KR100701672B1 - Genes of a korean isolate rbiv and a vaccine against rbiv - Google Patents

Abstract

The present invention includes a gene encoding a protein having immunogenicity of Korean sea bream iridovirus, a protein encoded by the gene, a synthetic peptide derived from the protein, a recombinant expression vector including the gene, and the recombinant vector. The present invention relates to a transformant, an inactivated vaccine prepared by inactivating the transformant, and a method of immunizing fish using the vaccine.

Sea bream iridovirus, inactivated vaccine

Description

GENES OF A KOREAN ISOLATE RBIV AND A VACCINE AGAINST RBIV}

1 is a restriction map of a recombinant expression vector pET28a / RBIV-KOR-TY1-B comprising the ORF B gene of RBIV-KOR-TY1.

2 is a SDS-PAGE photograph showing the degree of recombinant protein expression with and without treatment with the expression inducing agent IPTG 1 mM. LM is a low molecular weight size marker, HM is a high molecular weight size marker, lanes 1 to 5 show recombinant protein expression at 0, 2, 4, 6, and 18 hours after IPTG treatment, lanes 6 to 10 shows recombinant protein expression at 0, 2, 4, 6 and 18 hours after untreated IPTG.

Figure 3 is a SDS-PAGE picture showing the expression of recombinant protein by the treatment of the expression inducing agent IPTG. Lane 1 is a high molecular weight protein marker, lane 2 represents the protein expression of normal BL21 E. coli, lane 3 is the expression of recombinant protein when IPTG 1 mM treatment, lane 4 is recombinant protein when IPTG 5 mM treatment Expression, lane 5 is recombinant protein expression when IPTG 10 mM treatment, lane 6 is recombinant protein expression when IPTG 20 mM treatment, lane 7 is recombinant protein expression when 100 μg of IPTG 1 mM and kanamycin treatment, lane 8 Shows recombinant protein expression when 1 mM of IPTG and 100 µg of kanamycin were treated.

4 is a graph showing the effect of the recombinant inactivated vaccine according to the present invention.

The present invention is a rock bream iridovirus (RBIV) that infects domes and causes population death, and genes isolated from the Korean isolate RBIV-KOR-TY1, a recombinant expression vector comprising the genes, and the expression vector. It relates to a transformant comprising, inactivated vaccine prepared by the transformant and the like.

The dome is one of the high-end fish species, and its demand is high, and it is farmed in various parts of Asia including Japan, Korea, and China. In Japan and Korea, the sashimi food preference is high, and the consumption of aquaculture fish is increasing, and the aquaculture industry contributes greatly to the national economy, especially with the increase of exclusive protected areas. In particular, Korea is surrounded by the sea on three sides, and the East Coast, South Coast, West Coast, and Jeju Island have water quality and water temperature suitable for fish farming. However, the occurrence of mass mortality from viral diseases has been a major problem in dome farming.

Each year, tens of millions of domes are killed due to viral diseases caused by Iridovirus infection.In addition, once these viral diseases occur, they are very difficult to treat and economic losses are large. It has a huge economic impact on fish stocks and adversely affects fish stocks.

There are four genera in the genus Iridovirus: genus Iridovirus, genus Chloriridovirus, genus Ranavirus, and genus Lymphocystivirus. Iridoviruses that infect fish are of the genus Lanavirus and Lymphocytivirus. Among these, the iridovirus, which causes massive death of fish, is the iridovirus of the genus Lanavirus.

Iridovirus, which infects kidneys, spleen, nerve cells, or fish immune-related cells such as domes, perch, flounder, and defenses, causes massive mortality of these infected fish, which did not occur in Korea until 1997, Mass mortality of rock domes on caged farms in the South Coast began to occur (Sohn et al. J. Fish Path, 13 (2): 121-127, 2000), which has caused a lot of economic damage to fishermen to date.

The development of the fish Iridovirus vaccine is carried out by inoculating the tissues of fish infected with Iridovirus into GF (grant fin) cells and inoculating them with formalin (Nakajima et, al., DAO, 36). (1): 73-75, 1999), there have been no reports on the development of iridovirus vaccines using genes.

Accordingly, the present inventors have found genes and proteins that can be usefully used for the production of a prophylactic vaccine against iridoviruses, and a recombination vector comprising the genes, and a transformant transformed with the recombinant vector. The present invention was completed by inactivating the recombinant inactivated vaccine.

