KR100825503B1 - Vaccine composition for preventing infectious F. cysts in birds - Google Patents

Abstract

A vaccine composition is provided to show complete defense effect at low concentration, have excellent cross-defense capability on a heterogeneous infectious bursal disease(IBDV) and be easily mass-produced without a separate purification step. A vaccine composition for preventing IBDV comprises a recombinant VP2 or VP243 derived from SH/92 virus, which is a fragment A of the IBDV including VP2, VP4 and VP3. The composition further comprises a pharmaceutically acceptable adjuvant. In the composition, the recombinant protein is prepared by a method comprising the steps of: (a) preparing an expression vector including DNA encoding the VP2 protein or VP243 protein of an IBDV SH/92 virus strain; (b) introducing the expression vector into a host cell; and (c) culturing the host cell. Further, the expression vector is baculovirus and the host cell is insect cell.

Description

Vaccine composition for preventing infectious bursal disease

Figure 1 shows the results of electrophoresis on agarose gel after amplification of VP2 gene of IBDV by RT-PCR.

M: 1 kb DNA ladder (GibcoBRL)

Lane 1: SH / 92

Lane 2: Bur706,

Lane 3: Bursine 2

Lane 4: CEVAC

Lane 5: Negative Control

Figure 2 shows the results of electrophoresis on agarose gel after amplification of the VP243 gene of IBDV by PR-PCR.

M: 1 kb DNA ladder (GibcoBRL)

Lane 1: SH / 92

Lane 2: 1030

Lane 3: Blen

Lane 4: Bursine plus

Lane 5: Bur706

Lane 6: Bursine 2

Lane 7: CEVAC.

Figure 3 is a comparison of the base and amino acid sequence homology of the VP2 gene of IBDV.

4 shows the results of morphology of Sf 21 cells (× 200).

(a) Mok-infected Sf 21 cells

(b) Sf 21 cells infected with recombinant plasmid pBacPAK8 containing VP2 gene

(c) Sf 21 cells infected with recombinant plasmid pBacPAK8 containing VP243 gene

(d) cells infected with AcMNPV

5 shows the results of confirming the expression of recombinant protein VP2 or VP243 by immunofluorescent antibody method using polyclonal antibody (A) and monoclonal antibody (B) (× 200).

(A) (a) mok-infected cells; (b) cells infected with recombinant plasmid pBacPAK8 containing the VP2 gene; (c) cells infected with recombinant plasmid pBacPAK8 containing the VP243 gene.

(B) (a) mok-infected cells; (b) cells infected with wild type AcMNPV; (c) cells infected with recombinant plasmid pBacPAK8 containing VP243

Figure 6 shows the results of Western blot analysis using the polyclonal antibody (A) and monoclonal antibody (B) expression of recombinant protein VP2 or VP243.

(A) Lane 1: recombinant VP243 (cytoplasmic fraction), lane 2: preparative VP243 (supernatant), lane 3: recombinant VP2 (cytoplasmic fraction), lane 4: recombinant VP2 (supernatant), lane 5: BacPAK6 virus (cytoplasmic fraction) Lane 6: BacPAK6 virus (supernatant)

(B) Lane 1: recombinant VP2 (cytoplasmic fraction), lane 2: recombinant VP2 (supernatant), lane 3: recombinant P243 (cytoplasmic fraction), lane 4: recombinant VP243 (supernatant), lane 5: BacPAK6 virus (cytoplasmic fraction) Lane 6: BacPAK6 virus (supernatant)

7 shows the results of analyzing the expression levels of recombinant proteins VP2 and VP243 over time by dot-blot hybridization.

(A) and (B): use of polyclonal antibodies

(C): use of monoclonal antibody

Lanes a to d: cell lysates recovered on days 1, 2, 3 and 4 of culture, respectively

Figure 8 shows the size of the F bag according to the inoculation amount of the recombinant vaccine of the present invention in SPF chickens challenged with IBDV SH / 92.

(a) Negative Control: No Vaccine and Attack Vaccination

(b) Vaccination group: vaccination without vaccination

(c) Inoculated (2 × 10 ⁴ cells / does) and challenged with recombinant vaccine VP243

(d) Inoculated (2 × 10 ⁵ cells / does) and challenged with recombinant vaccine VP243

(e) Inoculated (2 × 10 ⁶ cells / does) and challenged with recombinant vaccine VP243

Figure 9 shows F cyst size (A) and hematoxylin-eosin stained fragments (B) following inoculation of the recombinant vaccines of the present invention in SPF chickens challenged with IBDV SH / 92.

(a) Attacking group

(b) VP2 inoculation group

(c) VP243 inoculation group

(d) Bursine-2 inoculation group

Figure 10 shows the size of the F bag according to the inoculation of the recombinant vaccine of the present invention in SPF chickens challenged with IBDV P4.

(a) negative control group

(b) attack group;

(c) BacPAK6 inoculation group

(d) VP2 inoculation group

(e) VP243 inoculation group

(f) IB + G + ND inoculation group

Figure 11 shows the size of the F bag according to the inoculation of the recombinant vaccine of the present invention in the practical application of IBDV SH / 92 challenge.

(a) negative control group

(b) attack group;

(c) IB + G + ND inoculation group

(d) VP2 inoculation group

(e) BacPAK6 inoculation group

(f) VP243 inoculation group

The present invention relates to a vaccine composition for preventing infectious F cystic disease of birds. More specifically, the present invention relates to a vaccine composition comprising recombinant protein VP2 or VP243 of avian infectious folliculosis virus.

Infectious bursal disease (hereinafter referred to as IBD) is an acute infectious disease of chickens that occurs worldwide and is caused by the IBD virus (hereinafter referred to as IBDV). IBDV is mainly caused by mortality with clinical symptoms such as feather inversion, diarrhea and depression, increased chances of secondary pathogens due to immunosuppression, and reduced immunity to other vaccines, mainly in chickens 3-6 weeks old. There is a significant economic loss in the poultry industry (Lukert et al., Avian Dis , 18: 243-250, 1974; Kibenge et al., Avian Dis , 32: 298-303, 1988).

IBDV is a bisegmented, double-stranded RNA virus belonging to the Avibirnavirus genus of Birnaviridae (Dobos et al., J Virol , 32: 593-). 605 1979; Muller et al., J Virol , 31: 584-589, 1979). IBDV consists of 51% VP2, 40% VP3, 6% VP4 and 3% VP1 (Dobos J Virol , 32: 593-605 1979). A large segment of the genome of IBDV, fragment A, has three ORFs, of which the large ORF is translated into a 110 kDa polyprotein (N-VPX-VP4-VP3-C) and then the viral protease VP4 (Birghan et al., EMBO J , 19: 114-123, 2000), which is divided into mature structural proteins VP2 (40kDa), VP3 (32kDa), and VP4 (28kDa) (Hudson et al., Nucleic Acids Res . 14: 5001). -5012, 1986; Spies et al., Virus Res , 8: 127-140, 1987; Bayliss et al., J Gen Virol , 71: 1303-1312, 1990). Small ORFs (435 bp) are expressed as VP5 (17 kDa) (Mundt et al., J Gen Virol , 76: 437-443, 1995). It consists of 145 amino acids. Section B, a small segment of the genome of IBDV, is known to be expressed as VP1 (98 kDa), a viral RNA-dependent RNA polymerase (Kibenge et al., J Gen Virol , 71: 569). -577, 1990; Bayliss et al., J Gen Virol , 71: 1303-1312, 1990).

The emergence of antigenic and very virulent IBDV (vvIBDV) strains since the 1980s has resulted in massive damage despite intensive vaccination efforts. Recently, many researches and developments have been made on new types of vaccines, including recombinant vaccines, using molecular biological methods to reduce the damage of IBD. However, despite the emergence of new and improved vaccines, IBDV is constantly changing in pathogenicity and antigenicity, so there is no common vaccine program for IBD so far.

Currently, various kinds of live and dead vaccines are used in Korea, but responses to these vaccinations are different in broilers, laying hens and breeders, and there is a problem that individual differences occur in these families.

