccct 2160

caacgcctat ggcgagattg agaatgtgag ctttagaagc accaagctcg ccactgcaca 2220

ccgacttggc ctcaagttgg ctggtcccgg cgcatttgac gtgaacaccg ggtccaactg 2280

ggcgacgttc atcaaacgtt ttcctcacaa tccacgcgac tgggacaggc tcccctacct 2340

aaaccttcca taccttccgc ccactgcagg acgccagtac gacctggcta tggccgcctc 2400

agagttcaag gagacccccg aactcgagag cgctgtcaga gccatggaag cagcagccaa 2460

cgtggaccca ctgttccaat ccgcgctcag cgtgttcatg tggctggaag agaatgggat 2520

tgtgaccgac atggccaact tcgcactcag cgacccgaac gcccatcgga tgcgcaactt 2580

tctcgcaaac gcaccacaag caggcagcaa gtcgcaaaga gccaagtacg ggacggcagg 2640

ctacggagta gaggcccggg gccccactcc agaggaagca cagagggaaa aagacacacg 2700

gatctcaaag aagatggaga ccatgggcat ctactttgca acaccagaat gggtagcact 2760

caatgggcac cgggggccaa gccccggcca gctaaagtac tggcagaaca cacgagaaat 2820

acctgatcca aacgaggact acctagacta cgtgcatgca gagaagagcc ggttggcatc 2880

agaagaacaa atcctaaggg cagctacgtc gatctacggg gctccaggac aggctgagcc 2940

accccaggcc ttcatagacg aagtcgccaa agtctatgaa atcaaccatg ggcgtggccc 3000

caaccaagag cagatgaaag atctgctcct gactgcgatg gagatgaagc atcgcaatcc 3060

caggcgggct ccaccaaagc ccaagccaaa acccaatgtt ccaacgcaga gaccccctgg 3120

tcggctgggc cgctggatca gggccgtctc tgatgaggac cttgagtgag gctcctggga 3180

gtctcccgac accacccgcg caggcgtgga caccaattcg gccatacaac atcccaaatt 3240

ggatccgttc gcgggtcccc 3260

<210> 2

<211> 2827

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggatacgatg ggtctgaccc tctgggagtc acgaattaac gtggctacta ggggtgacac 60