The present invention solves the problem of securing a high virus inactivation vaccine, a disadvantage of the existing fish iridovirus inactivated vaccine, and securing fish tissues for iridovirus infection for vaccine production due to a decrease in virus concentration according to virus passage, and the Korean-style sea bream iridovirus. An object of the present invention is to provide a prophylactic vaccine against dolphin iridovirus which is excellent in inducing an immune response against dolphin iridovirus, including the isolate RBIV-KOR-TY1.

In addition, the present invention can be used for the production of a prophylactic vaccine against the sea bream iridovirus as described above, iridovirus genes and proteins, peptides derived from the protein, recombinant vector containing the gene, transformation with the recombinant vector It is an object to provide a transformant and a vaccine prepared therefrom.

The present invention encodes the immunogenic proteins of iridovirus, and ORF A, B, C, D, E, F and Korean type of sea bream iridovirus RBIV-KOR-TY1, which can be used for the production of a prophylactic vaccine against iridovirus. Provide the G gene.

In addition, the present invention provides immunogenic proteins ORF A, B, C, D, E, F, encoded by the ORF A, B, C, D, E, F and G genes of the RBIV-KOR-TY1 of the present invention, and Provide G protein.

The invention also provides primer pairs used to clone the ORF A, B, C, D, E, F and G genes of the RBIV-KOR-TY1 of the invention.

The present invention also provides a method for amplifying the ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1 of the present invention.

The present invention also provides a recombinant expression vector comprising each of the ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1.

The present invention also provides a host cell transformed with an expression vector comprising each of the ORF A, B, C, D, E, F and G genes of the RBIV-KOR-TY1.

In addition, the present invention is a recombinant protein A, B, C, D, E encoded by the ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1 from the transformed host cell, respectively Provides a way to produce F and G. More specifically, the present invention In producing the recombinant proteins A, B, C, D, E, F, and G from the transformed host cell, it provides a method for mass production of the recombinant protein by optimizing the concentration and processing time of the expression inducing agent, etc. do.

The present invention also provides recombinant proteins ORF A, B, C, D, E, F, and G produced from the transformed host cell.

The present invention also provides a method for producing an inactivated vaccine by inactivating the transformed host cell of the present invention described above, and an inactivated vaccine produced thereby.

The present invention also provides a method of immunizing fish using an inactivated vaccine prepared by inactivating the transformed host cell described above.

The present invention also provides a kit comprising the inactivated vaccine of the present invention.

The present invention also provides polypeptide fragments that can be used for the production of prophylactic vaccines against iridoviruses.

Hereinafter, the present invention will be described in detail.

According to a first aspect of the present invention, the present invention encodes an immunogenic protein of iridovirus and is isolated from RBIV-KOR-TY1, a Korean sea-bream iridovirus, which can be used for the production of a prophylactic vaccine against iridovirus. ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1, and sequence homologs thereof.

The present inventors confirmed that RBIV-KOR-TY1, a Korean sea-bream iridovirus, contains more than 118 proteins, and has a genomic DNA size of 112,080 bp, which is different from fish iridovirus reported to date. The sequence of genomic DNA was determined and analyzed using a computer program to identify 118 possible ORFs. The amino acid sequences deduced from these ORFs were compared with GenBank's database using the BLAST program, and as a result, 29 ORFs having high homology with known proteins whose function was found to be homologous to the known sequences. 31 ORFs that do not have were identified (Do et al. Virology, 325: 351-363, 2004, incorporated herein by reference in its entirety). Their base sequence information was deposited with GenBank of NCBI under accession No. AY532606.

Based on the sequence and ORF information of the genomic DNA of RBIV-KOR-TY1 as described above, the inventors encode an immunogenic protein of Iridovirus to find a gene of Iridovirus useful for the preparation of a vaccine against Iridovirus. Of the 118 ORFs of KOR-TY1, firstly, a structural protein that is outside of the virus and is likely to induce an immune response of fish, and secondly, a protein expressed on the cell surface and adjacent to the cell where the virus proliferates. A protein that serves to bind tightly between normal cells, and third, a gene of the iridovirus protein that is likely to be present in the outer membrane of the host cell when the iridovirus protein is synthesized in the virus-infected cells.

To this end, the 118 ORF nucleotide sequences are transcribed into amino acid sequences and compared to the amino acid sequences registered to GenBank of the US Biological Information Center (NCBI) to date, seven genes satisfying the three conditions described above are identified. It was selected and named ORF A, B, C, D, E, F and G and these were used for vaccine preparation against the iridovirus of the present invention.