Recent studies on IBD vaccines tend to focus on DNA vaccines. DNA vaccines induce humoral immunity and cell-mediated immunity at the same time and use only a part of the virus's genes, including antigen-determining sites, so that the pathogenicity of the virus is unlikely to recover and is safe because it does not cause infection in individuals with reduced immunity. Many studies are being conducted because of the advantage. However, DNA vaccines that are not yet fully protective have not been developed, and have a disadvantage of being economically inefficient because they require a large amount of dose inoculation (tens of hundreds to hundreds of ug).

The inventors of the present invention, while studying to develop a more effective vaccine for preventing infectious F cystic disease, VP2 gene and VP243 gene (fragment A site) of the highly pathogenic vvIBDV SH / 92 strain isolated from Korea; Cloning the segment A region) and expressing it using a baculovirus expression system to prepare a recombinant protein and measuring the protective effect against IBD, confirming that the recombinant protein vaccine according to the present invention shows a complete protective effect. The invention has been completed.

Accordingly, an object of the present invention is to provide a vaccine composition for preventing infectious F cyst disease in birds comprising recombinant protein VP2 or VP243 derived from infectious bursal disease virus (IBDV) SH / 92 virus.

In order to achieve the object of the present invention as described above, the present invention provides a vaccine composition for preventing infectious F cystic disease of birds comprising a recombinant protein VP2 or VP243 derived from IBDV (infectious bursal disease virus) SH / 92 virus.

Hereinafter, the present invention will be described in detail.

The "vaccine composition" of the present invention is characterized by preventing subsequent pathogen infection by administering a protein antigen having a vaccine effect. In the present invention, the protein antigen refers to a VP2 protein or a VP243 protein derived from IBDV SH / 92 virus.

The "IBDV SH / 92 virus strain" is a virus isolated in Korea and is known as a very deadly IBDV showing 80% mortality in SPF chickens of 5 and 7 weeks of age (Kwon, et al., 37, pp637-47). , 1995).

The "VP2 protein" is an important structural protein of the IBDV virus and accounts for 51% of mature IBDV particle proteins. VP2 is produced from the preprotein VPX, which is composed of 454 amino acids. This protein is a major component of viral capsids and contains the structure-dependent epitopes necessary to produce viral neutralizing antibodies as host defense antigens. The VP2 protein contained in the vaccine composition of the present invention preferably has the amino acid sequence of SEQ ID NO: 8.

The "VP243 protein" refers to a segment A region of IBDV including not only VP2 protein but also VP3 and VP4 together. The VP3 protein is an important structural protein of IBDV and constitutes a capsid, which constitutes 40% of the mature virion protein and is composed of 289 amino acids.

The VP4 protein is a protein having a protease activity and is composed of 267 amino acids and accounts for 6% of the mature IBDV particle protein. VP4 is known to play an important role in the maturation of IBDV proteins.

The VP243 protein contained in the vaccine composition of the present invention preferably has the amino acid sequence of SEQ ID NO.

In the above, "algae" includes, but is not limited to, chickens, turkeys, geese, ducks, pheasants, quails, pigeons, ostriches, and the like, and preferably refers to chickens.

The VP2 protein or VP243 protein, which is an active ingredient of the vaccine composition of the present invention, can be obtained by extracting from nature or by using genetic recombination techniques. When using a genetic recombination technique can be obtained by the following method. The nucleic acid sequence encoding the antigenic protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the culturing the transformant, and then the antigenic protein is obtained from the transformant.

Preferably, the protein comprises the steps of: (a) preparing an expression vector comprising DNA encoding a VP2 protein or a VP243 protein of an IBDV SH / 92 virus strain;

(b) introducing the expression vector of step (a) into a host cell; And

(c) may be prepared by a method comprising culturing the host cell of step (b).

DNA encoding the VP2 protein or VP243 protein in step (a) is as shown in SEQ ID NO: 7 and SEQ ID NO: 9.

In an embodiment of the present invention, the DNA encoding the VP2 protein or the VP243 protein is a template of RNA isolated from SH / 92, which is an IBDV isolate, and is prepared by using a primer prepared based on the gene sequence of the IBDV standard strain. And VP243 genes were obtained by amplification, respectively, by RT-PCR (see Example 1).

The expression vector in the step (a) means a recombinant carrier (recombinant carrier) is inserted into the fragment of the heterologous DNA. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once the expression vector is in the host cell, it can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced. Preferably, the expression vector in the present invention refers to baculovirus. The baculovirus is characterized by being extremely toxic to certain insects but not pathogenic to vertebrates or plants. When an insect cell is infected by the virus, a polyhedrin protein is expressed. Since the polyhedrin gene is not essential for virus replication and production of extracellular virus particles (ECVs), an external gene is inserted instead of the gene. The desired recombinant protein is then produced by infecting the cells and then culturing the cells. In other words, the expression of the heterologous protein is induced by inserting the heterologous gene into the baculovirus genome at a position to be expressed under the control of the baculovirus regulatory element and infecting it with an insect cell line to culture the insect cell line under appropriate conditions.

Baculoviruses that can be used in the present invention can use a number of baculovirus species known in the art. Preferred virions include, but are not limited to, Autographa californica nuclear polyhedrosis virus (AcNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV) and Heliothis zea Spodoptera frugiperdanuclear polyhedrosis virus (HzNPV).

On the other hand, the baculovirus has a large gene size, so it is difficult to directly manipulate the gene, so that the baculovirus is genetically manipulated using a transfer vector. As a transfer vector, a plasmid present in E. coli may be used. The plasmid has a nucleotide sequence around a polyhedrin promoter of baculovirus around the place where an external gene enters, and thus, by homologous recombination, To get into the baculovirus. As the trans vector, those known in the art may be used, and examples thereof include pBacPAK8, pACYMI, pEV55, pAC373, pACRP, pEVIV, pEV51, and pVL941.

The host cell of step (b) is a plurality of continuously cultured insect cells, preferably Spodoptera frugiperda ( Sf ), Trichoplusia ni ( T.ni ), anti Anticarsa gemmitalis , Bombyx mori , Estigmene acrea , Heliothis virescens , Leucania separata , Lemantia disparta ( Lymantria dispar ), Malacasoma disstria , Mammestra brassicae , Manduca sexta , Plutella zylostella , Storofterra excigua ( Spodoptera exigua ) and Spodoptera littorlis may be used, and more preferably Spodoptera pruriferda may be used.

Introduction of the expression vector into the host cell in step (b) can be carried out by the following method. That is, the cultured host cell is co-transfected with the baculovirus trans vector and genomic wild type baculovirus DNA, and recombinantly produced to produce a viral genome having heterologous genes. Methods and conditions for causing cotransfection are well known in the art. For example, calcium phosphate co-precipitation (Graham, FL et al., J. Virology 52: 456-467, 1973), protamine (Wienhues, U. et al., DNA (NY) 6: 81-89, 1987), lipo Pectin and electroporation (Mann, SG and King, LA, J. Gen. Virology 70: 3501-3505, 1989).

In one embodiment of the present invention, the VP2 or VP243 gene derived from IBDV SH / 92 was inserted into pBacPAK8, a baculovirus transfer vector, and cotransfected into Sf cells together with linear baculovirus DNA (Example <2). -2>).

The host cell into which the expression vector is introduced in step (b) is cultured under medium and conditions suitable for expression of the recombinant protein.

Expression of the recombinant protein induced by the above method can be confirmed by a method known in the art. In one embodiment of the present invention, the shape comparison of transfected host cells (see Example <2-2>), immunofluorescent antibody method (see Example <2-3>), SDS-PAGE and Western blot analysis ( Example <2-4>) Dot blot hybridization (see Example <2-5>) and plaque analysis (see Example <2-6>) were used.

In addition, the present inventors investigated the amount of recombinant protein produced according to the culture time of the host cell using dot blot hybridization in one embodiment of the present invention (see Example <2-5>). On day 4, the expression level was found to be the highest (see FIG. 7).

In addition, the recombinant protein of the present invention may be prepared by a method further comprising the step of preparing a cell lysate by crushing the host cell cultured in the step (c).

In the present invention, a cell lysate prepared by crushing a cultured host cell without undergoing a separate purification process of the recombinant protein expressed in the host cell according to the method of the present invention is used as an active ingredient of the vaccine composition. . Therefore, according to the method of the present invention, the vaccine composition of the present invention can be easily produced in large quantities, which is very advantageous in terms of economics.