ccaccgctag ctgccacgtt agtggctcct cttcttgatg attctgccac catgagtgac 120

gtattcaaca gtccacaggc gcgaagcaaa atatcagcag cgtttggcat aaagcccaca 180

gctggacagg atgtggaaga actcttgatc cccaaggtct gggttccacc tgaggaccca 240

ctggcaagcc ccagtcgact tgccaaattc ctcagagaaa acggctacaa gattttgcag 300

ccgcggtctc tacccgagaa tgaggagtat gagaccgacc agatacttcc agacctagca 360

tggatgcgac agatagaggg ggcagttctg aagcccacct tgtctctccc cattggtgac 420

caagagtact tcccaaagta ttacccaact catcgcccga gcaaggagaa gcccaatgcg 480

tacccaccag acattgcatt actcaagcag atgatctacc tatttctcca gcttccggaa 540

gccacagagg gcctgaagga tgaagtgacc ctcctaactc aaaatatacg agacaaggcc 600

tacggaagtg ggacctacat ggggcaagcg accagacttg ttgccatgaa agaggttgcc 660

actgggagaa atccaaacaa ggatcctcta aaacttgggt acacttttga gagcatcgcg 720

cagttgcttg acatcaccct accagttggc ccacctggtg aggatgacaa gccctgggtg 780

ccactcacaa gagtgccgtc aaggatgcta gtgctgacgg gagacgtaga tggggacttt 840

gaggttgagg attacctccc gaaaatcaac ctcaagtcat caagtggact accatatgta 900

ggtcgcacta agggggaaac aattggcgag atgatagcca tctcaaacca attcctccgg 960

gagctatcag cgctgctgaa gcaaggtgca gggaccaaag ggtcaaacaa gaagaagcta 1020

ctcagcatgc tcagtgacta ttggtactta tcatgcgggc ttttgtttcc gaaggctgaa 1080

cggtatgaca aaagcacatg gcttaccaag acccggaaca tatggtcagc tccatccccg 1140

actcacctca tgatctccat gatcacctgg cccgtgatgt ccaacagccc taacaacgtg 1200

ttgaatattg aagggtgccc gtcactctac aagtttaacc cgtttagggg aggactaaat 1260

aggatcgttg agtggatact tgctccggaa gaacccaagg cccttgtata tgcagataac 1320

atatacatcg tccactctaa cacgtggtac tcaattgacc tagaaaaggg ggaggccaac 1380

tgcacacgtc aacacatgca ggcggctatg tactacatcc tcactagagg gtggtcagac 1440

aatggtgacc ccatgtttaa ccagacatgg gctacctttg ccatgaacat tgcccctgct 1500

ctagttgtgg actcctcgtg tctgataatg aacctgcaaa tcaagaccta tggtcaaggc 1560

agtgggaacg cagccacatt catcaacaac catctcttga gcaccctggt gcttgatcag 1620

tggaacttga tgaaacaacc cagcccagac agcgaggagt tcaagtcaat tgaggacaag 1680

ctaggtatta acttcaagat cgagaggtca attgatgaca tcaggggcaa gctaagacag 1740

cttgtccccc ttgcacaacc agggtatctg agtggaggag tcgaaccaga acactccagc 1800

ccaactgttg agcttgacct gctaggctgg tcagcaacat acagcaaaga tcttgggatc 1860

tatgtgcctg tacttgacaa ggaacgccta ttctgctcgg ctgcgtatcc caaaggggtc 1920

gagaacaaga gtctcaagtc caaagttggg atcgagcaag catacaaggt agttaggtat 1980

gaggcgttga gactggtagg tggttggaac tatccactcc tgaacaaagc ttgcaagaac 2040

aacgcaagcg ctgctcggcg gcatttggag gccaaggggt tcccactcga cgagttccta 2100

gccgagtggt ctgagctgtc agaattcggc gaggcctttg aaggcttcaa cataaagctg 2160

accgtaacac cagagatcct tgccgaactg aacaagccag tacccccaaa accccccaat 2220

gtcaacagac cagtcaacac cggaggtcta aaggctgtca gcaacgccct aaagaccggc 2280

cgctacagga atgaagccgg actgagtggc ctcgtccttc tagccacagc caggagccga 2340

ctgcaagacg cagtaaaggc caaagcagag gccgagaaac tccacaagtc caagccagac 2400

gaccccgatg cagactggtt tgaaagatcc gaaactctgt ctgaccttct ggagaaagcc 2460

gacatagcca gcaaggtcgc acactcagca ctcgtggaaa caagcgacgc tcttgaggcg 2520

gttcagtcaa cctctgtgta tactcccaaa tacccggagg tcaagaaccc acagaccgcc 2580

tccaaccccg ttgtcgggct ccacctgccc gccaagagag ccaccggtgt ccaggccgct 2640

cttctcggag caggaacgag caggccaatg gggatggagg ccccaacacg gtccaagaac 2700

gccgtaaaaa tggccaaaag gcggcaacgc caaaaggaga gccgccaata gccatgatgg 2760

gaaccactca agaagaggac actaacccca gaccccgtat ccccggcctt cgcctgcggg 2820

ggccccc 2827

<210> 3

<211> 3406

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgttaagcgt ctgatgagtc cgtgaggacg aaactatagg aaaggaattc ctatagtcgg 60