ORF A gene of RBIV-KOR-TY1 of the present invention selected through the above analysis has a nucleotide sequence of SEQ ID NO: 1 in the sequence list, ORF B gene has a nucleotide sequence of SEQ ID NO: 2, ORF C gene Has the nucleotide sequence of SEQ ID NO: 3, ORF D gene has the nucleotide sequence of SEQ ID NO: 4, ORF E gene has the nucleotide sequence of SEQ ID NO: 5, ORF F gene has the nucleotide sequence of SEQ ID NO: 6, ORF G The gene has the nucleotide sequence of SEQ ID NO: 7.

Meanwhile, SEQ ID NO: 1, 2, 3, 4, 5, having one or more base substitutions, deletions, insertions, and / or additions in the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 Sequence homologues having at least 70% sequence homology with the base sequence of 6, or 7 are also included in the present invention.
Also included in the present invention are complementary sequences that hybridize under stringent hybridization conditions with the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6 or 7.

Conditions for hybridization are disclosed, for example, in Current Protocols in Molecular Biology Vol. 1 (John Wiley and Sons, Inc.) or Molecular Cloning 2nd. Ed. (Sambrook et al. (1989)), for example 5 X. At SSC, 5 × Denhardt's solution, 1% SDS, 25 ° C. to 68 ° C., conditions such as several hours to overnight can be used. At this time, the hybridization temperature, more preferably 45 ℃ to 68 ℃ (without formamide) or 30 ℃ to 42 ℃ (50% formamide) is used, washing conditions are 0.2 X SSC, 46 ℃ to 68 ℃ Can be.

Anyone skilled in the art can obtain DNA hybridizing to the nucleotide sequences of the ORF A to G genes of the present invention by adjusting hybridization conditions such as salt concentration and temperature, and the complementary sequences thus obtained are included in the scope of the present invention.

That is, the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably relative to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 The sequence homolog of the ORF A to G genes of RBIV-KOR-TY1 having at least 95% sequence homology, and the complementary sequence of the nucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 Include.

According to a second aspect of the invention, the invention provides ORF A, which is an immunogenic protein of iridovirus, each encoded by the RBIV-KOR-TY1 ORF A, B, C, D, E, F and G genes of the invention, B, C, D, E, F and G proteins and their sequence homologs are provided.

The ORF A protein encoded by the ORF A gene of the present invention consists of 378 amino acids and has an estimated molecular weight of 40,731.48 Da and has an amino acid sequence of SEQ ID NO: 8. As a result of comparing the sequence homology with GenBank's known sequence database, it is assumed that the amino acid sequence homology is 95% or more with the amino acid transporter protein of ISKNV, an iridovirus derived from mandarin fish, and has the function of amino acid transporter.

The ORF B protein encoded by the ORF B gene consists of 453 amino acids and has an estimated molecular weight of 49,476.79 Da and has an amino acid sequence of SEQ ID NO: 9. Comparison of sequence homology with GenBank's known sequence database reveals 99% amino acid sequence homology with the major capsid protein of the red sea bream iridovirus (RSIV), presumably the major capsid protein.

The ORF C protein encoded by the ORF C gene consists of 515 amino acids and has an estimated molecular weight of 57,026.96 Da and has an amino acid sequence of SEQ ID NO: 10. Comparison of sequence homology with GenBank's known sequence database reveals an IgM heavy chain protein with at least 85% sequence homology with that of ISKNV.

The ORF D protein, encoded by the ORF D gene, consists of 805 amino acids with an estimated molecular weight of 87,332 Da and an amino acid sequence of SEQ ID NO: 11. A comparison of sequence homology with GenBank's known sequence database shows that it has a sequence homology of at least 90% with the sequence of the laminin binding protein of RSIV and is assumed to be a laminin binding protein.

The ORF E protein encoded by the ORF E gene consists of 275 amino acids and has an estimated molecular weight of 30,338.74 Da and has an amino acid sequence of SEQ ID NO: 12. Comparing sequence homology with GenBank's known sequence database, no sequence with a high sequence homology of 70% or more was found, and protein functional motifs were presumed to be prestilled membrane proteins.

The ORF F protein encoded by the ORF F gene consists of 258 amino acids and has an estimated molecular weight of 29,272.70 Da and has an amino acid sequence of SEQ ID NO: 13. Comparing sequence homology with GenBank's known sequence database, no sequence with homology was found.

The ORF G protein encoded by the ORF G gene consists of 454 amino acids and has an estimated molecular weight of 49,973.56 Da and has an amino acid sequence of SEQ ID NO: 14. Comparing sequence homology with GenBank's known sequence database, it is estimated to be ANKYRIN REPEATS having a sequence homology of at least 90% with that of ISKNV.

