KR101560793B1 - Avirulent infectious bursal disease variant virus and use as a vaccine - Google Patents

Abstract

The present invention relates to a novel non-pathogenic infectious pandemic virus (BP-IBDVac-II) having an antigenicity similar to that of an outdoor pathogenic infectious pandemic virus and having excellent proliferative activity and having excellent vaccine efficacy, a infectious pandemic vaccine containing the same, , The non-pathogenic infectious pandemic virus of the present invention can be usefully used for the preparation of a vaccine for the prevention of infectious pox, and the cell line for culturing infectious pandemic virus and the composition of the culture can be useful for developing a new vaccine.

Description

[0001] DESCRIPTION [0002] AVIRULENT INFECTIOUS BURSAL DISEASE VARIANT VIRUS AND USE AS A VACCINE [0003]

The present invention relates to a novel non-pathogenic infectious pandemic virus (BP-IBDVac-II) having similar antigenicity to an outdoor pathogenic infectious pandemic virus and having excellent proliferative activity and excellent vaccine efficacy, a infectious pandemic vaccine containing the same, .

Infectious bursal disease (IBD), also known as gumboro disease, is an infectious disease caused by the IBD virus (IBDV). IBDV belongs to the family Birnaviridae. The genome consists of two double-stranded RNA segments (A and B), VP5, VP2-VP4-VP3 gene in segment A, VP1 gene . The disease is a disease that causes immunosuppression by intensively destroying the Fabricius sac (Bursa of Fabricius: F-sac), which is the central organ of the humoral immune function of chicken. It is a disease that causes economical damage not only by acute but also secondary infection.

The IBD vaccine to prevent this disease is classified as virulent, middle poison, and mid poison plus (+) depending on the pathogenicity. When the live virus is infected, the poison does not cause atrophy of the follicle but the intermediate poison and mid poison + Resulting in weak or pronounced atrophy. The reason for the atrophy of the F sac is that the immature B-cells present in the F sac are killed by IBDV. Such immature B-cells will differentiate into antibodies that can bind to various pathogens, Lt; / RTI >

Among the methods of vaccination, there is an in ovo vaccination method in which the 18-day-old fetus under development is inoculated through the egg shell. This method is known to be the most effective vaccine method since it can be automated and mass- 18-day-old fetuses are susceptible to external stimuli, and if they are pathogenic to vaccine strains, they can not hatch and cause problems. Therefore, it is important to develop a vaccine with low virulence to enable in ovo vaccination.

In order to produce an economically viable vaccine, a suitable production system is required. In order to obtain viruses, high-pathogenic IBDV does not proliferate in the fetal cells or cell line. Therefore, the virus should be emulsified after susceptible SPF (specific pathogen free) Mutations of highly pathogenic IBDV and adaptive VP2 genes that are able to grow in cultured cells by adaptation to cell or premalignant cells are also known to be costly to produce by direct vaccine, but vaccines using such viruses have not been developed at present. In addition, IBDV, which is proliferative in fetal fibroblasts, has a relatively low proliferation rate compared to cost, so vaccine development with excellent proliferation in cultured cells is required.

The VP2 protein of IBDV has a hypervariable region consisting of amino acids 206-350 (Bayliss et al., 1990, Journal of General Virology 71: 1303-1312) and the antigenic mutants of the conventional standard VP2 protein Mutations (D213N / P222T and G318D / D323E) associated with antigenic mutations of the antigenic mutant in two hydrophilic moieties consisting of amino acids 212-224 and 314-324 amino acids were identified by comparison of the difference with the monoclonal antibody (Heine et al., 1991, Journal of General Virology, 72: 1835-1843). Recently, the VP2 protein protrusions, which constitute the outermost part of the IBDV virion, have been shown to have 8 beta-sheet (Coulibaly et al., Cell 2005, 120: 761-772) PCD, PDE, PEF, PFG, PGH, and PHI) that connect each beta-sheet with PB, PC, PD, PE, PF, PG, PH and PI.

IBDV production techniques using immunogenetics can be carried out by cloning D78 vaccinovirus genome segments A and B under the T7 RNA polymerase promoter, synthesizing positive sense RNA in vitro with T7 RNA polymerase and transforming Vero cell line (Mundt Genomic segments A and B were cloned between the T7 RNA polymerase promoter and the hepatitis delta virus ribozyme and the segment A and segment B plasmids were cloned into QM 5), and a recombinant virus was produced by infection with fowl pox virus expressing T7 RNA polymerase (Boot et al., 2000, Journal of Virology, 74: 6701-6711).

One embodiment of the present invention is to provide a novel hyperproliferative, outdoor pseudo-antigenic, non-pathogenic recombinant infectious pandemic virus (BP-IBDVac II) strain.

Another embodiment provides the use as a vaccine against the infectious pneumonia of the hyperplastic, outdoor primary pseudo-antigenic, non-pathogenic recombinant infectious pandemic virus (BP-IBDVac II) strain and / or its culture.

Another embodiment is to provide a method for culturing the non-pathogenic infectious pandemic virus (BP-IBDVac II) under optimal culture conditions.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The non-pathogenic infectious pandemic virus (hereinafter referred to as BP-IBDVac II), which is proliferative in the fetal fibroblast and various cell lines and has no pathogenicity to the SPF chicken and / or fetus of the present invention and which is similar to the outdoor pathogenic infectious disease virus ) Strain, its use as a vaccine, and a production method thereof.

The inventors of the present invention produced BP-IBDvac II, a recombinant virus substituted with alanine at position 321 with alanine at position 222 of BP-IBDVac, a non-pathogenic infectious pandemic virus, and characterized BP-IBDvac and antigen Although there is a difference and shows excellent proliferation, there is no pathogenicity to 18-day-old fetus and SPF chicken, and it is excellent in immunogenicity and can be used as in ovo vaccine or virulent vaccine as well as Sodok vaccine. And the atrophy of the F-sac, thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention provides a non-pathogenic infectious disease virus strain, which comprises a mutated VP2 protein which is synthesized as a mutated non-pathogenic infectious pandemic virus (BP-IBDVac II).

The non-pathogenic infectious pandemic virus strain (BP-IBDVac II) is a wild type non-pathogenic infectious pandemic virus (hereinafter referred to as BP-IBDvac) (Patent No. 10-1104911) obtained by inoculating a sample of outdoor samples into fibroblasts of fetal embryos (S222A), alanine at the 321st amino acid position was substituted with threonine (A321T), and the non-pathogenic infectious pandemic virus (BP-IBDVac II) is proliferating in fetal fibroblasts and various cell lines, and is a non-pathogenic infectious pneumonia virus similar to an outbreak of pathogenic infectious disease.

The VP2 protein of the non-pathogenic infectious disease virus (BP-IBDVac II) may be composed of the amino acid sequence of SEQ ID NO: 1.

The strain (BP-IBDVac II) may have the genomic sequence represented by the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3.

In an embodiment, the strain (BP-IBDVac II) may be a strain of Accession No. KCTC 11890BP.