atacgatcgg tctgaccccg ggggagtcac tcggggacag gccgacaagg ccctgttcca 120

ggttggaact cctccttcta cagtactatc attgatggtt agtagagatc agacaaacga 180

tcgcagcgat gacaaacctg caagatcaaa cccaacagat tgttccgttc atacggagcc 240

ttctgatgcc aacaaccgga ccggcgtcca ttccggacga caccctagag aagcacactc 300

tcaggtcaga gacctcgacc tacaatttga ctgtggggga cacagggtca gggctaattg 360

tctttttccc tggtttccct ggctcaattg tgggtgctca ctacacactg cagagcaatg 420

ggaactacaa gttcgatcag atgctcctga ctgcccagaa cctaccggcc agctacaact 480

actgcaggct agtgagtcgg agtctcacag tgaggtcaag cacactccct ggtggcgttt 540

atgcattaaa cggaaccata aacgccgtga ccttccaagg aagcctgagt gaactgacag 600

atgttagcta caatgggttg atgtctgcaa cggccaacat caacgacaaa attgggaacg 660

tcctagtagg ggaaggggta actgtcctaa gcttgcccac atcatatgat ctggggtatg 720

tgagactcgg tgaccccatt cccgctatag ggcttgaccc aaagatggta gcaacatgtg 780

acagcagtga caggcccaga gtctacacca taactgcagc caatgattac caattctcat 840

cacagtacca agcaggtgga gtgacaatta cactgttctc agcaaacatc gatgccatta 900

caagcctcag catcgggggg gaactcgtgt ttcaaacaag cgtccaaggc cttatactgg 960

gcgctaccat ctaccttata ggcttcgatg ggactgcggt aatcaccaga gctgtggccg 1020

caaacaatgg gctaacggcc ggcactgaca accttatgcc attcaatatt gtgataccga 1080

ccagcgagat aacccagcca atcacatcca tcaaactgga gatagttacc tccaaaagtg 1140

gtggtcaggc gggggatcag atgtcatggt cagcaagtgg gagcctagcg gtgacgatcc 1200

acggcggcaa ctatccaggg gccctccgtc ccgtcacact agtagcctac gaaagagtgg 1260

ctacaggatc tgtcgttacg gtcgctgggg tgagcaactt cgagctgatc ccaaatcctg 1320

aactagcaaa gaacctgatc acagaatacg gccgatttga cccaggagcc atgaactaca 1380

caaaattgat actgagtgag agggaccgtc ttggcatcaa gaccgtatgg ccaacaaggg 1440

agtacactga ctttcgcgag tacttcatgg aggtggccga cctcaactct cccctgaaga 1500

ttgcaggagc atttggcttc aaagacataa ttcgggcctt aaggaggata gctgtgccgg 1560

tggtctccac actgttccca cccgccgctc ctctggccca tgcaattggg gaaggtgtag 1620

actatcttct gggcgacgag gcacaggctg cttcaggaac tgctcgagcc gcgtcaggaa 1680

aagcaagagc tgcctcaggc cgcataaggc agctaaccct cgccgccgac aaggggtacg 1740

aggtagtcgc gaatctgttt caggtgcccc agaatcctgt agtcgacggg attctcgctt 1800

cacctgggat actccgcggc gcacacaacc tcgactgcgt gctgagagag ggtgccacgc 1860

tattccctgt ggtcatcacc acagtggaag atgccatgac acccaaagca ctgaacagca 1920

aaatgtttgc tgtcattgaa ggcgtgcgag aagacctcca acctccatct caaagaggat 1980

ccttcatacg aactctctcc ggacatagag tctatggata tgctccagat ggggtacttc 2040

cactggagac tgggagagat tacaccgtgg tcccaataga tgatgtctgg gacgacagca 2100

ttatgctgtc caaagacccc atacctccta ttgtgggaaa cagcgggaac ctcgccatag 2160

cttacatgga tgtgtttcga cccaaagtcc ccatccatgt agccatgacg ggagccctca 2220

acgcctatgg cgagattgag aatgtgagct ttagaagcac caagctcgcc actgcacacc 2280

gacttggcct caagttggct ggtcccggcg catttgacgt gaacaccggg tccaactggg 2340

cgacgttcat caaacgtttt cctcacaatc cacgcgactg ggacaggctc ccctacctaa 2400

accttccata ccttccgccc actgcaggac gccagtacga cctggctatg gccgcctcag 2460

agttcaagga gacccccgaa ctcgagagcg ctgtcagagc catggaagca gcagccaacg 2520

tggacccact gttccaatcc gcgctcagcg tgttcatgtg gctggaagag aatgggattg 2580

tgaccgacat ggccaacttc gcactcagcg acccgaacgc ccatcggatg cgcaactttc 2640

tcgcaaacgc accacaagca ggcagcaagt cgcaaagagc caagtacggg acggcaggct 2700

acggagtaga ggcccggggc cccactccag aggaagcaca gagggaaaaa gacacacgga 2760

tctcaaagaa gatggagacc atgggcatct actttgcaac accagaatgg gtagcactca 2820

atgggcaccg ggggccaagc cccggccagc taaagtactg gcagaacaca cgagaaatac 2880

ctgatccaaa cgaggactac ctagactacg tgcatgcaga gaagagccgg ttggcatcag 2940

aagaacaaat cctaagggca gctacgtcga tctacggggc tccaggacag gctgagccac 3000

cccaggcctt catagacgaa gtcgccaaag tctatgaaat caaccatggg cgtggcccca 3060

accaagagca gatgaaagat ctgctcctga ctgcgatgga gatgaagcat cgcaatccca 3120

ggcgggctcc accaaagccc aagccaaaac ccaatgttcc aacgcagaga ccccctggtc 3180

ggctgggccg ctggatcagg gccgtctctg atgaggacct tgagtgaggc tcctgggagt 3240

ctcccgacac cacccgcgca ggcgtggaca ccaattcggc catacaacat cccaaattgg 3300

atccgttcgc gggtccccgg gtcggcatgg catctccacc tcctcgcggt ccgacctggg 3360

catccgaagg aggacgcacg tccactcgga tggctaaggg agggcg 3406

<210> 4

<211> 2973

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgttaagcgt ctgatgagtc cgtgaggacg aaactatagg aaaggaattc ctatagtcgg 60