The present invention also provides amino acid sequences having substitution, deletion, insertion and / or addition of one or more amino acids in the amino acid sequences of SEQ ID NOs: 8 to 14 and having at least 70% sequence homology to the sequences, Proteins that have immunogenicity.

Preferably, the present invention is an amino acid having at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% homology to the amino acid sequence of SEQ ID NOS: 8-14 Include proteins having a sequence and immunogenicity of iridoviruses.

According to a third aspect of the invention, the invention is a primer used to amplify these genes by PCR from genomic DNA of RBIV-KOR-TY1 to obtain ORF A to G DNA of RBIV-KOR-TY1 of the invention. Provide a pair.

The inventors have determined that each of the ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1 selected for use in the preparation of a vaccine against iridovirus as described above may be used to amplify from genomic DNA. Primer pairs for the gene were devised.

The primer pairs are designed to have appropriate restriction enzyme sites at both ends, taking into account restriction enzyme sites in the expression vector to clone the amplified ORF A, B, C, D, E, F and G genes.

Preferred primer pairs of the present invention, considering the restriction sites in the preferred expression vector pET28a (Novagen, USA) used in the present invention, the forward primer has a Nhe I cleavage site, and the reverse primer It is designed to have a Hind III cleavage site.

However, primer pairs of the present invention may be designed to have a cleavage site corresponding to other restriction enzyme sites present in the vector pET28a in addition to the cleavage site described above, and such primer pairs are also included in the present invention.

In addition, the ORF A, B, C, D, E, F and G genes of the present invention can be cloned into various expression vectors as described below other than pET28a, and thus, various corresponding to restriction enzyme cleavage sites in such various vectors. It can be designed to have a cleavage site, and such primer pairs are also included in the present invention.

The base sequences of the preferred primer pairs for each of the ORF A, B, C, D, E, F and G genes are as follows:

ORF A: forward primer-5 'GCT AGC ATG GCA CCG TGT GTA CTA CAG TGT 3' (SEQ ID NO: 15)

        Reverse Primer-5 'AAG CTT TTA ATA CCC CTG TAA TTG TAT TTT 3' (SEQ ID NO: 16)

ORF B: forward primer-5 'GCT AGC ATG TCT GCA ATC TCA GGT G 3' (SEQ ID NO: 17)

         Reverse primer-5 'AAG CTT TTA CAG GAT AGG GAA GCC TGC 3' (SEQ ID NO: 18)

ORF C: forward primer-5 'GCT AGC TAC CGT GGG TAA GGC AGG TAA 3' (SEQ ID NO: 19)

        Reverse primer-5 'AAG CTT AGC TCA TCT ACG GCT ACT ATG 3' (SEQ ID NO: 20)

ORF D: forward primer-5 'GCT AGC TCA CCC ACG GTG TCA CCA CCA CCA 3' (SEQ ID NO: 21)

        Reverse primer-5 'AAG CTT TAC TCG TTA CCG TTG GGA CGA 3' (SEQ ID NO: 22)

ORF E: forward primer-5 'GCT AGC ATG AGT GCA ATA AAG GCA TGA TA 3' (SEQ ID NO: 23)

        Reverse primer-5 'AAG CTT GCC TTT GCA GAA TAC CTA TGT GAA 3' (SEQ ID NO: 24)

ORF F: forward primer-5 'GCT AGC TAC TAC CGG GAA GTC AAC ATA 3' (SEQ ID NO: 25)

        Reverse primer-5 'AAG CTT CTG TAG GAT CCG TTT TAT CTG 3' (SEQ ID NO: 26)

ORF G: forward primer-5 'GCT AGC ATG CAT GTA CAT GTG TCT GTT 3' (SEQ ID NO: 27)

        Reverse primer-5 'AAG CTT CTA CGT CTT ATC AGC TCA TCG 3' (SEQ ID NO: 28)

Also included in the present invention are primer pairs that include the sequence of a primer pair for each of the genes described above or a portion thereof and can be used for PCR amplification of ORF A, B, C, D, E, F and G genes.

 The primer of the present invention can be synthesized by an oligonucleotide synthesizer, and methods for synthesizing such oligonucleotides are known and used in the art, for example, Current Protocols in Molecular Biology Vol. 1 (John Wiley and Sons). , Inc.), Sambrook, J et al., Molecular Cloning, A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, 1.53 (1989) and the like.