In one embodiment of the present invention, in order to prepare a large amount of the vaccine composition of the present invention, the F150 flask was inoculated with Sf 21 cells and cultured, followed by recombinant VP2 virus or recombinant VP243 virus prepared according to the method of the present invention in each flask. After infection, the cells were harvested when the cytopathic effect was observed. The cells harvested above were centrifuged to obtain cell pellets, which were then disrupted to harvest supernatants and used as vaccines (see Example 3). This method allowed the production of a vaccine corresponding to 1000 doses in an F150 flask.

In addition, the vaccine composition of the present invention is a partially or completely purified VP2 or VP243 protein produced from a fixed host cell, host cell extract, or the host cell prepared by the method of the present invention or produced by chemical synthesis. It may be made of.

On the other hand, in previous studies, many researchers at home and abroad have expressed the antigen genes related to IBDV in prokaryotic and eukaryotic cells and inoculated them into chickens to evaluate their defense ability. However, the defensive force is not effective and does not have an effect on suppression of pathologic changes in F capsule (Lu et al., Article in Chinese, Sheng Wu Gong Cheng Xue Bao , 18: 472-476, 2002). (Azad et al., Virology . 149: 190-198, 1986) or have no cross-protection ability against heterologous viruses (Dybing et al., Avian Dis . 42: 80-91, 1998). There was this.

On the other hand, the vaccine composition of the present invention exhibits a perfect protective effect even at low concentrations, has a superior protective effect compared to commercially available vaccine compositions, and has a protective effect in both SPF (Specific pathogen free) and practical systems, and heterologous IBDV. It is characterized by its excellent cross-protection ability.

In one embodiment of the present invention, the protective ability of the recombinant protein VP2 of the present invention according to the inoculation amount was measured (see Example <4-1>). As a result, there was no significant difference in the protective effect of the vaccine of the present invention according to the inoculation amount, and even at low concentrations, no death occurred due to the challenge of strong pathogenic IBDV. It appeared to be very high, indicating a perfect protective effect (see Table 3 and FIG. 8).

In another embodiment of the present invention, the attenuated vaccine (bursine-2) (see Example 4-4) or the inactivated vaccine IB + G, which is commercially available for the protective effect of the recombinant vaccines VP2 and VP243 of the present invention. Measurements were made in comparison with the + ND mixed oil vaccine (see Examples <4-3> and Examples <4-4>). As a result, the recombinant proteins VP2 and VP243 of the present invention showed a complete protective effect, while the protective effect of a commercially available vaccine was found to be incomplete (see Tables 5 to 9).

In another embodiment of the present invention, the result of confirming whether the protective effects of the recombinant vaccines VP2 and VP243 of the present invention are shown in both SPF (Specific pathogen free) and practical systems (Examples <4-2> and Examples). <4-4>), the recombinant vaccine of the present invention was confirmed to exhibit a complete protective effect against IBDV in SPF system and practical use (see Table 5 and Table 10).

Furthermore, in another embodiment of the present invention, as a result of confirming that the recombinant vaccine of the present invention has a protective effect against heterologous IBDV (see Example 4-4), the recombinant vaccine of the present invention is directed to IBDV P4 which is heterologous IBDV. It was also confirmed that it has a perfect defense effect (see Table 8).

As described above, the recombinant vaccine of the present invention has several advantages in addition to excellent protective effect. First, it eliminates the risk of attenuated vaccine virus or inactivated vaccine. Second, because it consists only of specific proteins that induce neutralizing antibodies, the protein that suppresses the immune system possessed by the unique IBDV was completely removed. Third, the baculovirus expression system used in this study when new mutants occurred. Fourth, unlike the E. coli expression system, it is possible to generate IBDV-specific proteins because of its transcription or post-translational modification. Fifth, because it uses cell lysates, It is economical because there is no need for refining process and it is effective in small amount.

The vaccine composition for preventing infectious F cystic disease of the bird of the present invention may be administered alone, but preferably, is administered with an adjuvant.

Adjuvant is a substance that promotes an immune response to an antigen in a specific way during the initial activation of immune cells, and means an agent, a molecule, etc. that enhances immunity by increasing the activity of cells of the immune system, although it is not an immunogen to the host (Warren et. al., 1986, Annu. Rev. Immunol ,. 4: 369). Adjuvants that can be administered with the compositions of the present invention to enhance an immune response include any of a variety of adjuvants, and typical adjuvant includes Freund adjuvant, aluminum compound, muramyl The dipeptide (muramyl dipeptide), lipopolysaccharide (LPS), monophosphoryl lipid A, or kuyl A, etc. In the present invention, monophosphoryl lipid A was used as an adjuvant. It may be administered simultaneously with the prophylactic composition or sequentially at intervals of time.

The compositions of the present invention are formulated into suitable formulations with acceptable pharmaceutical carriers and administered by a variety of routes. The route of administration may be oral, intraperitoneal , intramuscular, subcutaneous, intradermal, topical, nasal, pulmonary, rectal. Preferably, it is formulated as an injection and administered by a method such as intramuscular injection, subcutaneous injection or intradermal injection. Injectables include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizer (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, microbial development Preservatives (for example, phenyl mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The composition of the present invention is administered in an immunologically effective amount. The term "immunologically effective amount" means an amount sufficient to produce a prophylactic effect and an amount not to cause side effects or serious or excessive immune responses, the exact dosage of which depends on the particular immunogen to be administered, from one to Multiple administrations are possible. In addition, the vaccine composition of the present invention can be administered in combination with a known vaccine. Preferably, the vaccine composition of the present invention may be inoculated 2-3 times at intervals of 3-4 weeks at a concentration of 10 ⁴ cells / 0.5 ml to 10 ⁵ cells / 0.5 ml per allowance.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1>

Cloning of the VP2 and VP243 Genes

<1-1> Viral RNA Extraction and Purification

RNA of IBDV was isolated according to known methods (Kwon et al, Avian Dis, 44: 691-696, 2000). The IBDV used in the experiment was obtained from the National Veterinary Research and Quarantine Service by vvIBDV (SH / 92) isolated from Korea in 1992. In this case, for comparison with the vvIBDV (SH / 92), domestic isolate vvIBDV 1030 (Seoul National University College of Veterinary Medicine) and vaccine strain Blen (Merial Select Inc), Bursine plus (Fort Dodge Animal Health, USA), Bur-706 (Attenuated vaccine strain, Merial), Bursine-2 (Intermediate vaccine strain, Fort Dodge Animal Health) and CEVAC (Intermediate plus vaccine strain, Sanofi) were used. The same amount of chloroform was added to the vvIBDV (SH / 92), and the mixture was mixed up and down five times. After centrifugation at 12,000 rpm for 2 minutes, the supernatant was transferred to a new tube. Sodium dodecyl sulfate; SDS, final concentration of 2% (w / v)] and proteinase K (proteinase K, final concentration of 250 µg / ml), followed by reaction at 55 ° C. for 5 minutes, followed by phenolic acid (acid phenol, pH 4.0). ) And chloroform: isoamyl alcohol (chloroform: isoamyl alcohol, 49: 1). The extracted RNA was purified according to the experimental method provided using the RNaid kit (BIO101). The purified RNA was mixed with 2x anhydrous ethanol and then left at -70 ° C for 3-4 hours or overnight, and then centrifuged at 4 ° C and 12,000 rpm for 25 minutes. After removing the supernatant, the precipitated RNA pellet was washed with 1 ml of ice-cold 70% ethanol, dried over Speed-Vac, and dissolved in 90% dimethyl sulphoxide (DMSO). After 5 minutes at 98 ℃ isolated RNA was treated for 2 minutes or more immediately on ice to pre-denaturate (double-strand RNA virus) and stored at -20 ℃ for RT-PCR Used as a template.

<1-2> Preparation of Primer

Based on the base sequence of GenBank accession no (D00499) of IBDV standard strain (STC), a number of forward and reverse primers suitable for amplifying VP2 gene and VP243 (VP2-VP4-VP3) gene were prepared. Especially taking into account that be cloned into the pBacPAK8 transfer vector to the baculovirus virus (Baculovirus transfer vector) tetragonal, wherein the primer is a reverse primer the Kpn I restriction site of the VP2 gene have been produced by inserting a EcoR I restriction site. And a forward primer of VP243 gene has reverse primer has a Kpn I site was constructed by inserting a Not I site. Primers used for RT-PCR of the present invention are shown in Table 1.