atacgatggg tctgaccctc tgggagtcac gaattaacgt ggctactagg ggtgacaccc 120

accgctagct gccacgttag tggctcctct tcttgatgat tctgccacca tgagtgacgt 180

attcaacagt ccacaggcgc gaagcaaaat atcagcagcg tttggcataa agcccacagc 240

tggacaggat gtggaagaac tcttgatccc caaggtctgg gttccacctg aggacccact 300

ggcaagcccc agtcgacttg ccaaattcct cagagaaaac ggctacaaga ttttgcagcc 360

gcggtctcta cccgagaatg aggagtatga gaccgaccag atacttccag acctagcatg 420

gatgcgacag atagaggggg cagttctgaa gcccaccttg tctctcccca ttggtgacca 480

agagtacttc ccaaagtatt acccaactca tcgcccgagc aaggagaagc ccaatgcgta 540

cccaccagac attgcattac tcaagcagat gatctaccta tttctccagc ttccggaagc 600

cacagagggc ctgaaggatg aagtgaccct cctaactcaa aatatacgag acaaggccta 660

cggaagtggg acctacatgg ggcaagcgac cagacttgtt gccatgaaag aggttgccac 720

tgggagaaat ccaaacaagg atcctctaaa acttgggtac acttttgaga gcatcgcgca 780

gttgcttgac atcaccctac cagttggccc acctggtgag gatgacaagc cctgggtgcc 840

actcacaaga gtgccgtcaa ggatgctagt gctgacggga gacgtagatg gggactttga 900

ggttgaggat tacctcccga aaatcaacct caagtcatca agtggactac catatgtagg 960

tcgcactaag ggggaaacaa ttggcgagat gatagccatc tcaaaccaat tcctccggga 1020

gctatcagcg ctgctgaagc aaggtgcagg gaccaaaggg tcaaacaaga agaagctact 1080

cagcatgctc agtgactatt ggtacttatc atgcgggctt ttgtttccga aggctgaacg 1140

gtatgacaaa agcacatggc ttaccaagac ccggaacata tggtcagctc catccccgac 1200

tcacctcatg atctccatga tcacctggcc cgtgatgtcc aacagcccta acaacgtgtt 1260

gaatattgaa gggtgcccgt cactctacaa gtttaacccg tttaggggag gactaaatag 1320

gatcgttgag tggatacttg ctccggaaga acccaaggcc cttgtatatg cagataacat 1380

atacatcgtc cactctaaca cgtggtactc aattgaccta gaaaaggggg aggccaactg 1440

cacacgtcaa cacatgcagg cggctatgta ctacatcctc actagagggt ggtcagacaa 1500

tggtgacccc atgtttaacc agacatgggc tacctttgcc atgaacattg cccctgctct 1560

agttgtggac tcctcgtgtc tgataatgaa cctgcaaatc aagacctatg gtcaaggcag 1620

tgggaacgca gccacattca tcaacaacca tctcttgagc accctggtgc ttgatcagtg 1680

gaacttgatg aaacaaccca gcccagacag cgaggagttc aagtcaattg aggacaagct 1740

aggtattaac ttcaagatcg agaggtcaat tgatgacatc aggggcaagc taagacagct 1800

tgtccccctt gcacaaccag ggtatctgag tggaggagtc gaaccagaac actccagccc 1860

aactgttgag cttgacctgc taggctggtc agcaacatac agcaaagatc ttgggatcta 1920

tgtgcctgta cttgacaagg aacgcctatt ctgctcggct gcgtatccca aaggggtcga 1980

gaacaagagt ctcaagtcca aagttgggat cgagcaagca tacaaggtag ttaggtatga 2040

ggcgttgaga ctggtaggtg gttggaacta tccactcctg aacaaagctt gcaagaacaa 2100

cgcaagcgct gctcggcggc atttggaggc caaggggttc ccactcgacg agttcctagc 2160

cgagtggtct gagctgtcag aattcggcga ggcctttgaa ggcttcaaca taaagctgac 2220

cgtaacacca gagatccttg ccgaactgaa caagccagta cccccaaaac cccccaatgt 2280

caacagacca gtcaacaccg gaggtctaaa ggctgtcagc aacgccctaa agaccggccg 2340

ctacaggaat gaagccggac tgagtggcct cgtccttcta gccacagcca ggagccgact 2400

gcaagacgca gtaaaggcca aagcagaggc cgagaaactc cacaagtcca agccagacga 2460

ccccgatgca gactggtttg aaagatccga aactctgtct gaccttctgg agaaagccga 2520

catagccagc aaggtcgcac actcagcact cgtggaaaca agcgacgctc ttgaggcggt 2580

tcagtcaacc tctgtgtata ctcccaaata cccggaggtc aagaacccac agaccgcctc 2640

caaccccgtt gtcgggctcc acctgcccgc caagagagcc accggtgtcc aggccgctct 2700

tctcggagca ggaacgagca ggccaatggg gatggaggcc ccaacacggt ccaagaacgc 2760

cgtaaaaatg gccaaaaggc ggcaacgcca aaaggagagc cgccaatagc catgatggga 2820

accactcaag aagaggacac taaccccaga ccccgtatcc ccggccttcg cctgcggggg 2880

cccccgggtc ggcatggcat ctccacctcc tcgcggtccg acctgggcat ccgaaggagg 2940

acgcacgtcc actcggatgg ctaagggagg gcg 2973

<210> 5

<211> 2973

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgttaagcgt ctgatgagtc cgtgaggacg aaactatagg aaaggaattc ctatagtcgg 60