The proliferation of BP-IBDVac II with S222A mutation and V4-126 virus with S222A mutation only in PK-15 cell line showed that the proliferation of BP-IBDVac II with the A321T mutation in addition to the S222A mutation was associated with the S222A mutation Gt; V4-126 < / RTI >

As a result of BP-IBDVac II antigenic test, antihypertensives for BP-IBDVac showed low antigenic association index for BP-IBDVac II compared to BP-IBDVac II, while antisera for BP-IBDVac II was BP-IBDVac , Respectively. Antigenic changes of BP-IBDVac II by S222A and A321T mutations were recognized, and BP-IBDVac II was also effective against non-mutated viruses.

Antibodies against BP-IBDVac were lower in BP-IBDVac compared to BP-IBDVac II, but the antinociceptive index was lower in BP-IBDVac II than in BP-IBDVac II. Showed high neutralizing antibody. Thus, the antigenic changes of BP-IBDVac II due to S222A and A321T mutations were recognized, and BP-IBDVac II was also shown to be superior to the mutant virus.

As a result of evaluating the virulence of the virus, the antibody was not detected in the negative control but neutralized antibody of 7.0 in the test group inoculated with BP-IBDVac II showed efficacy as the virus vaccine of the virus BP-IBDVac II of the present invention , And the efficacy of BP-IBDVac II as a Sadok oil vaccine was evaluated by AGP test and virus neutralization test. As a result, more than 93% of test vaccines were tested in immunodiffusion test and 100% antibody was detected in ELISA test. It was evaluated as an effective vaccine that meets the criteria for testing.

Thus, the non-pathogenic infectious pandemic virus (BP-IBDVac II) strain of the present invention has excellent proliferative activity and shows a difference in antigenicity from the BP-IBDVac strain and is safe enough to be used as an in ovo vaccine. .

Thus, another embodiment provides a vaccine composition for infectious pneumonia comprising the non-pathogenic infectious virulence virus (BP-IBDVac II) strain and / or the virus-free cell-free or cell-containing culture.

In an embodiment, the vaccine may be a live virus vaccine, a sadox vaccine and / or an in ovo vaccine.

The culture may be a cell-free or cell-containing culture obtained by culturing the non-pathogenic infectious disease virus (BP-IBDVac II).

The non-pathogenic infectious killing virus (BP-IBDVac II) strain of the present invention is preferably selected from the group consisting of a fetal bovine extract, preferably a fetal bovine extract of 16 to 21 days, more preferably a fetal bovine extract , The growth rate was excellent. Further, based embryo fibroblasts (preferably from 10 to system embryo fibroblasts 12 days of age), and / or various mammalian derived cells, (for example, ST (swine testis, CRL-1746 ^TM) cells, or PK-15 (porcine kidney-15, CCL-33 ^TM ) cells.

Therefore, the culture product can be obtained by culturing the non-pathogenic infectious pandemic virus (BP-IBDVac II) strain at 10 to 12 days of fetal fibroblast, ST cell, or PK-15 cell and / Lt; RTI ID = 0.0 > 1, < / RTI >

The fetal bovine extract may be obtained by cutting out fetal bovine fetuses at 16 to 21 days, preferably 18 to 21 days, and extracting the active ingredient by adding a buffer solution to the fetal bovine fetuses. The 21-day-old is meant to include the fetus just before hatching.

It is preferable that the fetus is removed by removing egg yolk because it can increase the culture efficiency. In addition, the size of the fetus can be appropriately adjusted, for example, but not limited to, width x width x height each 0.1-10 mm x 0.1-10 mm x 0.1-10 mm.

The truncated fetus may be extracted from a solvent selected from the group consisting of phosphate buffered saline, saline, and balanced salt solution. The extraction temperature may be about 0 ° C to room temperature (about 25 ° C), preferably about refrigeration temperature, such as about 2 to 8 ° C, and the extraction time may be about For about 1 to 12 hours, preferably about 3 to 10 hours, but is not limited thereto. The amount of the solvent used is preferably 1: 0.5 to 1.5 (fetus: solvent), preferably approximately the same amount, based on the weight of the chopped fetus.

Considering the culture efficiency, the amount of the fetal bovine serum to be added is 0.1 to 50% by weight, preferably 1 to 20% by weight, more preferably 3 to 15% by weight, for example, 5 to 10% by weight .

The vaccine composition of the present invention may further comprise conventional preservatives, stabilizers, and / or additives.

Another embodiment provides a method for culturing the non-pathogenic infectious pandemic virus (BP-IBDVac II).

More specifically, the culturing method is characterized in that the non-pathogenic infectious disease virus strain is cultured in fetal fibroblasts, ST cells, or PK-15 cells at 5 to 15 days, preferably 10 to 12 days, Lt; RTI ID = 0.0 > of: < / RTI >

As described above, the novel hyperproliferative, outdoor pseudo-antigenic, non-pathogenic infectious pandemic virus (BP-IBDVac II) of the present invention is useful not only for preparing a vaccine for the prevention of infectious disease, Culture cell lines and culture compositions can be useful for developing new vaccines.

Figure 1 is a schematic diagram of the pRGIBD-A plasmid.
Figure 2 is a schematic diagram of the pRGIBD-B plasmid.
Figure 3 shows the process of BP-IBDVac II generation using reverse genetics.
FIG. 4 shows fluorescence microscopic observation of confirmation of proliferation of recombinant IBDV (BP-IBDVac II and V4-126) in fetal fibroblasts using indirect fluorescent antibody method.
FIG. 5A shows the nucleotide sequence of the mutant portion of BP-IBDVac II, and FIG. 5B shows the mutant partial nucleotide sequence of V4-126.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example]

Example  1. Using reverse genetics BP - IBDVac II Produce

1-1. IBDV  Vector Generation for Reverse Genetics

IBDV Segment A and IBDV Segment B transfer vectors were prepared. The primers used are shown in Table 1 below.