Primer sequence used for RT-PCR primer order Remarks SEQ ID NO: IBDVP2-F1 5'-AGCT GGTACC ATG ACAAACCTG-3 ' Forward primer ( Kpn I site) for amplification of VP2 or VP243 One IBDVP2-F2 5'-ATAT GGTACC AAACGATCGCAGCGATGACAAAC-3 ' Forward primer ( Kpn I site) for amplification of VP2 or VP243 2 IBDVP2-R1 5'-GACT GAATTC TCA AGCTTTTCCTGA-3 ' Reverse primer for amplification of VP2 ( EcoR I site) 3 IBDVP2-R2 5'-GACT GAATTC TCATGCTCTTGCTTTTCCTGACG-3 ' Reverse primer for amplification of VP2 ( EcoR I site) 4 IBDVP243-R1 5'-TAA GCCGCCGC TTTGGGATGTTGT-3 ' Reverse primer for amplification of VP243 ( Not I site) 5 IBDVP243-R2 5'-ATTA GCGGCCGCC AATTTGGGATGTTGTAAGG-3 ' Reverse primer for amplification of VP243 (Not I site) 6

<1-3> RT-PCR

5 μl of random primer, 0.5 mM deoxynucleotide triphosphates (dNTPs) and 5 μl of sterile distilled water (DDW) were added to 5 μl of IBDV RNA extracted in Example <1-1> and reacted at 65 ° C. for 5 minutes. After the reaction, 5X buffer, 0.005M dithiothreitol (DTT), and 40 U of RNasin (Invitrogen) were added and reacted at 25 ° C for 10 minutes and at 42 ° C for 2 minutes. Then add 200 U of Superscript ^TM Ⅱ RNase H-RT (Invitrogen), react at 50 ° C. for 50 minutes at 42 ° C., and 15 minutes at 70 ° C., and add 2 U of ribonuclease H (Invitrogen). IBDV cDNA was synthesized by reaction at 20 ° C. for 10 minutes at 85 ° C.

PCR was performed using 5 μl of 10 × PCR buffer, 2.5 mM of MgCl ₂ , 0.4 mM of dNTPs, 20 pmole primer, and 2U Expand High Fidelity PCR System (Roche) at a total volume of 50 μl. After adding 3 μl, initial denaturation was performed at 95 ° C. for 5 minutes, and the procedure was repeated 35 times for 30 seconds at 95 ° C., 30 seconds at 51 ° C., 2 minutes at 72 ° C., and final elongation at 72 ° C. for 7 minutes. Was carried out. When amplifying VP243, 5 μl of template DNA was added and initial denaturation was performed at 95 ° C. for 5 minutes, and the procedure was repeated 37 times for 30 seconds at 95 ° C., 30 seconds at 51 ° C., and 4 minutes at 68 ° C. Stretching was performed at 68 ° C. for 15 minutes. The amplified PCR product was observed after electrophoresis on 1% agarose gel containing ethidium bromide (10 mg / ml, Sigma), and λ Hind III ladder (GibcoBRL) was used as a marker.

As a result, when the VP2 gene was amplified by RT / PCR, PCR products of about 1,540bp were generated in SH / 92 isolates and three vaccine strains (Bur-706, Bursine-2, and CEVAC) (FIG. 1). When the VP243 gene was amplified by RT / PCR, PCR products of 3,148bp size were detected in domestic isolates SH / 92, vaccine Bursine2, and CEVAC strains, and very weakly in 1030 strains (FIG. 2).

<1-4> Cloning of the VP2 and VP243 Genes

The VP2 and VP243 genes amplified by RT-PCR were electrophoresed on a 1% agarose gel, and then the bands displayed on the gel were cut and DNA was extracted using a Geneclean turbo kit (BIO 101). . In order to identify the nucleotide sequences of the amplified VP2 and VP243 genes, the DNA was cloned into PCR 2.1 TOPO vector (Invitrogen) and analyzed for nucleotide sequences. After analysis of the nucleotide sequence was cloned into PCR 2.1 TOPO by the VP2 and VP243 gene inserted into a vector treated with Kpn I and EcoR Ⅰ or Kpn I and Not Ⅰ restriction enzyme after pBacPAK8 vector treated with the same restriction enzyme (Clontech) . After selecting clones that were exactly recombinant plasmids and incubating them in 1L LB broth, a large amount of recombinant DNA was obtained using the QIAfilter ^TM plasmid maxi kit (QIAGEN), and λ Hind was used to determine the concentration of plasmid DNA. DNA concentration was determined by electrophoresis with III to compare densities, and used as a material for transfection. In addition, the nucleotide sequence near the restriction enzyme was analyzed to determine whether it was inserted correctly. Sequencing was performed using DNASIS version 7.0 software (Hitachi software engineering Co.). The base and amino acid sequences determined were compared with the previously published IBDV.

The base sequence and amino acid sequence of the VP2 gene of vvIBDV SH92 strain are as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and the base sequence and amino acid sequence of the VP243 gene are as shown in SEQ ID NO: 9 and SEQ ID NO: 10. In addition, the homology between the nucleotide sequence and amino acid sequence of the VP2 gene of vvIBDV SH92 strain was compared with that of the known IBDV sequence. It showed homology of ˜96.5% (FIG. 3).

<Example 2>

Expression of VP2 and V243 Recombinant Proteins Using a Baculovirus Expression System

<2-1> Sf Culture of 21 Cells

Expression of the structural proteins VP2 and VP243 genes of IBDV was carried out in accordance with the method provided by the manufacturer using a baculovirus expression system (Baculovirus expression system, Clontech and Invitrogen). The culture of baculovirus used Spodoptera frugiperda 21, Sf 21 cells. The cells were grown in Grace's insect medium (GibcoBRL) with 1% antibiotics (100X antibiotics-antimicotics, GibcoBRL) and 10% fetal bovine serum (FBS, GibcoBRL). Incubated at 27 ° C.

<2-2> Cotransfection

Sf 21 cells were co-transfected with pBacPAK8 transfer vector plasmid with IBDV VP2 and VP243 genes and linear baculovirus DNA. That is, Sf 21 cells cultured in Example <2-1> were dispensed into 2 × 10 ⁶ cells in three 60 mm tissue culture plates so that the cell density was 50-70%. . The Sf 21 cells were then incubated at 27 ° C. in Grace's insect medium (GibcoBRL) with 10% immobilized FBS and antibiotics. Then, 16 μl (1 μg / μl,> 4 μg) of pBacPAK8 transfer vector plasmid DNA into which the VP2 or VP243 gene of IBDV prepared in Example <1-4> was inserted, 10 μl of linear AcMNPV (Invitrogen) DNA, 1 ml of Grace's insect medium (no FBS) and 20 µl of Cell infection reagent were added to a microcentrifuge tube, mixed for 10 seconds, and allowed to stand for 15 minutes to prepare a DNA mixture. Meanwhile, the medium of Sf 21 cells to be transfected was discarded, washed three times with Grace insect medium without FBS, and the medium was completely removed. The prepared DNA mixture was slowly added in 3-5 drops. Four hours later, fresh Grace insect medium with FBS was added. In this case, cells not inoculated with the virus were used as a negative control, and cells inoculated with 10 μl of wild-type AcMNPV virus were used as the positive control. After transfection, the cells were cultured at 27 ° C. for 4 days to compare the shape of the transfected Sf 21 cells under a microscope.

Experimental results showed that the positive control group inoculated with AcMNPV virus formed many occlusion bodies by the polyhedrin gene of the virus, whereas in Sf 21 cells transfected with recombinant plasmid containing VP2 or VP243 Unlike normal Sf 21 cells, the morphology of the cells was observed to be deformed and have an irregular shape (FIG. 4).