atacgatggg tctgaccctc tgggagtcac gaattaacgt ggctactagg ggtgacaccc 120

accgctagct gccacgttag tggctcctct tcttgatgat tctgccacca tgagtgacat 180

tttcaacagt ccacaggcgc gaagcacgat ctcagcagcg ttcggcataa agcctactgc 240

tggacaagac gtggaagaac tcttgatccc taaagtttgg gtgccacctg aggatccgct 300

tgccagccct agtcgactgg caaagttcct cagagagaac ggctacaaag ttttgcagcc 360

gcggtctctg cccgagaatg aggagtatga gaccgaccaa atactcccag acttagcatg 420

gatgcgacag atagaagggg ctgttttaaa acccactcta tctctcccta ttggagatca 480

ggagtacttc ccaaagtact acccaacaca tcgccctagc aaggagaagc ccaatgcgta 540

cccgccagac atcgcactac tcaagcagat gatttacctg tttctccagg ttccagaggc 600

caacgagggc ctaaaggatg aagtaaccct cttgacccaa aacataaggg acaaggccta 660

tggaagtggg acctacatgg ggcaagcgac cagacttgtt gccatgaaag aggttgccac 720

tgggagaaat ccaaacaagg atcctctaaa acttgggtac acttttgaga gcatcgcgca 780

gttgcttgac atcaccctac cagttggccc acctggtgag gatgacaagc cctgggtgcc 840

actcacaaga gtgccgtcaa ggatgctagt gctgacggga gacgtagatg gggactttga 900

ggttgaggat tacctcccga aaatcaacct caagtcatca agtggactac catatgtagg 960

tcgcactaag ggggaaacaa ttggcgagat gatagccatc tcaaaccaat tcctccggga 1020

gctatcagcg ctgctgaagc aaggtgcagg gaccaaaggg tcaaacaaga agaagctact 1080

cagcatgctc agtgactatt ggtacttatc atgcgggctt ttgtttccga aggctgaacg 1140

gtatgacaaa agcacatggc ttaccaagac ccggaacata tggtcagctc catccccgac 1200

tcacctcatg atctccatga tcacctggcc cgtgatgtcc aacagcccta acaacgtgtt 1260

gaatattgaa gggtgcccgt cactctacaa gtttaacccg tttaggggag gactaaatag 1320

gatcgttgag tggatacttg ctccggaaga acccaaggcc cttgtatatg cagataacat 1380

atacatcgtc cactctaaca cgtggtactc aattgaccta gaaaaggggg aggccaactg 1440

cacacgtcaa cacatgcagg cggctatgta ctacatcctc actagagggt ggtcagacaa 1500

tggtgacccc atgtttaacc agacatgggc tacctttgcc atgaacattg cccctgctct 1560

agttgtggac tcctcgtgtc tgataatgaa cctgcaaatc aagacctatg gtcaaggcag 1620

tgggaacgca gccacattca tcaacaacca tctcttgagc accctggtgc ttgatcagtg 1680

gaacttgatg aaacaaccca gcccagacag cgaggagttc aagtcaattg aggacaagct 1740

aggtattaac ttcaagatcg agaggtcaat tgatgacatc aggggcaagc taagacagct 1800

tgtccccctt gcacaaccag ggtatctgag tggaggagtc gaaccagaac actccagccc 1860

aactgttgag cttgacctgc taggctggtc agcaacatac agcaaagatc ttgggatcta 1920

tgtgcctgta cttgacaagg aacgcctatt ctgctcggct gcgtatccca aaggggtcga 1980

gaacaagagt ctcaagtcca aagttgggat cgagcaagca tacaaggtag ttaggtatga 2040

ggcgttgaga ctggtaggtg gttggaacta tccactcctg aacaaagctt gcaagaacaa 2100

cgcaagcgct gctcggcggc atttggaggc caaggggttc ccactcgacg agttcctagc 2160

cgagtggtct gagctgtcag aattcggcga ggcctttgaa ggcttcaaca taaagctgac 2220

cgtaacacca gagatccttg ccgaactgaa caagccagta cccccaaaac cccccaatgt 2280

caacagacca gtcaacaccg gaggtctaaa ggctgtcagc aacgccctaa agaccggccg 2340

ctacaggaat gaagccggac tgagtggcct cgtccttcta gccacagcca ggagccgact 2400

gcaagacgca gtaaaggcca aagcagaggc cgagaaactc cacaagtcca agccagacga 2460

ccccgatgca gactggtttg aaagatccga aactctgtct gaccttctgg agaaagccga 2520

catagccagc aaggtcgcac actcagcact cgtggaaaca agcgacgctc ttgaggcggt 2580

tcagtcaacc tctgtgtata ctcccaaata cccggaggtc aagaacccac agaccgcctc 2640

caaccccgtt gtcgggctcc acctgcccgc caagagagcc accggtgtcc aggccgctct 2700

tctcggagca ggaacgagca ggccaatggg gatggaggcc ccaacacggt ccaagaacgc 2760

cgtaaaaatg gccaaaaggc ggcaacgcca aaaggagagc cgccaatagc catgatggga 2820

accactcaag aagaggacac taaccccaga ccccgtatcc ccggccttcg cctgcggggg 2880

cccccgggtc ggcatggcat ctccacctcc tcgcggtccg acctgggcat ccgaaggagg 2940

acgcacgtcc actcggatgg ctaagggagg gcg 2973

<210> 6

<211> 3260

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccuaugcuag ccagacuggg gcccccucag ugagccccug uccggcuguu ccgggacaag 60