primer SEQ ID NO: Base sequence CMV-Not1-F 4 5'-GCGGCCGC-GTTGACATTGATTATTGACT-3 ' CMV-Sac1-R 5 5'-CTCGAGACGAATATATCTGGAGGGTGG-3 ' M13R 6 5'-CACACAGGAAACAGCTATGACCAT -3 ' BGHpA-F (EcoRI) 7 5'-GAGAGAATTCTCGACTGTGCCTTCTAGTTG-3 ' BGHpA-R (KpnI) 8 5'-GAGAGGTACCCCATAGAGCCCACCGCATCC-3 ' CMV-IBD-A5 9 5'-GAGCTCGTTTAGTGAACCGGGATACGATCGGTCTGACC-3 ' Sac1 222-R 10 5'-CAGAGCTCTCCCCCAATGCTGAG-3 ' SacI 321-F 11 5'-GAGAGCTCGTGTTCCATACAAGCGT-3 ' SpeI 321-R 12 5'-ACTCTTTCGTAGGCTACTAGTGTGAC-3 ' IBDA-3E-BsmBI 13 5'-GACGTCTCTGGGGACCCGCGAACGGATCCAAT-3 ' IBDA-XbaI-3E 14 5'-GGACTATCTAGACTACGTGCATGC-3 ' HDV-IBDA-3E-BsmBI 15 5'-GACGTCTCTCCCCGGGTCGGCATGGCATCTCCA-3 ' HDV-EcoRI-R 16 5'-GAGAGAATTCGCTCTCCCTTAGCCATCCGA-3 ' P3 17 5'-GCCCAGAGTCTACACCATAACTGC-3 ' SA-VP-R 18 5'-ATAGCGTGGCACCCTCTCT-3 ' SA-VP-1F 19 5'-ATGCAGGACGCCAGTACCAC-3 ' S222A-F 20 5'-ATCACAGTACCAAGCAGGTGGGGTAAC-3 ' P2 21 5'-TCAGGATTTGGGATCAGC-3 ' A321T-R 22 5'-CATCTGGTCCCCTGTCTGACCGCCACT-3 ' A321T-F 23 5'-CAAAAGTGGCGGTCAGACAGGGGAGGA-3 ' Spe 321-R 24 5'-ACTCTTTCGTAGGCTACTAGTGTGAC-3 ' SB-F 25 5'-GGATACGATGGGTCTGACCC-3 ' SB-VP1R 26 5'-TGTCATCAATGGACCTCTCA-3 ' SB-VPIF 27 5'-GAATGCAGCCACGTTCATCA-3 ' SB-R 28 5'-GGGGGCCCCCGCAGGCGAA-3 ' M13F 29 5'-GTAAAACGACGGCCAGT-3 ' BGHpA-F (BamHI) 30 5'-GAGAGGATCCTCGACTGTGCCTTCTAGTTG-3 ' BGHpA-R (NdeI) 31 5'-GAGACATATGTCAGAAGCCATAGAGCCCAC-3 ' IBD B-3E-ApaI 32 5'-ATATGGGCCCCCGGGTCGGCATGGCATCTCCACCT-3 ' BGHpA-R-ApaI 33 5'-ATATGGGCCCCCATAGAGCCCACCGCATCCCC-3 ' HDV-IBD B-3E-ApaI F 34 5'-ATATGGGCCCCCGGGTCGGCATGGCATCTCCACCT-3 '

One) IBDV Segment  A pre-production vector production

IBDV Segment A after transfer amplify the CMV promoter as a promoter for mRNA transferred to a vector produced by CMV-Not1-F / CMV- Sac1-R primers in pcDNA6 using Platinum ^® PCR SuperMix High Fidelity kit (invitrogen) Manufacturer The clones were cloned by the TOPO TA cloning kit according to the experimental method and the clones were selected by sequencing with the M13R primer.

BGHpA fragment, a transcriptional termination and poly A tailing sequence, was amplified from pcDNA6 / V5-His-A with BGHpA-F (EcoRI) and BGHpA-R (KpnI) primers (condition: 1) 94 ° C for 5 minutes, 2) 94 ° C 20 50 ° C for 15 sec., 72 ° C for 30 sec., 3) 72 ° C for 5 min., And 2) for 27 cycles. The clone was selected using the CMV promoter.

IBDV genome segment A was divided into four fragments (SegAl, SegA2, SegA3 and SegA3E), and CMV-IBD-A5 / Sac1 222-R primer (SegA), SacI 321-F / SpeI 321-R primer 3E-BsmBI / IBD A-XbaI-3E primer (SegA3) and IBD A-3E-BsmBI / IBD A-3E-BsmBI primer (SegA3E), and clones were selected using the CMV promoter Clones were selected as the method.

PCR was performed using HDV-IBD A-3E-BsmBI and HDV-EcoRI-R primers from pTMH plasmid (1) at 94 ° C for 5 minutes, 2 ) Was amplified at 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 30 seconds (27 cycles), and 3) 72 ° C for 5 minutes) and clones were selected using the CMV promoter. The plasmids of the selected clones were purified to digest KPnI / EcoRI for pA, EcoRI / BsmBI for HDV, and KpnI / BsmBI for SegA3E according to the manufacturer's method. After harvesting each gene fragment, agarose gel electrophoresis After centrifugation, the cells were purified using a high purity purification kit, MinElute Gel Extraction Kit (QIAGEN). The purified pA, HDV and SegA3E were reacted with T4 DNA ligase (NEB) at 16 ° C for 1 hour, transformed into E. coli DH5α, plated on LB agar supplemented with Ampicillin, cultured at 37 ° C The transformed white-colony was inoculated into TB broth supplemented with ampicillin and cultured. The PCR product (condition: 1) was incubated at 94 ° C for 5 minutes, and 2) 94 ° C with IBD A-XbaI-3E / BGHpA-R (KpnI) primer 3) / BGHpA-R ((B)) was extracted from the culture solution in which the amplification product was detected, and the plasmid was extracted from the culture solution KpnI) primers to determine the correct clones.

SegA2 and SegA3 were cloned, and digested with Nru1 / Not1. Plasmid fragments containing SegA2 fragment and SegA3 were reacted for 1 hour at 16 ° C using T4 DNA ligase (NEB), and then E. coli DH5α After transformation, the cells were plated on LB agar supplemented with Ampicillin. After culturing at 37 ° C, the transformed white-colony was inoculated on TB broth supplemented with ampicillin, and incubated. After reaction, the primers of P3 and SA-VP- 2) 94 ° C for 20 seconds, 50 ° C for 20 seconds, 72 ° C for 1 minute, 3) 72 ° C for 5 minutes The above 2) was carried out with 35 cycles, and the culture solution in which the amplified product was detected The plasmid was extracted from P3 and SA-VP-R to determine the exact clone.

A plasmid containing SegA2-SegA3 and a plasmid containing SegA3E-HDV-pA were digested with XbaI and reacted with T4 DNA ligase (NEB) for 1 hour at 16 ° C, transformed into E. coli DH5α and amplified with Ampicillin After incubation at 37 ° C, the transformed white-colony was inoculated into TB broth supplemented with ampicillin and cultured. The cells were stained with SA-VP-1F and BGHpA-F (EcoRI) (Condition 1) 94 ° C for 5 minutes, 2) 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 50 seconds, and 2) for 27 cycles. The plasmid was extracted from the culture solution in which the amplified product was detected, -1F and BGHpA-F (EcoRI) primers to determine the correct clones.

In order to ligate the CMV promoter part to the SegA2-SegA3-SegA3E-HDV-pA plasmid, the plasmid in which the CMV promoter was cloned and the SegA2-SegA3-SegA3E-HDV-pA plasmid were digested with Sac1 / Not1 restriction enzyme and T4 DNA ligase NEB) for 1 hour at 16 ° C, transformed into E. coli DH5α, and streaked on LB agar supplemented with Ampicillin. After culturing at 37 ° C, the transformed white-colony was transformed with TB broth supplemented with ampicillin 2) 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 50 seconds, 2) PCR with CMV-IBD-A5 and Sac1 222-R primers (condition 1) Was performed with 25 cycles. The plasmid was extracted from the culture solution in which the amplified product was detected, and the clone was selected by determining the base sequence with CMV-IBD-A5 and Sac1 222-R primers.