According to a fourth aspect of the present invention, the present invention provides a method for obtaining the ORF A to G genes of RBIV-KOR-TY1 using the oligonucleotide primer pairs of the present invention.

The method of the present invention comprises RBIV-KOR-TY1 whole genomic DNA as a template, amplification of each of the RBIV-KOR-TY1 ORF A to G genes by PCR using the oligonucleotide primer pairs of the present invention, and amplification Isolating each gene.

Viral genomic DNA separation, PCR (polymerase chain reaction) and amplified gene isolation methods used in the method of the present invention are known and used in the art, for example, Sambrook, J et al., Molecular Cloning, A The Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, 1.53 (1989) and the like.

In short, amplification of genes by PCR forms a PCR reaction with the entire genomic DNA of the virus as a template and the appropriate polymerase and primer pairs, which are denatured, annealed and extended in a PCR reactor. ) By reaction.

Polymerases useful in the methods of the present invention are EX Taq polymerases, but are not limited thereto, and suitable reaction temperatures and reaction times for denaturation, annealing and extension reactions can be readily determined and used by those skilled in the art. Preferably, the denaturation reaction of the method of the present invention is carried out for 45 seconds at 94 ℃, the annealing reaction for 45 seconds at 60 ℃, the extension reaction is carried out for 45 seconds at 72 ℃.

Each gene to be cloned into the expression vector can be obtained by treating the amplified DNA with an appropriate restriction enzyme, preferably Nhe I and Hind III, electrophoresing on an agarose gel to identify the amplified DNA band and purifying the DNA therefrom. have.

The ORF A-G genes of RBIV-KOR-TY1 obtained by the method of the present invention can be used for the production of recombinant expression vectors to be used to produce recombinant ORF A-G proteins.

According to a fifth aspect of the invention, the invention provides recombinant expression vectors comprising each of the ORF A to G genes of RBIV-KOR-TY1.

A vector for preparing a recombinant expression vector by inserting the ORF A to G genes of RBIV-KOR-TY1 of the present invention, wherein the gene sequence is inserted into the vector and then the vector is infected into the host to express the desired gene in the host cell. Any expression vector capable of producing a desired protein can be used. Such expression vectors include one or more selectable markers, promoter sequences, and the like, and examples of expression vectors include, but are not limited to, pET28c, pGEX-4T-1, pET28a, and the like. PET28a (commercially available from Novagen, USA) is particularly preferred.

Methods for inserting the ORF A to G genes of RBIV-KOR-TY1 of the present invention into vectors such as plasmids are described, for example, in Sambrook, J et al., Molecular Cloning, A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory , 1.53 (1989).

Briefly summarizing the preparation of the recombinant expression vector used in the present invention, each of the ORF A to G genes of RBIV-KOR-TY1 was amplified by PCR using oligonucleotide primer pairs of the present invention described above, and The ORF A to G genes are each digested by digesting the expression vector with the same restriction enzyme and ligation of each of the genes to the expression vector using an appropriate ligase, e.g., a T4 DNA ligase. Recombinant expression vectors A to G are prepared.

As an example of the recombinant expression vector of the present invention, a vector pET28a / RBIV-KOR-TY1-B comprising an ORF B gene is shown in FIG. 1.

The recombinant expression vectors A to G thus prepared can be used for transformation of host cells.

According to a sixth aspect of the invention, the invention provides host cells A to G transformed with recombinant expression vectors A to G comprising each of the ORF A to G genes of RBIV-KOR-TY1.

The host cell that can be used in the present invention may be a prokaryotic cell suitable for the recombinant expression vector of the present invention and can be transformed, and E. coli strains commonly used in the art may be used, but are not limited thereto. Do not. Preferably E. coli (M15, JM109, BL21, etc.) are used in the present invention as host cells.

As a method for introducing the recombinant vectors A to G of the present invention into a host cell, for example, methods known in the art such as calcium phosphate, electroporation, and chemical treatment such as PEG may be used. These methods are disclosed, for example, in Sambrook, J et al., Molecular Cloning, A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, 1.53 (1989) and the like.

Preferably, the calcium phosphate method is used in the present invention, and briefly summarized, the host cell and the recombinant expression vector are mixed and reacted on ice for 30 minutes, and then treated at 90 ° C. for 90 seconds to apply a thermal shock, followed by 90 on ice. Leave for a second. Subsequently, the transformants are incubated at 37 ° C. for about 1 hour and then plated on a plate containing antibiotics, followed by culturing, and the transformants containing the recombinant expression vector are selected.