<2-3> Identification of expressed protein using immunofluorescent antibody method

In order to confirm the expression of recombinant protein VP2 or VP243 it was confirmed by the following method. After dispensing one sterilized cover glass per well into a 6 well plate, Sf 21 cells were dispensed at about 2.5 × 10 ⁵ cells / 2 ml per well. After incubation at 27 ° C. for 2 hours, the medium was removed, and then inoculated into each well with BacPAK6 virus or 10-fold diluted recombinant baculovirus VP2 and VP243, followed by adsorption at room temperature for 1 hour. At this time, as a negative control, only medium was adsorbed. After removing the virus solution, 2 ml of Grace's medium was dispensed for each well and cultured at 27 ° C. for 3-4 days to observe cytopathic effect (CPE). IFA staining of Sf 21 cells was performed when CPE was observed. Sf 21 cells were removed from the attached cover glass (dry) at room temperature for 20 minutes and fixed for 10 minutes with ice-cold 100% methanol. Methanol was removed, dried, and reacted at 37 ° C. for 1 hour after the addition of IBDV VP2 monoclonal antibody or positive antibody diluted 500-1000 times with PBS (phosphate buffered saline). The IBDV positive sera used was measured by ELISA in serum collected from the group recovered from IBDV infection, and the sera used after partial infection were used. Monoclonal antibodies were three types of monoclonal antibodies (R63, B29, B69) was used. Anti-mouse IgG-FITC conjugate (anti-chicken IgG-FITC conjugate) diluted 500-1000 fold after 3-5 washes with PBS or anti-chicken IgG-FITC conjugate, After addition of Sigma), the reaction was carried out at 37 ° C for 1 hour. After washing 3-5 times with PBS to remove the secondary antibody, the solution was completely removed, the mounting buffer (mounting buffer) was accumulated on the cover glass and placed on a slide and photographed with a fluorescence microscope.

As a result, strong fluorescence was observed in Sf 21 cells infected with the recombinant BacPAK8 plasmid carrying the VP2 or VP243 gene and the fluorescence was evenly distributed throughout the protein-expressed cells. On the other hand, Sf 21 cells, the Sf 21 cells were infected with only Sf 21 cells infected with AcMNPV and BacPAK6 were not observed at all a specific fluorescence (Fig. 5).

<2-4> Identification of the expressed protein by SDS-PAGE and Western blot

In Example <2-2>, the cell culture medium having the cytopathic effect (CPE) confirmed after transfection was collected, and then the cells and the supernatant were separated by centrifugation and expressed through SDS-PAGE and Western blot hybridization. Protein was identified. To more easily identify the expressed protein, Sf21 cells were cultured in two F150 flasks for mass cultivation of the virus and recombinant VP2 virus and recombinant VP243 virus were detected at 80% confluency. The cells were harvested at 4 days after infection and incubation at 27 ° C. After separating the cells and the supernatant, the cells were washed twice with PBS, concentrated for 1/10 and left on ice using a microtip probe, using sonication (40-60% power, 1min tuch, 3times, soniprep). Each sample was mixed with 3X SDS-PAGE loading buffer [6% SDS, 10% 2-mercaptoethanol, 60% glycerol, 0.3M Tris (pH6.8), 0.06% bromophenol blue] and then at 100 ° C. After boiling for 5 minutes, electrophoresis was performed. After electrophoresis, the gel was blotted onto a nitrocellulose membrane and then the membrane was blocked with 5% scheme milk. Monoclonal antibodies (100-fold dilution) or polyclonal antibodies (1000-fold dilution) were used as primary antibodies. The polyclonal antibody was measured by ELISA of serum collected from the group recovered from IBDV infected and recovered, and the serum used after partial infection was used as the monoclonal antibody, monoclonal antibody B29 distributed by Genobiotech. HRP-conjugated anti-mouse IgG (or whole molecule), or anti-chicken IgG (secondary molecule) was used as a secondary antibody, and the membrane was washed and then detected by adding 4-chloro-1-naphthol.

As a result, Western blot analysis using IBDV positive serum showed that 45 KDa VP2 protein was detected in both supernatant and cell lysate (FIG. 6A). In the supernatant, the intensity of the band was observed faintly compared to the cell lysate. In contrast, no similar bands were observed in the supernatants and cell lysates of mock-infected cells and BacPAK6 infected cells. Bands similar in size to VP3 were observed in both supernatants and cell lysates infected with VP2 and VP243 recombinant baculosviruses in the vicinity of about 32 KDa. On the other hand, when Western blot analysis was performed using monoclonal antibody B29, a 32KDa size band was observed only in cell lysates of transfected cells with VP243 (FIG. 6B).

<2-5> dot Blot  Hybridization method Dot blot hybridization Analysis of expressed protein using

Dot blot hybridization was performed to observe the expression level of the recombinant protein according to the culture time. After inoculating baculoviruses containing VP2 and VP243 genes into Sf 21 cells grown in 6-well plates, incubating for 1, 2, 3, and 4 days, and then harvesting the supernatants and cells in each well. After centrifugation at 3,000 rpm for 10 minutes, the supernatant and the cells were harvested separately. Precipitated cells were suspended in 0.5 ml PBS (0.15M, pH7.2) and left on ice and then subjected to sonication (40-60% power, 1min tuch) using a microtip probe. , 3times, soniprep, manufacturer). Each cell lysate was adsorbed onto a nitrocellulose membrane using a dot blotter and detected by the same method as Western blot hybridization.

As a result, hybridization using IBDV positive serum as the primary antibody showed the highest expression level of VP2 or VP243 on day 4 of culture (A and B of FIG. 7). On the other hand, when hybridized using monoclonal antibody B29 as the primary antibody, only VP243 was observed to have the highest expression level on day 4 of culture (FIG. 7C).

<Example 3>

Preparation of Genetically Recombinant IBDV Vaccine

In order to mass culture the recombinant IBDV vaccine, the F150 flask was inoculated with Sf 21 cells at 6fla10 ⁶ cells / flask, followed by incubation at 27 ° C. Cells were harvested when cytotoxicity was observed after infecting each flask with the recombinant VP2 virus or recombinant VP243 virus prepared in Example <2-2> at the time of 80% confluency. The cytopathic effect was observed in the same manner as in Example <2-2>. The cells harvested above were centrifuged at 3,000 rpm for 10 minutes, then the supernatant was discarded. The cell pellet was washed once with sterile 0.15 M PBS (pH 7.2) and then suspended in 4 ml PBS again and then iced. The microtip probe was used to perform sonication ((40-60% power, 1min tuch, 3times, soniprep) in the state of standing in. Then, the supernatant was harvested by centrifugation at 1500rpm for 5 minutes. 3 ml of the harvested supernatant and 7 ml of MONTANIDE ISA70 (SEPPIC) were mixed, passed through 50 times using a 17 gauge needle, and used as a vaccine while being stored at 4 ° C.

<Example 4>

Determination of the Protective Effect of Recombinant IBDV Vaccine

<4-1> Effect of Recombinant Vaccine on Inoculation

According to the invention To measure the protective effect of the recombinant IBD vaccine, the vaccine was challenged with the same strong pathogenic IBDV after vaccination in SPF chickens, followed by mortality, B / B ratio, B / B ratio less than 2, and antibody against IBDV. , F size, histological lesions were analyzed.

The recombinant vaccine VP243 prepared in Example 3 was inoculated subcutaneously at 3 and 4 weeks by dividing the inoculation amount of the allowable recombinant VP243 vaccine into 2 × 10 ⁴ , 2 × 10 ⁵ and 2 × 10 ⁶ cells / dose, respectively. At this time, the volume of one dose was adjusted to 0.5 ml. Two weeks after the second vaccination, vvIBDV SH / 92 (available from the National Veterinary Medical Quarantine Service) was challenged orally by 1 × 10 ^4.8 EID ₅₀ / water. As a control group, there was a negative control group that was not vaccinated and not challenged at all, and a challenge control group which had not been vaccinated but received only vaccination. . Two weeks after the second inoculation, IBDV SH / 92 strains were challenged with 1 × 10 ^4.8 EID ₅₀ / male oral and mortality was observed for 10 days. Each experimental group and control group are shown in Table 2.