A plasmid was constructed by SeqA1 cloning of Sac1 restriction site and IBDV SegA1 cloning in order to construct S222A type of antigen recognition site. PCR was performed with CMV-IBD-A5 and S222A-F / Sac1 222-R primers OE (Overlap extension) PCR (Condition 1) 94 ° C after performing 2 cycles of 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 40 seconds, 3) 72 ° C for 5 minutes, 5 minutes, 2) 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 40 seconds, and 2) for 25 cycles.

The PCR product was cloned with the TOPO TA cloning kit, and the clone was selected using the CMV promoter to select the clone.

Two plasmids were digested with SacI restriction enzyme to ligate SegA1 (S222A) fragment to CMV-SegA2-SegA3-SeaA3E-HDV-pA plasmid and reacted as described above. PCR was performed with P3 / P2 primer (condition: 1) 2) 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 30 seconds, 3) 72 ° C for 5 minutes The above 2) was performed with 33 cycles, and the plasmid was extracted from the culture solution in which the amplified product was detected, The nucleotide sequence was determined to select an accurate clone (completion of S222A-A321A).

PCR was performed using Sac1 321-F / A321T-R and A321T-F / Spe 321-R primers (conditions: 1) at 94 ° C for 5 minutes, and 2) 94 C for 15 seconds, 53 for 10 seconds, 72 for 40 seconds, and 2) for 25 cycles.

The PCR product was cloned with the TOPO TA cloning kit, and the clone was selected using the CMV promoter as described above to select an exact clone. The SegA2 (A321T) cloned plasmid was ligated to SeaA3 in the same manner as SegA2 described above, and the remaining portions were ligated in the same manner to complete a segmentA clone for producing a recombinant virus having the S222A-A321T mutation (see FIG.

2) IBDV Segment  Producing Vector Using B

To prepare the Segment B transcription vector for IBDV reverse genetics, segment B of virus was divided into two fragments (SegB1 and SegB2), and the SB-VP / SB-VP1R primer (SegB) and the SB-VPIF / SB- ) And cloned into the TOPO TA cloning kit.

SeqB1 and CMV were digested with KpnI / SacI / EcoRI and KpnI / SacI restriction enzymes and reacted as described above. PCR was carried out using M13F / M13R primer (condition 1) at 94 ° C for 5 minutes, 2) 94 ° C for 20 seconds , 50 캜 for 20 seconds, 72 캜 for 2 minutes, and 2) for 35 cycles.

VPI / SB-VP1R primer (condition: 1) at 94 ° C for 5 minutes, 2) at 94 ° C for 20 seconds, and with SephB1-CMV and SegB2, 50 ° C for 20 seconds, 72 ° C for 2 minutes, and 2) was performed with 35 cycles. The nucleotide sequence was determined with M13R primer and an accurate clone was selected.

The pA-HDV for CMV-Segment B was constructed as follows. The pA gene fragment was subjected to PCR (condition 1) at 94 ° C for 5 minutes, 2) at 72 ° C for 20 seconds, at 55 ° C for 20 seconds, at 72 ° C for 15 seconds, using the BGHpA-F (BamHI) and BGHpA- 2) was performed with 27 cycles. The correct clone was selected in the same manner as the Segment A vector.

For pA and HDV linkage, the plasmid containing the pA gene and the pTMH plasmid containing the HDV nucleotide sequence were digested with the BamH1 / Nde1 restriction enzyme according to the manufacturer's instructions to select the correct clone. 2) 94 ° C for 20 seconds, 50 ° C for 20 seconds, 72 ° C for 30 seconds, 3) PCR with the HDV-IBD B-3E-ApaI / BGHpA-R-ApaI primer ) 72 ° C for 5 min. The above 2) was performed with 25 cycles. Apa1 recognition sites were made at both ends of the clone, TOPO TA was cloned, and clones were selected as in the clone selection method using the CMV promoter.

To ligate CMV-SegB1-SegB2 with HDVpA, each plasmid was treated with Apa1 restriction enzyme and reacted according to the above method. 2) 94 ° C for 20 seconds, 50 ° C for 15 seconds, 72 ° C for 30 seconds, and 2) for 35 cycles) using the HDV-IBD B-3E-ApaI F / M13F primer An accurate clone was selected to complete the segment B transfection vector for IBDV reverse genetics (see FIG. 2).

1-2. Recombination IBDV Produce

The IBDV reverse genetics vector pRGIBD-A (S222A) or pRGIBD-A (S222A-A321T) and pRGIBD-B prepared in Example 1-1 were mixed at a ratio of 1: 1 as shown in FIG. 3, (ATCC CRL-12203), which was grown at 80% in a 6-well plate by mixing with liposome at a ratio as given by the manufacturer (ROCHE, Indianapolis, USA). (S222A mutant strains V4-126 and S222A-A321T BP-2) were inoculated into SPF-type chicken embryo fibroblast cells (CEF) after culturing the cells at 37 ° C for 2 to 3 days. IBDVac II).

Indirect fluorescent antibody method was performed to confirm intracellular proliferation of mosaic IBD virus. Fetal fibroblasts were co-infected with mosaic IBD virus (BP-IBDVac II and V4-126) on a 96-well plate and cultured for 72 hours. The medium was removed and 100% methanol was added to fix the cells and virus. After freezing in a freezer for 10 minutes, PBS containing 3% FBS was added, and blocking reaction was performed at room temperature for 120 minutes. The IBDV vaccine diluted 1:80 in blocking buffer was added to each well, incubated at room temperature for 90 minutes, washed three times with PBS, and labeled with an anti-chicken IgG antibody Diluted (1: 100), added to each well, and cultured at room temperature for 90 minutes. After completion of the reaction, the cells were washed three times with PBS solution and observed with a fluorescence microscope at a magnification of 40 to 200 times. The results are shown in FIG.

FIG. 4 shows fluorescence microscopic observation of confirmation of proliferation of recombinant IBDV (BP-IBDVac II and V4-126) in fetal fibroblasts using the indirect fluorescent antibody method.

As shown in FIG. 4, specific fluorescence was not observed in the well treated with the primary antibody of SPF serum, but when IBDV immunized serum was treated, specific fluorescence was observed to confirm the virus proliferation.

1-3. Sequencing

The RNA extracted from each mosaic IBDV-infected fetal fibroblast culture was incubated at 70 ° C for 10 minutes, and the secondary structure of the RNA was loosened. The RNA was purified using a LongRange 2Step RT-PCR Kit (QIAGEN, Hilden, Germany) 1 μl of Oligo-dT primer (20 μM), 4 μl of 5 × LongRange reverse transcriptase buffer, 2 μl of 10 mM dNTPs (2.5 mM each), 0.2 μl of LongRange RNase inhibitor (4 U / μl) and 1 μl of LongRange reverse transcriptase The mixture was incubated at 42 ° C for 90 minutes and reacted at 85 ° C for 5 minutes to synthesize cDNA. Platinum PCR SuperMix High Fidelity kit (recombinant Taq DNA polymerase, Pyrococcus species GB-D thermostable polymerase, and Platinum Taq Antibody, 66mM Tris Incubation was carried out at 95 ° C for 5 minutes before starting the indicated number of PCR cycles using -SO ₄ (pH 8.9), 19.8 mM (NH ₄ ) ₂ SO ₄ 2.4 mM MgSO ₄ 220 μM dNTPs, After the cycle, samples were elongated at least 3 x PCR cycles Incubation temperature, or in the kidney, ONE-STEP RT-PCR kit (QIAGEN) by the RNA in the 5X RT-PCR buffer 20㎕ volume OneStep (Tris-Cl, KCl used in the final volume 20㎕, (NH ₄₎ for a period of _{two SO-, 12.5mM MgCl 2, DTT;} pH 8.7) 4㎕, RT-PCR Enzyme Mix (Omniscript ® ^® with Sensiscript RT mixture), 10mM dNTP s (2.5mM each) incubated at 50 ℃ 30 minutes to use the 2㎕ , Followed by incubation at 95 ° C for 15 minutes before starting the indicated number of PCR cycles, and after PCR cycles, samples were incubated at extension temperature for at least 3 x PCR cycles of extension time. The PCR fragments were purified by agarose gel electrophoresis, purified using a high purity purification kit, MinElute Gel Extraction Kit (QIAGEN), and sequenced. The results are shown in FIGS. 5A and 5B.