According to a seventh aspect of the invention, the invention comprises culturing the transformed host cells A to G in an appropriate nutrient medium from the transformed host cells A to G of the RBIV-KOR-TY1 of the invention. It provides a method for mass production of recombinant proteins A, B, C, D, E, F and G.

Production of recombinant protein from transformed host cells is generally accomplished by culturing the transformed host cell under cultivation conditions suitable for the production of the recombinant protein in a nutrient medium containing a nutrient source such as a carbon source necessary for the host cell to grow.

In the method of the present invention, the nutrient medium for culturing the transformant is selected according to the host cell used in the present invention, such as E. coli and the like. In the case of E. coli , LB medium and the like are generally used.

In the method of the present invention, the culture conditions for the transformant should be selected to produce a large amount of the recombinant proteins A, B, C, D, E, F and G of the present invention, the inventors of the IPTG is an expression inducer The concentration of (isopropylthio-β-D-galactoside), the treatment time and the concentration of the antibiotic were examined to determine the appropriate culture conditions for mass production of the recombinant protein of the present invention.

As a result, suitable culture conditions of transformed host cells A to G for mass production of recombinant proteins A to G of the present invention were treated with IPTG 0.5 mM to 20 mM when the OD600 value of the transformant culture reached 0.6. It was confirmed to incubate for at least 4 hours later. Preferably, the optimum culture conditions were confirmed to incubate 4-6 hours after treatment with IPTG 1 mM ~ 10 mM. However, culture conditions somewhat outside this range can also be used in the present invention and included in the present invention as long as it induces mass production of the recombinant protein of the present invention.

According to an eighth aspect of the present invention, the present invention provides recombinant proteins A to G produced by culturing the transgenic host cells A to G of the present invention as described above.

As described above, the desired recombinant protein can be obtained by culturing the transformed host cell under suitable culture conditions, and then separating and purifying the desired recombinant protein from the culture.

Methods for isolating and purifying recombinant proteins from transformed cell cultures are well known in the art and, briefly, transformed cells are collected by centrifugation of the cell culture and suspended in suitable buffers such as sterile PBS and the like. Cells are disrupted by methods such as ultrasonication and centrifugation to obtain the contents of the host cells, and then the desired proteins are separated by known methods such as affinity chromatography, ion exchange chromatography, gel filtration and isoelectric point chromatography. Can be purified.

The recombinant proteins A to G of the present invention obtained by the above separation and purification can be used for the production of a vaccine for immunizing fish.

According to a ninth aspect of the present invention, the present invention is inactivated transgenic host cells A to G which are each transformed with the recombinant expression vectors A to G of the present invention to mass produce the recombinant proteins A to G of the present invention, respectively. Methods of preparing Inactivated Vaccines A to G, and Inactivated Vaccines A to G prepared by the methods are provided. The present invention also provides a combination of vaccines comprising at least two of the inactivated vaccines A to G.

The method for preparing an inactivated vaccine of the present invention comprises treating the transformed host cells A to G of the present invention with an inactivating agent to eliminate the possibility of infection of the host cell.

Methods of making inactivated vaccines are known in the art, and preferably, the method of the present invention comprises the steps of culturing the transformed host cell of the present invention to express the recombinant protein and using the transformed host cell culture as an inactivating agent. Processing. More preferably, the method of the present invention comprises the steps of culturing the transformed host cell of the present invention to express a recombinant protein, obtaining a cell pellet by centrifugation of the host cell culture, and treating the suspension of the pellet with an inactivating agent. Steps.

As a general deactivator, for example, formalin, glutaraldehyde, β-propionlactone, and the like are used. The mixture is added and mixed before or after preparing the vaccine stock solution. When formalin is used, the addition amount is about 0.0004 to 0.7% (v / v), the inactivation temperature is about 2 ° C to 37 ° C, and the inactivation time is about 2 days to 180 days.

For the inactivation vaccine preparation method of the present invention, any of the known inactivating agents as described above can be used to treat the transgenic host cells A to G of the present invention, preferably formalin. The amount of formalin added to the method of the present invention is preferably 0.1% to 0.7% (v / v), more preferably 0.5%. Preferable inactivating agent treatment time for the method of the present invention is 3 days or more, more preferably 3 days. Preferred inactivation treatment temperatures for the process of the invention are 4 ° C.

The recombinant inactivated vaccines A to G of the present invention are obtained by treating transformed host cells A to G of the present invention with an inactivating agent according to the method of the present invention as described above.

The recombinant inactivated vaccines A to G of the present invention thus obtained may be used alone or in various combinations to immunize fish including sea bream.