guccaaccuu gaggaggaag augucaugau aguaacuacc aaucaucucu agucuguuug 120

cuagcgucgc uacuguuugg acguucuagu uuggguuguc uaacaaggca aguaugccuc 180

ggaagacuac gguuguuggc cuggccgcag guaaggccug cugugggauc ucuucgugug 240

agaguccagu cucuggagcu ggauguuaaa cugacacccc cuguguccca gucccgauua 300

acagaaaaag ggaccaaagg gaccgaguua acacccacga gugaugugug acgucucguu 360

acccuugaug uucaagcuag ucuacgagga cugacggguc uuggauggcc ggucgauguu 420

gaugacgucc gaucacucag ccucagagug ucacuccagu ucgugugagg gaccaccgca 480

aauacguaau uugccuuggu auuugcggca cuggaagguu ccuucggacu cacuugacug 540

ucuacaaucg auguuaccca acuacagacg uugccgguug uaguugcugu uuuaacccuu 600

gcaggaucau ccccuucccc auugacagga uucgaacggg uguaguauac uagaccccau 660

acacucugag ccacuggggu aagggcgaua ucccgaacug gguuucuacc aucguuguac 720

acugucguca cuguccgggu cucagaugug guauugacgu cgguuacuaa ugguuaagag 780

uagugucaug guucguccac cucacuguua augugacaag agucguuugu agcuacggua 840

auguucggag ucguagcccc cccuugagca caaaguuugu ucgcagguuc cggaauauga 900

cccgcgaugg uagauggaau auccgaagcu acccugacgc cauuaguggu cucgacaccg 960

gcguuuguua cccgauugcc ggccgugacu guuggaauac gguaaguuau aacacuaugg 1020

cuggucgcuc uauugggucg guuaguguag guaguuugac cucuaucaau ggagguuuuc 1080

accaccaguc cgcccccuag ucuacaguac cagucguuca cccucggauc gccacugcua 1140

ggugccgccg uugauagguc cccgggaggc agggcagugu gaucaucgga ugcuuucuca 1200

ccgauguccu agacagcaau gccagcgacc ccacucguug aagcucgacu aggguuuagg 1260

acuugaucgu uucuuggacu agugucuuau gccggcuaaa cuggguccuc gguacuugau 1320

guguuuuaac uaugacucac ucucccuggc agaaccguag uucuggcaua ccgguuguuc 1380

ccucauguga cugaaagcgc ucaugaagua ccuccaccgg cuggaguuga gaggggacuu 1440

cuaacguccu cguaaaccga aguuucugua uuaagcccgg aauuccuccu aucgacacgg 1500

ccaccagagg ugugacaagg gugggcggcg aggagaccgg guacguuaac cccuuccaca 1560

ucugauagaa gacccgcugc uccguguccg acgaaguccu ugacgagcuc ggcgcagucc 1620

uuuucguucu cgacggaguc cggcguauuc cgucgauugg gagcggcggc uguuccccau 1680

gcuccaucag cgcuuagaca aaguccacgg ggucuuagga caucagcugc ccuaagagcg 1740

aaguggaccc uaugaggcgc cgcguguguu ggagcugacg cacgacucuc ucccacggug 1800

cgauaaggga caccaguagu ggugucaccu ucuacgguac uguggguuuc gugacuuguc 1860

guuuuacaaa cgacaguaac uuccgcacgc ucuucuggag guuggaggua gaguuucucc 1920

uaggaaguau gcuugagaga ggccuguauc ucagauaccu auacgagguc uaccccauga 1980

aggugaccuc ugacccucuc uaauguggca ccaggguuau cuacuacaga cccugcuguc 2040

guaauacgac agguuucugg gguauggagg auaacacccu uugucgcccu uggagcggua 2100

ucgaauguac cuacacaaag cuggguuuca gggguaggua caucgguacu gcccucggga 2160

guugcggaua ccgcucuaac ucuuacacuc gaaaucuucg ugguucgagc ggugacgugu 2220

ggcugaaccg gaguucaacc gaccagggcc gcguaaacug cacuuguggc ccagguugac 2280

ccgcugcaag uaguuugcaa aaggaguguu aggugcgcug acccuguccg aggggaugga 2340

uuuggaaggu auggaaggcg ggugacgucc ugcggucaug cuggaccgau accggcggag 2400

ucucaaguuc cucugggggc uugagcucuc gcgacagucu cgguaccuuc gucgucgguu 2460

gcaccugggu gacaagguua ggcgcgaguc gcacaaguac accgaccuuc ucuuacccua 2520

acacuggcug uaccgguuga agcgugaguc gcugggcuug cggguagccu acgcguugaa 2580

agagcguuug cgugguguuc guccgucguu cagcguuucu cgguucaugc ccugccgucc 2640

gaugccucau cuccgggccc cggggugagg ucuccuucgu gucucccuuu uucugugugc 2700

cuagaguuuc uucuaccucu gguacccgua gaugaaacgu uguggucuua cccaucguga 2760

guuacccgug gcccccgguu cggggccggu cgauuucaug accgucuugu gugcucuuua 2820

uggacuaggu uugcuccuga uggaucugau gcacguacgu cucuucucgg ccaaccguag 2880

ucuucuuguu uaggauuccc gucgaugcag cuagaugccc cgagguccug uccgacucgg 2940

ugggguccgg aaguaucugc uucagcgguu ucagauacuu uaguugguac ccgcaccggg 3000

guugguucuc gucuacuuuc uagacgagga cugacgcuac cucuacuucg uagcguuagg 3060

guccgcccga ggugguuucg gguucgguuu uggguuacaa gguugcgucu cugggggacc 3120

agccgacccg gcgaccuagu cccggcagag acuacuccug gaacucacuc cgaggacccu 3180

cagagggcug uggugggcgc guccgcaccu gugguuaagc cgguauguug uaggguuuaa 3240

ccuaggcaag cgcccagggg 3260

<210> 7

<211> 2827

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccuaugcuac ccagacuggg agacccucag ugcuuaauug caccgaugau ccccacugug 60