Experimental group and control group group Vaccination week Number of chickens used in experiment Negative control 3, 4 (PBS inoculation) 7 Attack group 3, 4 (PBS inoculation) 7 VP243 (2 × 10 ⁴ cell) 3, 4 7 VP243 (2 × 10 ⁵ cell) 3, 4 7 VP243 (2 × 10 ⁶ cell) 3, 4 7

Whole blood was collected through the jugular vein before challenge challenge and 10 days after challenge challenge to determine antibody titers to IBDV from each experimental and control group. The antibody titer against IBDV was measured by ELISA kit (IDEXX, USA), and it was determined that the antibody titer against IBDV is 396 or more. In addition, the chickens of all groups that survived without death were euthanized by cervical spinal dislocation method and the body weight and F-segment weight were measured to investigate the B / B ratio. That is, all live chickens were culled, and then the weight and weight of the F sac were measured, and then the B / B ratio [(F sac weight) / (weight) × 1000] was calculated and the number of B / B ratios less than 2 was measured. . Individuals with a B / B ratio <2 are evidence of atrophy of the F capsule and indirectly indicate that the inoculated virus is present in the F capsule (Martinez-Torrecuadrada et al., Vaccine, 21: 3342-3350, 2003) . The extracted F sac was fixed in 10% formalin solution, and then stained with hematoxylin-eosin (H-E) to observe histological changes.

The results of all experiments were analyzed using Statistical package SAS 8.01 (SAS Institute, 2000). One-way ANOVA and Tukey's tests were used to assess the difference between B / B ratio and ELISA antibody titer. It was. The significance was judged as P <0.05.

As a result, the survival rate up to 10 days after vaccination showed 80% mortality in the vaccination group but no death in the vaccination group (Table 3).

In addition, the B / B ratio was similar in the vaccinated group and the negative control group regardless of the inoculation amount of the VP243 recombinant protein (P <0.05). As a result of measuring the population with a B / B ratio of less than 2, the surviving group also severely contracted one survivor. However, in the vaccinated group, no individuals with B / B ratio less than 2 were observed regardless of the inoculation amount (Table 3 and FIG. 8).

As a result of measuring antibody titer for IBDV, almost no antibody was detected before challenge in the challenge group, but the antibody titer surviving after challenge was 26,603, which was high. The group inoculated with VP243 protein showed high titers of 10,769-13,386 prior to challenge and high antibody titers of 11,847-16,417 after challenge. Attack significant difference in titers of the inoculation before inoculum size is did not indicate inoculation after which the the 2 × 10 ⁴ cells / high Antibody able than the group inoculated in an amount of 2 × 10 ⁶ cells / dose a group significance inoculated with a dose VP243 Is shown (Table 3).

Taken together, no significant differences were observed in the protective effect according to the dose. Therefore, after the experiment, the inoculation amount of VP2 or VP243 was uniformized to 2 × 10 ⁴ cells / dose to analyze the protective effect.

Protective Effect of Recombinant Vaccine VP243 on Different Inoculation Volumes group Mortality (%) B / B ratio ¹⁾ Number of chickens with B / B ratio less than 2 ELISA antibody ¹⁾ Before inoculation After challenge Negative control 0/5 (0) 5.37 ± 0.84 ^a 0/5 (0) <396 ^a (0/5) ²⁾ <396 ^a (0/5) Attack group 4/5 (80) 1.27 ^b 1/1 (100) <396 ^a (0/5) 26603 ^b (1/1) VP243 (2 × 10 ⁴ cells) 0/5 (0) 5.55 ± 1.10 ^a 0/5 (100) 10769 ± 2849 ^c (5/5) 11193 ± 2028 ^c 5/5) VP243 (2 × 10 ⁴ cells) 0/5 (0) 5.36 ± 0.86 ^a 0/5 (100) 12677 ± 3419 ^cd (5/5) 11847 ± 4556 ^cd (5/5) VP243 (2 × 10 ⁶ cells) 0/5 (0) 4.38 ± 0.83 ^a 0/5 (100) 13386 ± 632 ^cd (5/5) 16417 ± 964 ^d (5/5)

1) Mean ± Standard Deviation

2) Number of positive serum samples / number of tested serum samples

* Different characters mean that there is a significant difference at p <0.05

<4-2> SPF ( Specific Pathogen Free ) From chicken  Recombinant vaccines VP2  And V243 Measures the effectiveness of

In order to confirm the protective effect of the recombinant vaccines VP2 and VP243 against IBDV, the VP2 and VP243 recombinant vaccine prepared in Example 3 (2 × 10 ⁴ cells / dose / 0.5ml) were used in SPF chickens at 6 and 7 weeks, respectively. 0.5 ml each was inoculated subcutaneously. As a positive control, a commercially available attenuated bursine-2 vaccine (Fort Dodge Animal Health) was administered orally twice at the same age to the recommended dose. As a control group, there was a negative control which had not been vaccinated and challenged at all, and a challenge control group which had not been vaccinated but received only vaccination, and they received 0.5 ml of PBS instead of vaccine. Subcutaneous inoculation. Two weeks after the second vaccination, vvIBDV SH / 92 was orally challenged with 1 × 10 ^4.8 EID ₅₀ / water for 10 days and mortality was observed. Each experimental group and control group are shown in Table 4. The antibody to IBDV, the B: B ratio and the observation of histological changes of the F capsule were measured in the same manner as in Example <4-1>, and the experimental results were statistically treated in the same manner.

Experimental group and control group group Vaccination week Number of chickens used in experiment Negative control 6, 7 (PBS inoculation) 3 Attack group 6, 7 (PBS inoculation) 10 VP2 6, 7 10 VP243 6, 7 10 Bursine-2 6, 7 9

As a result, mortality up to 10 days after vaccination showed 90% (9/10) mortality in the vaccination group, whereas no mortality occurred in the group vaccinated with Bursine-2, recombinant VP2 vaccine and recombinant VP243 vaccine. It did not appear (Table 5).

The B / B ratio of the groups vaccinated with the recombinant VP2 vaccine and the recombinant VP243 vaccine was similar to that of the negative control group. These results indicate that SPF chickens vaccinated with the recombinant VP2 vaccine and recombinant VP243 vaccine had little damage despite the challenge of vvIBDV. On the other hand, the B / B ratios of the challenge and Bursine-2 inoculated groups were similar. In addition, the group vaccinated with Bursine-2 was observed similar survival rate compared to the recombinant vaccinated group of the present invention, but the F bag was shown to be damaged by the challenge virus (Table 5).

Histopathologic findings showed no B / B less than 2 in the negative control, recombinant VP2 and VP243 vaccine groups, but 100% in the challenge group and 55.6% in the Bursine-2 inoculation group. Was observed. This suggests that the vaccinated F capsule was contracted by the vaccinated vvIBDV in the challenge group and the positive control group, and the presence of vvIBDV in the contracted F capsule. In particular, in challenge group, atrophy of excretory cavity, increase of intervesicular settlement tissue, increase of RE cells in medulla, cell necrosis and phagocytosis, bleeding of superficial epithelial cells were observed. In Bursine-2 vaccination group, these findings were partially weak or moderate (Table 6). However, in the group vaccinated with the recombinant VP2 and VP243 vaccines, little or no such finding was observed (FIG. 9). From the above experimental results, it was confirmed that the recombinant VP2 and VP243 vaccines of the present invention have excellent protection against IBDV.

As a result of measuring the titer of IBDV in each experimental group and control group, almost no antibody was detected prior to challenge, whereas one antibody surviving after challenge was 21,524. The recombinant VP2 vaccination group had moderate titers of 1,514 before challenge and 2,218 after vaccination. Recombinant VP243 vaccination showed the highest antibody titer of 9,196 before challenge and decreased titer after vaccination. Bursine-2 vaccination group showed higher titers similar to VP243 inoculation group and decreased in titer after challenge (Table 5).

In conclusion, the positive control group, Bursine-2, inhibited chicken mortality, but the B / B ratio was significantly similar to that of challenge group. The presence of IBDV in the sac was confirmed indirectly. Therefore, it was confirmed that Bursine-2, which has been put into practical use, does not completely defend against vvIBDV. In comparison, the vaccine composition of the present invention was confirmed to exhibit a complete protective effect.