Combinations of inactivated vaccines that can be used in the present invention include at least two of inactivated vaccines A to G, preferably, a combination of inactivated vaccines B and C, a combination of inactivated vaccines B and D, an inactivated vaccine Combinations of A, B and E and combinations of inactivated vaccines B, F and G, which combinations are based on and similar to recombinant protein B, taking into account the function of the recombinant protein produced by the transformant. As a combination in the manner of adding a functioning protein, various combinations other than the four combinations exemplified above are also included in the vaccine combination of the present invention. More preferably, the combination of inactivated vaccines of the present invention is a combination of inactivated vaccines B, F and G.

As described above, the inactivated vaccines A to G of the present invention were administered to dolphin fry at a concentration twice the dose for immunizing fish, and the mortality was observed. It became.

According to a tenth aspect of the present invention, the present invention provides a method for immunizing fish, comprising administering to the fish the inactivated vaccines A to G or a combination thereof.

Fishes that can be immunized with the inactivated vaccine of the present invention include domes, perches, flounders and defenses, among which domes are preferred, and stone domes are particularly preferred.

The method of immunizing fish using the inactivated vaccine of the present invention comprises administering the vaccine of the present invention to the fish using intraperitoneal, intramuscular, or subcutaneous inoculation or infiltration, oral administration and the like. Preferably, the inactivated vaccine of the present invention is administered via intramuscular injection.

Combinations of inactivated vaccines of the invention that can be used in the immune methods of the invention include at least one of inactivated vaccines A to G, preferably include at least two of inactivated vaccines A to G, more preferably Preferably inactivated vaccines B and C, inactivated vaccines B and D, inactivated vaccines A, B and E, or inactivated vaccines B, F and G, most preferably inactivated vaccines B, F and G. It includes.

When immunizing fish using at least two or more of the inactivated vaccines A to G of the present invention, the two or more inactivated vaccines are preferably mixed before being administered to the fish and then administered to the fish.

When immunizing fish, the dose of the inactivated vaccine of the present invention is preferably administered from 0.01 ml to 0.5 ml per dose so that the amount of recombinant protein is 200 μg (200 μg recombinant protein / kg body weight) per kilogram of fish.

On the other hand, according to the eleventh aspect of the present invention, the present invention provides a kit comprising the inactivated vaccine of the present invention.

The kit of the present invention comprises at least one of the inactivated vaccines A to G of the present invention, and preferably includes two or more inactivated vaccines. Preferably, the kits of the invention comprise inactivated vaccines B and C, inactivated vaccines B and D, inactivated vaccines A, B, and E or inactivated vaccines B, F and G, most preferably present Kits of the invention include inactivated vaccines B, F and G.

Kits of the invention may include instructions for the administration of the inactivated vaccine included in the kit.

When two or more inactivated vaccines are included in the kit of the present invention, the inactivated vaccines A to G of the present invention may be included in the kit as a premixed form, or alternatively separately contained in each separate container in the kit. It may be. If the inactivated vaccines are contained separately in separate containers, the user can mix and use each inactivated vaccine according to the instructions in the kit.

According to a twelfth aspect of the present invention, the present invention provides a peptide having a part of the amino acid sequence of the ORF A to G proteins of the present invention and having immunogenicity of iridoviruses.

The peptide of the present invention is analyzed by analyzing the six amino acid sequences encoded by the ORF A, C to G genes of RBIV-KOR-TY1 of the present invention, first, high antigenicity, second, exposed to the surface, third, hydrophilic It obtained by selecting the part which satisfy | fills three things, such as a characteristic.

The peptide of the present invention may be natural or synthetic, and the synthetic peptide may be synthesized using a peptide synthesizer or the like.

According to the invention the peptide sequence selected for each ORF protein of the invention is as follows:

ORF A: AWPLFDDRMRVDQCRBI (SEQ ID NO 29)

ORF C: TLQSHKQLYSDYRTEC (SEQ ID NO: 30)

        APPDRDDDRWTVPTGCRBIV (SEQ ID NO 31)

ORF D: PTEPEPEEPEEEEDEC (SEQ ID NO: 32)

        TEPEPEEPEEEEDEYC (SEQ ID NO: 33)

ORF E: QVDRSGAKVPSEAPC (SEQ ID NO 34)

        CAMRSRDPLYDKVK (SEQ ID NO 35)

ORF F: YARARGSTHIRRQKVCRBIV (SEQ ID NO 36)

ORF G: CSPETRNSNFKHSVTY (SEQ ID NO 37)

        DMSHITKEMALNTKQHTCRBIV (SEQ ID NO: 38)

Such peptide of the present invention has excellent immunogenicity and can be usefully used for the production of a vaccine against iridovirus.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are set forth for illustrative purposes only and are not intended to limit the scope of the present invention.