FIG. 5A shows the nucleotide sequence of the mutant portion of BP-IBDVac II, and FIG. 5B shows the mutant partial nucleotide sequence of V4-126.

As shown in FIGS. 5A and 5B, it was confirmed that codon No. 321 was substituted with alanine (GCA) and threonine (ACA) in alanine (GCA) in serine (TCA) and 222 in codon BP-IBDVac2.

Example  2. Using Reverse Genetics BP - IBDVac II Produce

2-1. BP - IBDVac II of in ovo  Evaluation of vaccine efficacy and safety

To investigate the efficacy and safety of BP-IBDVac II in ovo vaccine, we injected 1 × 10 ⁶ TCID ₅₀ BP-IBDVac II in 18-day-old SPF (specific pathogen free) And the results are shown in Table 2 below.

Table 2 shows the hatching rates of the control and 1 × 10 ⁶ TCID ₅₀ inoculation groups for the impact on the hatching rate of BP-IBDVac II.

Test group Hatching rate (frequency) Control group 90% (9/10) 1x10 ⁶ TCID ₅₀ group 100% (10/10)

As shown in Table 2, 90% of the control (non-inoculated) and 100% of the 1x10 ⁶ TCID ₅₀ inoculated group showed hatching rate.

Hatching chicks were harvested and blood samples were collected at 14 days of age. Serum samples were collected and tested for virus neutralization. For the virus neutralization test, serum was immobilized at 56 ° C for 30 minutes. The serum was binarized with PBS in a test tube, transferred to a 96-well plate in an amount of 100 μl each, and the same amount of virus (BP-IBDVac II) after addition of the TCID ₅₀ / 25㎕), neutralized for 1 hour at 37 ℃. 25 占 퐇 of neutralizing material and 100 占 퐇 of suspension solution suspended in fibroblast cultured in SPF fetus at 10 to 12 days of age were inoculated into each well. After incubation at 37 ° C for 4 days, complete neutralization was carried out and the reciprocal of the highest dilution factor, in which the cytopathic effect was not exhibited at all, was neutralized. The results are shown in Table 3.

Table 3 shows the in ovo vaccine efficacy of BP-IBDVac II, showing the virus-neutralizing antibodies in the control and 1x10 ⁶ TCID ₅₀ inoculated groups.

Test group Virus neutralizing antibody (mean + standard deviation) Control group 0.00 1x10 ⁶ TCID ₅₀ group 6.0 ± 1.6

As shown in Table 3, no antibodies were detected in the control group, but BP-IBDVac II 1 × 10 ⁶ TCID ₅₀ inoculation group showed neutralizing antibodies at an average of 6.0 at 14 days, and the virus BP-IBDVac II of the present invention showed efficacy as an in ovo vaccine .

In order to investigate the atrophy and weight gain of F-sac, hatching chicks were sacrificed at 21 days and BB-ratio [(F-sac weight (g) / weight (g)] x1000] The results are shown in Table 4 below.

Table 4 shows the effect of BP-IBDVac II on F-sac and body weight. BB ratio and mean body weight of control group and 1 × 10 ⁶ TCID ₅₀ group are shown.

Test group BB ratio
(3 weeks old; mean ± SD) Average weight
(3 weeks old; g) Control group 5.56 ± 1.06 168 ± 20 1x10 ⁶ TCID ₅₀ group 5.02 ± 1.25 167 ± 20

As shown in Table 4, there was no significant difference in the BB-ratio and the mean body weight in the BP-IBDVac II inoculation group compared to the control (P> 0.01), BP-IBDVac II did not inactivate the F- And it is confirmed that it is safe.

In conclusion, as shown in Table 3 and Table 4, it can be seen that the virus BP-IBDVac II of the present invention is excellent in efficacy as an in ovo vaccine, and at the same time, secures safety.

2-2. BP - IBDVac II Cell proliferation

To confirm the effect of A321T mutation on cell proliferation, the proliferation of BP-IBDVac II with S222A and A321T mutations and the recombinant virus V4-126 with S222A mutation alone were compared in PK-15 cell line. PK-15 cells were suspended in RPMI-1640 medium (5% FBS, 4 × 10 ⁵ / ml) and added to 1 ml of each 6-well culture container. . The cells were suspended in RPMI-1640 medium (500 μl) containing no serum. Three wells were added to each well. The cells were inoculated at 37 ° C for 1 hour, and then 1.3 ml of RPMI-1640 medium and 200 μl of FBS were added thereto for 7 days. The viral titer (TCID ₅₀ ) was measured by inoculating 25 μl of the virus of the same dilution in 6 wells to the fetal fibroblasts cultured in a 96-well culture container by 10-fold dilution of harvested virus, (Reed, LJ & Muench, H. American Journal of Hygiene, 27: 493-497, 1938).

Table 5 shows a comparison of PK-15 cell proliferation of BP-IBDVac II.

virus moi Virus titer
(TCID ₅₀ / ml, log10) BP-IBDVAc II 0.1 1.9x10 ⁹ V4-126 0.1 1.3x10 ⁸ BP-IBDVAc II 0.01 7.1 x 10 ⁷ V4-126 0.01 8.6 x ^{10 8}

As shown in Table 5, BP-IBDVac II exhibited a titer of 1.9 × ^{10 9} TCID ₅₀ / ml in PK-15 cell line and 7.1 × ^{10 7} TCID ₅₀ / ml in 0.01 moi when inoculated with 0.1moi, and V4-126 (S222A ) Showed 1.3 × ^{10 8} TCID ₅₀ / ml at 0.1 moi and 8.6 × ^{10 8} TCID ₅₀ / ml at 0.01 moi. Therefore, it was confirmed that the proliferation of BP-IBDVac II with the A321T mutation in addition to the S222A mutation was higher than that of the V4-126 mutant with only the S222A mutation.