Protective Effect of Recombinant VP2 and VP243 Vaccines against vvIBDV SH / 92 group Mortality (%) B / B ratio ^{1 )} Number of chickens with B / B ratio less than 2 ELISA antibody ¹⁾ Before inoculation After challenge Negative control 0/3 (0) 4.04 ± 1.00 ^a 0/10 (0) <396 ^a (0/3) ²⁾ <396 ^a (0/3) Attack group 9/10 (90) 1.03 ^b 1/1 (100) <396 ^a (0/10) 21524 ^b (1/1) VP2 inoculation group 0/10 (0) 4.69 ± 1.13 ^a 0/10 (0) 11346 ± 2363 ^d (10/10) 12756 ± 1609 ^cd (10/10) VP243 inoculation group 0/10 (0) 4.63 ± 1.04 ^a 0/10 (0) 13412 ± 1432 ^cd (10/10) 14003 ± 1226 ^c (10/10) Bursine-2 inoculation group 0/9 (0) 1.87 ± 1.14 ^b 5/9 (55.6) 2904 ± 1835 ^e (9/9) 3832 ± 2706 ^e (9/9)

1) Mean ± Standard Deviation

2) Number of positive serum samples / number of tested serum samples

* Different characters indicate a significant difference at p <0.05.

Histopathological Findings in Experimental and Control Groups group Negative control VP2 inoculation group VP243 inoculation group Bursine-2 inoculation group Case No. One 10 10 9 Defecation atrophy 1/1 (100%) 0/10 (0.0%) 0/10 (0.0%) 7/9 (77.8%) Very weak 1/9 weak 1/9 Secondary 4/9 Severe 1/1 1/9 Intervesicle connective tissue 1/1 (100%) 0/10 (0.0%) 0/10 (0.0%) 7/9 (77.8%) Very weak 2/9 weak 4/9 Secondary 1/9 Reticuloendothelial proliferation 1/1 (100%) 10/10 (100%) 10/10 (100%) 9/9 (100%) Very weak 2/10 weak 8/10 10/10 2/9 Secondary 7/9 Severe 1/1 Necrosis and phagocytosis of lymphocytes 1/1 (100%) 10/10 (100%) 10/10 (100%) 0/9 (100%) Very weak 4/10 weak 6/10 10/10 5/9 Secondary 4/9 Severe 1/1 Proliferation of superficial epithelial cells 1/1 (100%) 0/10 (0.0%) 0/10 (0.0%) 8/9 (88.9%) weak 3/9 Secondary 1/1 5/9 bleeding 0/10 (100%) 0/10 (0.0%) 0/10 (0.0%) 1/9 (11.1%) Secondary 1/9

<4-3> Effect of Recombinant Vaccine on Heterologous IBDV

It was confirmed that the recombinant vaccine of the present invention has a protective effect against other types of IBDV other than the vvIBDV SH / 92 strain. To this end, the recombinant VP2 vaccine and the VP243 vaccine of the present invention were inoculated subcutaneously by 0.5 ml at 3 weeks and 4 weeks for SPF chickens aged 3 weeks. As a positive control, a commercially available inactivating vaccine, IB + G + ND mixed oil vaccine (Intervet), was inoculated into muscle twice at the same age of 0.5 ml twice. As a control group, there was a negative control group that was not vaccinated and not challenged at all, and a challenge control group which had not been vaccinated but received only vaccination, and subcutaneously received 0.5 ml of PBS per dose instead of the vaccine. It was. In addition, after transfection of BacPAK6 to Sf 21 cells in the same manner as the recombinant VP2 vaccine and VP243 vaccine of the present invention, cell lysates were extracted and inoculated into chickens of the same age. One week after the second vaccination, IBDV P4 strain (prepared from the National Veterinary Research and Quarantine Service) (1 × 10 ^4.8 EID ₅₀ / water) was orally challenged with 100 μl of mortality and observed mortality for 10 days. The antibody to IBDV, the B / B ratio and the histological change of the F capsule was measured in the same manner as in Example <4-1> and the experimental results were statistically treated in the same manner. Each experimental group and control group are as shown in Table 7.

Experimental group and control group group Vaccination week Number of chickens Negative control 3, 4 (PBS inoculation) 10 Inoculation Control 3, 4 (PBS inoculation) 10 BacPAK6 inoculation group 3, 4 10 VP2 inoculation group 3, 4 10 VP243 inoculation group 3, 4 10 IB + G + ND Inoculation Group 3, 4 10

As a result, mortality up to 10 days after challenge was shown to be 10% and 30% mortality in the inoculated control group and BacPAK6 inoculated group, respectively, whereas IB + ND + G oil vaccine, recombinant VP2 vaccine and recombinant VP243 vaccine were inoculated. One group had no mortality (Table 8).

In addition, after the euthanasia of all the surviving chickens without euthanasia by cervical spinal bone dissection, the B / B ratio was measured by measuring the body weight and F-segment weight, and the B / B ratio of the group vaccinated with the recombinant VP2 and VP243 vaccines. Was found to be similar to the negative control. The positive control group also showed similar B / B ratio. The B / B ratios of the challenge and BacPAK6 inoculated groups were similar, but these values were much lower than those in the negative control, positive control, VP2 or VP243 groups. As a result of observing populations with a B / B ratio of less than 2, 100% of the inoculated and BacPAK6 inoculated groups were found to have contracted all of the surviving chicken F capsules. Appeared (FIG. 10). However, in the group inoculated with VP2 and VP243, no individuals with B / B ratio less than 2 were observed. There was no difference in mortality and B / B ratio between the recombinant vaccines VP2 and VP243.

In the inoculated group, almost no antibody was detected before vaccination, whereas the mean antibody titers surviving after vaccination were 22,965, which was the highest in all groups. Recombinant VP2 vaccination group showed 1,1470 before challenge and 15,631 after vaccination showed an increase in titer. Recombinant VP243 vaccination group showed a lower antibody titer of 7,900 before vaccination than VP2 vaccination group, but increased titer (16,653) after vaccination. In the IB + G + ND vaccinated group, the titer was similar to that of the BacPAK6 vaccinated group and the VP243 vaccinated group before challenge, and the titer increased after challenge. Therefore, the recombinant VP2 and VP243 vaccines of the present invention were confirmed to have an excellent protective effect against heterologous IBDV.

Overall, the protective effect of the recombinant vaccines VP2 and VP243 of the present invention was compared with the IB-G-ND mixed oil vaccine used as the inactivating vaccine control group, but showed similar effects in mortality and B / B ratio. The B / B ratio was less than 2% and 20% showed that it did not completely defend against atrophy of F capsule. Therefore, it can be seen that the protective effect against heterologous IBDV is more effective than the recombinant vaccine of the present invention.

Protective Effect of Recombinant VP2 and VP243 Vaccines against vvIBDV P4 group Mortality (%) B / B ratio ¹⁾ Number of chickens with B / B ratio less than 2 ELISA Antibodies ^{1 )} Before inoculation After challenge Negative control 0/10 (0) 4.37 ± 0.73 ^a 0/10 (0) <396 ^a (0/10) ²⁾ <396 ^a (0/10) Attack group 1/10 (10) 1.32 ± 0.20 ^b 9/9 (100) <396 ^a (0/10) 22956 ± 6152 ^b (9/9) BacPAK6 inoculation group 3/10 (30) 1.32 ± 0.34 ^b 7/7 (100) 3510 ± 1250 ^e (10/10) 22124 ± 3951 ^b (7/7) VP2 inoculation group 0/10 (0) 4.91 ± 0.78 ^a 0/10 (0) 11470 ± 2430 ^cd (10/10) 15631 ± 8292 ^bc (10/10) VP243 inoculation group 0/10 (0) 5.37 ± 1.41 ^a 0/10 (0) 7900 ± 4197 ^de (10/10) 16653 ± 2481 ^bc (10/10) IB + G + ND Inoculation Group 0/10 (0) 3.87 ± 1.86 ^a 2/10 (20) 3983 ± 6476 ^e (10/10) 16642 ± 1451 ^bc (10/10)

1) Mean ± Standard Deviation

2) Number of positive serum samples / number of tested serum samples

* Different characters indicate a significant difference at p <0.05.