gguggcgauc gacggugcaa ucaccgagga gaagaacuac uaagacggug guacucacug 120

uaaaaguugu cagguguccg cgcuucgugc uagagucguc gcaagccgua uuucggauga 180

cgaccuguuc ugcaccuucu ugagaacuag ggauuucaaa cccacggugg acuccuaggc 240

gaacggucgg gaucagcuga ccguuucaag gagucucucu ugccgauguu ucaaaacguc 300

ggcgccagag acgggcucuu acuccucaua cucuggcugg uuuaugaggg ucugaaucgu 360

accuacgcug ucuaucuucc ccgacaaaau uuugggugag auagagaggg auaaccucua 420

guccucauga aggguuucau gauggguugu guagcgggau cguuccucuu cggguuacgc 480

augggcgguc uguagcguga ugaguucguc uacuaaaugg acaaagaggu ccaaggucuc 540

cgguugcucc cggauuuccu acuucauugg gagaacuggg uuuuguauuc ccuguuccgg 600

auaccuucac ccuggaugua ccccguucgc uggucugaac aacgguacuu ucuccaacgg 660

ugacccucuu uagguuuguu ccuaggagau uuugaaccca ugugaaaacu cucguagcgc 720

gucaacgaac uguaguggga uggucaaccg gguggaccac uccuacuguu cgggacccac 780

ggugaguguu cucacggcag uuccuacgau cacgacugcc cucugcaucu accccugaaa 840

cuccaacucc uaauggaggg cuuuuaguug gaguucagua guucaccuga ugguauacau 900

ccagcgugau ucccccuuug uuaaccgcuc uacuaucggu agaguuuggu uaaggaggcc 960

cucgauaguc gcgacgacuu cguuccacgu cccugguuuc ccaguuuguu cuucuucgau 1020

gagucguacg agucacugau aaccaugaau aguacgcccg aaaacaaagg cuuccgacuu 1080

gccauacugu uuucguguac cgaaugguuc ugggccuugu auaccagucg agguaggggc 1140

ugaguggagu acuagaggua cuaguggacc gggcacuaca gguugucggg auuguugcac 1200

aacuuauaac uucccacggg cagugagaug uucaaauugg gcaaaucccc uccugauuua 1260

uccuagcaac ucaccuauga acgaggccuu cuuggguucc gggaacauau acgucuauug 1320

uauauguagc aggugagauu gugcaccaug aguuaacugg aucuuuuccc ccuccgguug 1380

acgugugcag uuguguacgu ccgccgauac augauguagg agugaucucc caccagucug 1440

uuaccacugg gguacaaauu ggucuguacc cgauggaaac gguacuugua acggggacga 1500

gaucaacacc ugaggagcac agacuauuac uuggacguuu aguucuggau accaguuccg 1560

ucacccuugc gucgguguaa guaguuguug guagagaacu cgugggacca cgaacuaguc 1620

accuugaacu acuuuguugg gucgggucug ucgcuccuca aguucaguua acuccuguuc 1680

gauccauaau ugaaguucua gcucuccagu uaacuacugu aguccccguu cgauucuguc 1740

gaacaggggg aacguguugg ucccauagac ucaccuccuc agcuuggucu ugugaggucg 1800

gguugacaac ucgaacugga cgauccgacc agucguugua ugucguuucu agaacccuag 1860

auacacggac augaacuguu ccuugcggau aagacgagcc gacgcauagg guuuccccag 1920

cucuuguucu cagaguucag guuucaaccc uagcucguuc guauguucca ucaauccaua 1980

cuccgcaacu cugaccaucc accaaccuug auaggugagg acuuguuucg aacguucuug 2040

uugcguucgc gacgagccgc cguaaaccuc cgguucccca agggugagcu gcucaaggau 2100

cggcucacca gacucgacag ucuuaagccg cuccggaaac uuccgaaguu guauuucgac 2160

uggcauugug gucucuagga acggcuugac uuguucgguc auggggguuu ugggggguua 2220