2-3. BP - IBDVac II Antigenicity

In order to investigate the antigenic changes caused by S222A and A321T mutations, BP-IBDVac II virus was mutated with BP-IBDVac without mutation, but the same BP-IBDVac II virus was inoculated with 1x10 ⁶ TCID ₅₀ And the separated sera were mixed to measure cross-virus neutralizing antibodies. The results are shown in Table 6. < tb >< TABLE >

Table 6 shows the antigenicity comparison of BP-IBDVac and BP-IBDVac II.

virus Antiserum Anti-BP-IBDVac (antigenic association index%) Anti-BP-IBDVac II (antigenic association index%) BP-IBDVac 5.7 ± 2.5 (100) 5.0 ± 2.2 (116) BP-IBDVac II 2.8 ± 1.0 (49) 4.3 ± 1.5 (100)

As shown in Table 6, the antigenicity index of BP-IBDVac was 49% lower than that of BP-IBDVac compared to BP-IBDVac II due to its low neutralizing antibody. However, the antiserum against BP-IBDVac II showed a relatively high neutralizing antibody against BP-IBDVac. Thus, the antigenic changes of BP-IBDVac II by S222A and A321T mutations were recognized, and BP-IBDVac II was also effective against viruses without mutations.

2-4. BP - IBDVac II of An autism vaccine  efficacy

In order to evaluate the effect of BP-IBDVac II on the virulence of vaccine, BP-IBDVac II was intraperitoneally inoculated with 1.0 × 10 ⁷ TCID ₅₀ in 8 of 21-day-old SPF 13 water obtained by self-hatching SPF (SPAFAS, USA) Were counted as a negative control. After 3 weeks, blood was collected and the virus was neutralized by the neutralization test. The results are shown in Table 7.

Test group Virus neutralizing antibody titer 0 One 2 3 4 5 6 7 8 9 10 11 12 Mean ± SD BP-IBDVac II One 3 One One 2 7.0 ± 1.5 Negative control group 5 0

As shown in Table 7, no antibody was detected in the negative control group, but in the test group inoculated with BP-IBDVac II, the neutralizing antibody showed an average of 7.0, confirming the efficacy of the virus BP-IBDVac II of the present invention as a live vaccine .

2-5. BP - IBDVac Sadok Oil Vaccine  efficacy

To determine the efficacy of BP-IBDVac as a sadok oil vaccine, three batches of Sadox oil vaccine, BP-IBDVac II, were injected into a 15-week-old six-week-old SPF test system obtained by self-hatching the SPF strain (SPAFAS, USA) AGP test and virus neutralization test were performed with the serum collected 3 weeks after inoculation of 500 ㎕, and the neutralizing antibody level was measured. Fifteen (15) were isolated from the control group.

BP-IBDVac II Inoculated with 0.2% formaldehyde, the culture medium obtained by culturing BP-IBDVac for 7 days in fetal fibroblasts was inoculated with 10% of inactivated virus culture medium with 20% PBS and ISA70 oil (SEPPIC, Cosmetics / Pharmacy Division, Paris, France) was added to prepare a batch of three water-in-oil emulsion test vaccines. The immunodiffusion test was carried out using the VDPro ^® Eyebee Age ID Kit of GenoBio, Inc., which is similar to the method of Hirai et al. But using recombinant VP2 protein of D78 strain as an antigen (K. Hirai, S. Shimakura and M. Hirose. Immunodiffusion Reaction to Avian Infectious Bursal Virus. Avian Diseases, 16 (4): 961-964), and the results are shown in Table 8.

Lot No Test Factor Route of administration Dose Immunodiffusion test
(Positive number / inoculation number) Potency
(ELISA titer) IBDK01 15 Intramuscular injection 0.5ml 14/15 6593 IBDK02 15 Intramuscular injection 0.5ml 14/15 5003 IBDK03 15 Intramuscular injection 0.5ml 15/15 4951 Uninoculated control 15 - - 0/15 313

As a result, all of the test vaccines were evaluated as an effective vaccine, which was more than 93% in the immunodiffusion test and 100% antibody in the ELISA test, thus meeting the national test standards as the Sadox vaccine.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Korea Biotechnology Research Institute KCTC11890BP 20110309