<4-4> Protective effect of recombinant vaccine in practical field

In the practical field, the protective effect of the recombinant VP2 and VP243 vaccines of the present invention was investigated. To this end, three vaccinated chicks were inoculated at 3, 5, and 7 weeks of age in hatched chicks on farms receiving IBD vaccine. Negative control group and challenge group were included and they were inoculated subcutaneously with 0.5 ml of PBS. In addition, BacPAK6 was inoculated three times by inoculating BacPAK6 instead of the recombinant vaccine of the present invention, and as a positive control, IB + G + ND, which is a commercial inactivating vaccine, was inoculated three times in the muscle by 0.5ml according to the recommended administration method. Two weeks after the third vaccination, IBDV SH / 92 was orally challenged with 1 × 10 ^4.8 EID ₅₀ / water for 10 days and mortality was observed. The antibody to IBDV, histological changes of B: B ratio and F sac were measured in the same manner as in Example <4-1>, and the experimental results were statistically treated in the same manner. Each experimental group and control group are as shown in Table 9.

Experimental group and control group group Vaccination week Number of chicks Negative control 3, 5, 7 (PBS inoculation) 10 Attack group 3, 5, 7 (PBS inoculation) 10 BacPAK6 inoculation group 3, 5, 7 10 VP2 inoculation group 3, 5, 7 10 VP243 inoculation group 3, 5, 7 10 IB + G + ND Inoculation Group 3, 5, 7 10

As a result, mortality was 50% mortality in challenge group but no death in BacPAK6, IB + G + ND vaccine, recombinant VP2 vaccine and recombinant VP243 vaccine group (Table 10). .

 The B: B ratio was similar for the IB + G + ND vaccinated group, the recombinant VP2 vaccine and the recombinant VP243 vaccinated group, as compared to the negative control group not vaccinated with IBDV. However, the challenge group and the BacPAK6 group were significantly lower than the negative control group (P <0.05). In the group vaccinated with the IB + G + ND vaccine, 10% showed atrophy as a result of confirming the degree of atrophy of F capsule (FIG. 11). This means that F sac was damaged by challenge virus. There was no difference in mortality and B / B ratio between the recombinant VP2 vaccine and VP243 vaccine of the present invention.

As a result of measuring antibody titer, in the inoculation group, almost no antibody was detected before inoculation, whereas the average antibody titers of five survivors after inoculation were 28,351, which is similar to the mean antibody titer of the BacPAK6 group (28,846). Indicated. The recombinant VP2 vaccination group had a high titer of 19,729 before challenge and 16,821 after vaccination. Recombinant VP243 vaccine showed the highest antibody titer (19,899) prior to challenge and increased titer after inoculation. In the IB + G + ND vaccinated group, the titer was lower than the VP2 and VP243 vaccinated groups before challenge, and the titer increased after challenge.

From the above experimental results, it can be seen that the recombinant VP2 vaccine and recombinant VP243 vaccine of the present invention show excellent protective effect in practical applications.

Protective Effect of Recombinant Vaccine in Practical Applications group % Mortality B / B ratio ¹⁾ Number of chickens with B / B ratio less than 2 ELISA Antibodies ^{1 )} Before inoculation After challenge Negative control 0/10 (0) 3.90 ± 0.68 ^a 0/10 (0) <396 ^a (0/10) ²⁾ <396 ^a (0/10) Attack group 5/10 (50) 1.42 ± 0.29 ^b 5/5 (100) <396 ^a (0/10) 28351 ± 5946 ^b (5/5) BacPAK6 inoculation group 0/10 (0) 1.32 ± 0.28 ^b 10/10 (100) 13941 ± 1291 ^d (10/10) 28846 ± 2572 ^b (10/10) VP2 inoculation group 0/10 (0) 4.55 ± 0.67 ^a 0/10 (0) 19729 ± 2004 ^c (10/10) 16821 ± 3748 ^cd (10/10) VP243 inoculation group 0/10 (0) 4.39 ± 0.65 ^a 0/10 (0) 19899 ± 1946 ^c (10/10) 20333 ± 1886 ^c (10/10) IB + G + ND Inoculation Group 0/10 (0) 4.49 ± 1.34 ^a 1/10 (10) 7234 ± 4214 ^e (10/10) 12888 ± 7561 ^d (10/10)

1) Mean ± Standard Deviation

2) Number of positive serum samples / number of tested serum samples

* Different characters indicate a significant difference at p <0.05.

<Example 5>

Comparison of Effect of Recombinant Protein Vaccine and DNA Vaccine of the Invention on vvIBDV SH / 92

The effect of recombinant protein vaccines of the present invention and known DNA vaccines on vvIBDV SH / 92 strains was compared. To this end, the recombinant protein vaccine of the present invention or a known DNA vaccine (Korean Patent No. 568193) is inoculated into SPF chickens and then challenged with the same strongly pathogenic IBDV. Investigate.

The inoculated doses of the recombinant protein vaccines VP2 and VP243 of the present invention were divided into 2 × 10 ⁴ cells / dose and inoculated subcutaneously at 3 and 4 weeks. At this time, the volume of one dose was adjusted to 0.5 ml. In addition, the DNA vaccine was inoculated 100 μg each into muscle and abdominal cavity.

As a result of the experiment, the protective effect of the recombinant protein vaccine of the present invention was found to be superior to the DNA vaccine. In addition, the recombinant protein vaccine of the present invention was confirmed to exhibit this effect in a very small amount (10 ⁴ cells). Therefore, the recombinant protein vaccine of the present invention was found to be very excellent in terms of economics compared to the DNA vaccine.

Comparison of Effect of DNA Vaccine and Recombinant Protein Vaccine of the Invention on vvIBDV SH / 92 group Mortality (%) B / B ratio ¹⁾ ELISA antibody ¹⁾ Before inoculation After challenge Negative control 0/10 (0) 4.04 ± 1.00 ^a <396 ^a (0/10) ²⁾ <396 ^a (0/3) Attack group 9/10 (90) 1.03 ^b <396 ^a (0/10) 21524 ^b (1/1) DNA vaccine VP2 inoculation group 5/10 (50) 2.22 ± 0.76 ^ab <396 ^a (0/10) 4449 ± 2998 ^bc (5/5) DNA vaccine VP243 inoculation group 3/10 (30) 2.22 ± 0.90 ^ab <396 ^a (0/10) 13982 ± 6801 ^cd (7/7) Protein vaccine VP2 inoculation group 0/10 (0) 4.69 ± 1.13 ^a 11346 ± 2363 ^d (10/10) 2756 ± 1609 ^cd (10/10) Protein vaccine VP243 inoculation group 0/10 (0) 4.63 ± 1.04 ^a 13412 ± 1432 ^cd (10/10) 14003 ± 1226 ^c (10/10)

1) Mean ± Standard Deviation

2) Number of positive serum samples / number of tested serum samples

* Different characters indicate a significant difference at p <0.05.

As described above, the vaccine composition of the present invention exhibits a perfect protective effect even at low concentrations, has a better protective effect than the known vaccine composition, and exhibits a protective effect in both SPF (Specific pathogen free) and practical systems. Excellent cross protection against heterogeneous IBDV. In addition, there is an effect that can be easily produced in large quantities without undergoing a separate purification process.

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Claims (9)

Infectious bursal disease virus (IBDV) A vaccine composition for preventing infectious follicular disease of birds comprising a recombinant protein VP2 or VP243 (segment A of IBDV including VP2, VP4 and VP3) derived from SH / 92 virus. The vaccine composition of claim 1, further comprising a pharmaceutically acceptable adjuvant. The method of claim 1, wherein the recombinant protein comprises: (a) preparing an expression vector comprising DNA encoding a VP2 protein or VP243 protein (segment A of IBDV including VP2, VP4 and VP3) of an IBDV SH / 92 virus strain; step; And (b) introducing the expression vector of step (a) into a host cell; And (c) The vaccine composition, characterized in that prepared by the method comprising the step of culturing the host cell of step (b). The vaccine composition according to claim 3, further comprising preparing a cell lysate by crushing the host cell cultured in the step (c). The vaccine composition according to claim 3, wherein the expression vector of step (a) is baculovirus. The vaccine composition according to claim 3, wherein the host cell of step (b) is an insect cell. The vaccine composition of claim 3, wherein the culturing of the host cell in step (c) is performed for 4 days. The vaccine composition of claim 1, wherein said bird is a chicken. The vaccine composition according to claim 1, wherein the birds are inoculated 2 to 3 times at intervals of 3 to 4 weeks at a concentration of 10 ⁴ cells / 0.5ml to 10 ⁵ cells / 0.5ml.

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