caguugucug gucaguugug gccuccagau uuccgacagu cguugcggga uuucuggccg 2280

gcgauguccu uacuucggcc ugacucaccg gagcaggaag aucggugucg guccucggcu 2340

gacguucugc gucauuuccg guuucgucuc cggcucuuug agguguucag guucggucug 2400

cuggggcuac gucugaccaa acuuucuagg cuuugagaca gacuggaaga ccucuuucgg 2460

cuguaucggu cguuccagcg ugugagucgu gagcaccuuu guucgcugcg agaacuccgc 2520

caagucaguu ggagacacau augaggguuu augggccucc aguucuuggg ugucuggcgg 2580

agguuggggc aacagcccga gguggacggg cgguucucuc gguggccaca gguccggcga 2640

gaagagccuc guccuugcuc guccgguuac cccuaccucc gggguugugc cagguucuug 2700

cggcauuuuu accgguuuuc cgccguugcg guuuuccucu cggcgguuau cgguacuacc 2760

cuuggugagu ucuucuccug ugauuggggu cuggggcaua ggggccggaa gcggacgccc 2820

ccggggg 2827

Claims

1. A recombinant infectious bursal disease virus is a recombinant virus obtained by replacing RNA capable of being translated to obtain a VP1 protein N-terminal structural domain in chicken infectious bursal disease virus with RNA shown from 112 th to 612 th site of 5' end of a sequence 7 in a sequence table;

the chicken infectious bursal disease virus is an infectious bursal disease virus NN1172 strain.

2. A recombinant infectious bursal disease virus is prepared by cotransfecting chicken embryo fibroblast with a vector A containing a specific fragment A and a vector B containing a specific fragment B, and rescuing the obtained recombinant virus by using a reverse genetics technology;

the cDNA corresponding to the chicken infectious bursal disease virus genome B segment is shown as 59-2885 bits from the 5' end of the sequence 5 in the sequence table;

3. The recombinant infectious bursal disease virus of claim 2, wherein:

the specific fragment A sequentially has a coding sequence of hammerhead nuclease HamRz, cDNA corresponding to the chicken infectious bursal disease virus genome A segment and a coding sequence of hepatitis delta virus ribozyme HdvRz from the 5 'end to the 3' end;

4. Use of a recombinant infectious bursal disease virus of claim 1 or 2 for the preparation of an infectious bursal disease virus vaccine.

5. Use of a recombinant infectious bursal disease virus of claim 1 or 2 for the preparation of a product for the prevention and/or treatment of infectious bursal disease virus.

6. An infectious bursal disease virus vaccine, the active ingredient of which is the recombinant infectious bursal disease virus of claim 1 or 2.

7. A product comprising as an active ingredient the recombinant infectious bursal disease virus of claim 1 or; the product is used for preventing and/or treating infectious bursal disease virus.