<110> BioPoA, Inc.          KBNP, INC. <120> AVIRULENT INFECTIOUS BURSAL DISEASE VARIANT VIRUS AND USE ASA          VACCINE <130> DPP20134224KR <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> BP-IBDVac2 VP2 <400> 1 Met Thr Asn Leu Thr Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg   1 5 10 15 Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr              20 25 30 Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr          35 40 45 Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro      50 55 60 Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr  65 70 75 80 Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr                  85 90 95 Asn Tyr Cys Arg Leu Val Ser Ser Ser Leu Thr Val Arg Ser Ser Thr             100 105 110 Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr         115 120 125 Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu     130 135 140 Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val 145 150 155 160 Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly                 165 170 175 Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys             180 185 190 Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile         195 200 205 Thr Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Ala Gly Gly     210 215 220 Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu 225 230 235 240 Ser Ile Gly Gly Glu Leu Val Phe His Thr Ser Val His Gly Leu Ala                 245 250 255 Leu Asp Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Thr Val Ile             260 265 270 Thr Arg Ala Val Ala Ser Asp Asn Gly Leu Thr Thr Gly Ile Asp Asn         275 280 285 Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro     290 295 300 Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln 305 310 315 320 Thr Gly Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr                 325 330 335 Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val             340 345 350 Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val         355 360 365 Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val     370 375 380 Thr Gly Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu 385 390 395 400 Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr                 405 410 415 Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu             420 425 430 Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile         435 440 445 Arg Ala Ile Arg     450 <210> 2 <211> 3259 <212> DNA <213> Artificial Sequence <220> <223> BP-IBDVac2 segment A <400> 2 gatacgatcg gtctgacccc ggaggagtca cccggggaca ggccgtcaag gctttgttcc 60 aggatggaac tcctccttct acaacgctat cattgatggt tagtagagat cagacaaacg 120 atcgcagcga tgacaaacct gacagatcaa acccaacaga ttgttccgtt catacggagc 180 cttctgatgc caacaaccgg accggcgtcc attccggacg acaccctgga gaagcacact 240 ctcaggtcag agacctcgac ctacaatttg actgtggggg acacagggtc agggctaatt 300 gtctttttcc ctggattccc tggctcaatt gtgggtgctc actacacact gcagagcaat 360 gggaactaca agttcgatca gatgctcctg actgcccaga acctaccggc cagttacaac 420 tactgcaggc tagtgagtcg gagtctcaca gtgaggtcaa gcacactccc tggtggcgtt 480 tatgcactaa acggcaccat aaacgccgtg accttccaag gaagcctgag tgaactgaca 540 gatgttagct acaatgggtt gatgtctgca acggccaaca tcaacgacaa aattgggaat 600 gtcctggtag gggagggggt caccgtcctc agcttaccca catcatatga tcttgggtat 660 gtgaggcttg gtgaccccat tcctgctata gggcttgacc caaaaatggt agccacatgt 720 gacagcagtg acaggcccag agtctacacc ataactgcag ccgatgatta ccaattctca 780 tcacagtacc aagcaggtgg ggtaacaatc acactgttct cagccaacat tgatgctatc 840 acaagcctca gcattggggg agagctcgtg ttccatacaa gcgtccatgg ccttgcactg 900 gacgccacca tctaccttat aggctttgat gggactacag taatcaccag agctgtggcc 960 tcagacaatg ggctgactac cggcatcgac aatcttatgc cattcaatct tgtgattcca 1020 accaacgaga taacccagcc aatcacatcc atcaaactgg agatagtgac ctccaaaagt 1080 ggcggtcaga caggggacca gatgtcatgg tcggcaagtg ggagcctagc agtgacaatc 1140 catggtggca actatccagg ggccctccgt cccgtcacac tagtagccta cgaaagagtg 1200 gcaacaggat ccgtcgttac ggtcgccggg gtgagcaact tcgagctgat cccaaatcct 1260 gaactagcaa agaacctggt tacagaatac ggccgatttg acccaggagc catgaactac 1320 acaaaattga tactgagtga gagggaccgt cttggcatca agaccgtctg gccaacaagg 1380 gagtacactg actttcgtga gtacttcatg gaggtggccg acctcaactc tcccctgaag 1440 attgcaggag catttggctt caaagacata atccgggcca taaggaggat agctgtgccg 1500 gtggtctcta cattgttccc acctgcagct cccctagccc ttgcaattgg ggaaggtgta 1560 gactacctgc tgggcgatga ggcacaggct gcttcaggaa ctgctcgagc cgcgtcagga 1620 aaagcaagag ctgcctcagg ccgtataagg cagctaactc tcgccgctga caaggggtac 1680 gaggttgtcg cgaatctatt ccaggtgccc cagaatcccg tagtcgacgg gattcttgct 1740 tcacctgggg tactccgcgg cgcacataac ctcgactgcg tgctaagaga gggtgccacg 1800 ctattccctg tggtcattac gacagtggaa gacgccatga cacccaaagc actgaacagc 1860 aaaatgtttg ctgtcattga aggtgtgcga gaagacctcc aacctccatc tcaacgagga 1920 tccttcatac gaaccctctc cggacacaga gtctatggat atgctccaga tggggtactt 1980 ccactggaga ctgggagaga ctacaccgtt gtcccaatag atgatgtatg ggacgacagc 2040 attatgctgt ccaaagaccc catacctcct attgtgggaa acagtggcaa cctagccata 2100 gcttacatgg atgtgtttcg acccaaagtc cccatccatg tggccatgac aggagccctc 2160 aatgcttgtg gcgagattga gaaagtaagc tttagaagca ccaagctcgc cactgcacac 2220 cgacttggcc tcaagttggc tggtcccgga gcattcgatg taaacaccgg gcccaattgg 2280 gcaacgttca tcaaacgttt ccctcacaat ccacgtgact gggacaggct cccctacctc 2340 aaccttccat accttccacc caatgcagga cgccagtacc accttgccat ggctgcatca 2400 gagttcaaag agacccctga actcgagagc gccgtcagag ccatggaagc agcagccaac 2460 gtggacccac tattccaatc tgcaatcagt gtgttcatgt ggctggaaga gaatgggatt 2520 gtgactgaca tggccaactt cgcactcagc gacccgaacg cccatcggat gcgaaatttt 2580 cttgcaaacg caccacaagc aggcagcaag tcgcaacggg ccaagtacgg gacagcaggc 2640 tacggagtgg aggcccgggg ccccacacca gaggaagcac agagggaaaa agacacacgg 2700 atctcaaaga agatggagac catgggcatc tactttgcaa caccagaatg ggtagcactc 2760 aatgggcacc gagggccaag ccctggccag ctaaagtact ggcagaacac acgagaaata 2820 ccggacccaa acgaggacta tctagactac gtgcatgcag agaagagccg gttggcatca 2880 gaagaacaaa tccaaagggc agctacgtcg atctacgggg ctccaggaca ggcagagcca 2940 ccccaagctt tcatagacga agttgccaaa gtctatgaaa tcaaccatgg acgtggccca 3000 aaccaagagc agatgaaaga tctgctcttg actgcgatgg agatgaagca tcgcaatccc 3060 aggcgggctc caccaaagcc caagccaaaa cccaatgctc caacaccgag accccctggt 3120 cggctgggcc gctggatcag gactgtctct gatgaggacc ttgagtgagg ctcctgggag 3180 tctcccgaca ccacccgcgc aggtgtggac accaattcgg ccttacaaca tcccaaattg 3240 gatccgttcg cgggtcccc 3259 <210> 3 <211> 2827 <212> DNA <213> Artificial Sequence <220> <223> BP-IBDVac2 segment B <400> 3 ggatacgatg ggtctgaccc tctgggagtc acgaattaac atggctacta ggggcgatgc 60 ccgccgctaa ttgccatgtt agtggctcct cttcttgatg attctgccac catgagtgac 120 attttcaaca gtccacaggc gcgaagcaag atctcagcag cgttcggcat aaagcctact 180 gctggacaag acgtggaaga actcttgatc cctaaagtct gggtgccacc tgaggatccg 240 cttgccagcc ctagtcgact ggcaaagttc ctcagagaga acggctacaa agttttgcag 300 ccacggtctc tgcccgagaa tgaggagtat gagaccgacc aaatactccc agacttagca 360 tggatgcgac agatagaagg ggctgtttta aaacctactc tatctctccc cattggagac 420 caggagtact tcccaaagta ctacccaaca catcgcccta gcaaggagaa gaccaatgcg 480 tacccgccag acatcgcact actcaagcag atgatttacc tgtttctcca ggttccagag 540 gccaacgagg gcctaaagga tgaagtaacc ctcctgaccc aaaatataag ggataaggcc 600 tatggaagtg ggacctacat ggggcaagca actcgacttg tagccatgaa ggaggttgcc 660 actgggagaa acccgaacaa ggatcctcta aaacttgggt acacttttga gagcatcgcg 720 cagctgcttg acatcacact accggtaggc ccacccggtg aggatgacaa gccctgggtg 780 ccactcacaa gagtgccgtc gcggatgttg gtgctgacgg gagacgtaga tggcgacttt 840 gaggttgaag attaccttcc caaaatcaac ctcaagtcat caagtggact accatatgta 900 ggtcgcacca aaggagagac aattggcgag atgatagcta tctcaaacca gtttctcaga 960 gagctatcaa cactgttgaa gcaaggtgca gggacaaagg ggtcaaacaa gaagaagcta 1020 ctcagcatgt taagtgacta ttggtactta tcatgcgggc ttttgtttcc aaaggctgaa 1080 aggtacgaca aaagcacatg gctcaccaaa acccggaaca tatggtcagc tccatcccca 1140 acacacctca tgatctccat gatcacctgg cccgtgatgt ccaacagccc aaacaacgtg 1200 ttgaacattg aagggtgtcc atcactctac aaattcaacc cgttcagagg agggttgaac 1260 aggatcgtcg agtggatatt ggctccggaa gaacccaagg ctcttgtata tgcggacaac 1320 atatacattg tccactcaaa cacgtggtac tcaattgacc tagagaaggg cgaggcaaac 1380 tgcacacgcc aacacatgca agccgcaatg tactacatcc tcaccagagg gtggtcagac 1440 aacggcgacc caatgttcaa tcaaacatgg gccacctttg caatgaacat tgcccctgct 1500 ctagtggtag actcatcgtg cctgataatg aacctgcaaa ttaagaccta tggtcaaggc 1560 agtgggaatg cagccacgtt catcaataac cacctcttga gcacgctagt gcttgaccag 1620 tggaacctga tgagacagcc cagaccagac agcgaggagt tcaaatcaat tgaggacaag 1680 ctaggcatca acttcaagat tgagaggtcc attgatgaca tcaggggcaa gctgagacag 1740 cttgtccccc ttgcacaacc agggtacctg agtggggggg ttgaaccaga acaatccagc 1800 ccaactgttg agcttgacct actagggtgg tcagctacat acagcaaaga tctcgggatc 1860 tatgtgccgg tgcttgacaa ggaacgccta ttttgttctg cggcgtatcc caagggagta 1920 gagaacaaga gtctcaagtc taaagtcggg atcgagcagg catacaaggt agtcaggtat 1980 gaggcgttga ggttggtagg tggttggaac tacccactcc tgaacaaagc ctgcaagaat 2040 aacgcaggcg ctgctcggcg gcatctggag gccaaggggt tcccactcga cgagttccta 2100 gccgagtggt ccgagctgtc agagttcggt gaggccttcg aaggcttcaa tatcaagctg 2160 accgtaacat ctgagagcct agccgaactg aacaagccag taccccccaa gcccccaaat 2220 gtcaacagac cagtcaacac tgggggactc aaggcagtca gcaacgccct taagaccggt 2280 cggtacagga acgaagccgg actgagtggt ctcgtccttc tagccacagc aaggagccgt 2340 ctgcaagacg cagttaaggc caaggcagaa gccgagaaac tccacaagtc caagccagac 2400 gaccccgatg cagactggtt tgaaagatca gaaactctgt cagaccttct ggagaaagcc 2460 gacatcgcca gtaaggtcgc ccactcagca ctcgtggaaa caagcgacgc tcttgaagca 2520 gttcagtcga cttccgtgta cacccccaag tacccagaag tcaagaaccc acagaccgcc 2580 tccaaccccg ttgttgggct ccacctgccc gccaagagag ccaccggtgt ccaggccgct 2640 cttctcggag caggaacgag ccgaccaatg gggatggagg ccccaacacg gtccaagaac 2700 gccgtgaaaa tggccaaacg gcggcaacgc caaaaagaga gccgccaata gccatgatgg 2760 gaaccactca agaagaggac actaatccca gaccccgtat ccccggcctt cgcctgcggg 2820 ggccccc 2827 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CMV-Not1-F (primer) <400> 4 gcggccgcgt tgacattgat tattgact 28 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CMV-Sac1-R (primer) <400> 5 ctcgagacga atatatctgg agggtgg 27 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> M13R (primer) <400> 6 cacacaggaa acagctatga ccat 24 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> BGHpA-F (EcoRI) (primer) <400> 7 gagagaattc tcgactgtgc cttctagttg 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> BGHpA-R (KpnI) (primer) <400> 8 gagaggtacc ccatagagcc caccgcatcc 30 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> CMV-IBD-A5 (primer) <400> 9 gagctcgttt agtgaaccgg gatacgatcg gtctgacc 38 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Sac1 222-R (primer) <400> 10 cagagctctc ccccaatgct gag 23 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SacI 321-F (primer) <400> 11 gagagctcgt gttccataca agcgt 25 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SpeI 321-R (primer) <400> 12 actctttcgt aggctactag tgtgac 26 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> IBDA-3E-BsmBI (primer) <400> 13 gacgtctctg gggacccgcg aacggatcca at 32 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> IBDA-XbaI-3E (primer) <400> 14 ggactatcta gactacgtgc atgc 24 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> IBDA-XbaI-3E (primer) <400> 15 gacgtctctc cccgggtcgg catggcatct cca 33 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> HDV-EcoRI-R (primer) <400> 16 gagagaattc gctctccctt agccatccga 30 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > P3 (primer) <400> 17 gcccagagtc tacaccataa ctgc 24 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SA-VP-R (primer) <400> 18 atagcgtggc accctctct 19 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SA-VP-1F (primer) <400> 19 atgcaggacg ccagtaccac 20 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> S222A-F (primer) <400> 20 atcacagtac caagcaggtg gggtaac 27 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > P2 (primer) <400> 21 tcaggatttg ggatcagc 18 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A321T-R (primer) <400> 22 catctggtcc cctgtctgac cgccact 27 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > A321T-F (primer) <400> 23 caaaagtggc ggtcagacag gggagga 27 <210> 24 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Spe 321-R (primer) <400> 24 actctttcgt aggctactag tgtgac 26 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SB-F (primer) <400> 25 ggatacgatg ggtctgaccc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SB-VP1R (primer) <400> 26 tgtcatcaat ggacctctca 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SB-VPIF (primer) <400> 27 gaatgcagcc acgttcatca 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SB-R (primer) <400> 28 gggggccccc gcaggcgaa 19 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13F (primer) <400> 29 gtaaaacgac ggccagt 17 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> BGHpA-F (BamHI) (primer) <400> 30 gagaggatcc tcgactgtgc cttctagttg 30 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> BGHpA-R (NdeI) (primer) <400> 31 gagacatatg tcagaagcca tagagcccac 30 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> IBD B-3E-ApaI (primer) <400> 32 atatgggccc ccgggtcggc atggcatctc cacct 35 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> BGHpA-R-ApaI (primer) <400> 33 atatgggccc ccatagagcc caccgcatcc cc 32 <210> 34 <211> 35 <212> DNA <213> Artificial Sequence <220> HDV-IBD B-3E-ApaI F (primer) <400> 34 atatgggccc ccgggtcggc atggcatctc cacct 35

Claims (9)

As non-pathogenic infectious pandemic virus (BP-IBDVac II)
Wherein the VP2 protein to be synthesized is composed of the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the virus strain is a genus of the non-pathogenic infectious disease virus, wherein the genomic nucleotide sequence is represented by SEQ ID NO: 2 or SEQ ID NO: 3. The non-pathogenic infectious disease virus strain according to claim 1, which is deposited at KCTC 11890BP. 4. A vaccine composition for infectious pneumonia, comprising a non-pathogenic infectious disease virus strain according to any one of claims 1 to 3, or a cell-free or cell-containing culture of said strain. 5. The method of claim 4,
The vaccine composition may be in the form of a live vaccine or a vaccine,
Vaccine composition. 5. The method of claim 4,
The vaccine composition is an in ovo vaccine,
Vaccine composition. 5. The method of claim 4,
Wherein the culture is obtained by culturing the non-pathogenic infectious disease virus strain in a culture medium containing a fetal bovine extract at 16 to 21 days of age. 5. The method of claim 4,
Wherein the culture product is obtained by culturing the non-pathogenic infectious disease virus strain in a fetal fibroblast, ST (swine testis) cell, or PK-15 (porcine kidney-15) cell at 10 to 12 days of age. 5. The method of claim 4,
The culture is obtained by culturing the non-pathogenic infectious disease virus strain in a culture medium containing fetal bovine extract at 16 to 21 days in fetal fibroblast, ST cell, or PK-15 cells at 10 to 12 days of age , Vaccine composition.

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