Example

Example 1

Genomic DNA Sequencing and Sequence Analysis of RBIV-TY

1) Viral DNA Isolation

Virus samples used in the present invention were obtained from sea bream farmed in Tongyeong, Gyeongsangnam-do. GF cells were grown in BME (Basal Medium Eagle) medium supplemented with 10% fetal bovine serum, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Viral stock was amplified by infection of GF cells. Infected cells with cytopathic effect were collected and frozen at -20 ° C. Frozen cells were thawed, centrifuged at 3500 x g for 10 minutes at 4 ° C to remove cell debris, and the cell free supernatant was centrifuged at 30000 x g for 30 minutes at 4 ° C. Virus precipitate was suspended in TNE buffer (0.01 M Tris-HCl, Ph 8.0, 0.1 M NaCl, 0.001 M EDTA) and incubated with DNase I and RNase A for 15 minutes at 37 ° C. The virus was purified by centrifugation in a SW40 Ti rotor (Beckman) with a 20-50% (w / w) sucrose gradient at 60000 x g for 2 hours at 4 ° C. Virus bands were removed and diluted with PBS buffer, and virion pellets were collected after centrifugation at 60000 x g for 1 hour at 4 ° C. Virion was incubated with 0.5 mg / ml proteinase K and 0.5% SDS at 55 ° C. for 3 hours to extract viral DNA. The DNA was extracted with phenol-chloroform and ethanol precipitated (Molecular Cloning 2nd. Ed. (Sambrook et al. (1989)).

2) PCR amplification and DNA sequencing of viral DNA

PCR was performed to amplify the whole genomic DNA of RBIV-KOR-TY1. Primers for PCR were designed from nucleotide sequences in the GenBank / EMBL database of ISKNV. Over 120 PCR primer pairs were designed to make the average PCR product 1000 to 1200 bp long. The template was genomic DNA of RBIV-KOR-TY1. Gene amplification reaction conditions were as follows: 1 cycle at 94 ° C. for 5 minutes; 35 cycles at 92 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute; And 1 cycle at 72 ° C. for 5 minutes. There was a slight change in annealing temperature depending on the PCR primer pairs. The amplified PCR product was cloned into pGEM-T vector (Promega) and sequenced by an automated sequence analyzer (Applied Biosystems, Inc., USA).

3) DNA sequence analysis

The genomic DNA sequences obtained above and the amino acid sequences deduced therefrom were compared with the GenBank / EMBL database using the BLAST program. Sequences were sequenced using CLUSTAL W (Thompson et al., Nucleic Aicds Res. 22, 4673-4680, 1994) and then flowed into TreeView (Page, RD, Comput. Appl. Biosci. 12, 357-358, 1996). Was constructed. ORFs were identified on the basis of starting with methionine codons and consisting of more than 50 codons and not being located within a larger ORF. As a result, 118 ORFs were identified, of which 29 had high homology with known sequences and 31 could not identify sequences with homology (Do et, al. Virology, 325: 351-363). , 2004, incorporated herein by reference in its entirety). The nucleotide sequence data obtained here were deposited with GenBank under accession number AY532606.

Example 2

ORF A, B, C, D, E, F, and G Gene Selection of RBIV-KOR-TY1

Based on the ORF data obtained from the genome sequence analysis of RBIV-KOR-TY1 in Example 1, 7 genes available for vaccine production were selected from 118 ORFs using an ORF analysis program (DNAsisMAX, Hitachi, Japan). ORF A, B, C, D, E, F and G.

The ORF A gene of the present invention selected through the above analysis has the nucleotide sequence of SEQ ID NO: 1 (1,137 base pairs), the ORF B gene has the nucleotide sequence of SEQ ID NO: 2 (1,362 base pairs), and ORF C The gene has a nucleotide sequence of SEQ ID NO: 3 (1,548 base pairs), the ORF D gene has a nucleotide sequence of SEQ ID NO: 4 (2,418 base pairs), and the ORF E gene has a nucleotide sequence of SEQ ID NO: 5 (828 base pairs) The ORF F gene has the nucleotide sequence of SEQ ID NO: 6 (777 base pairs), and the ORF G gene has the nucleotide sequence of SEQ ID NO: 7 (1,365 base pairs).

Example 3

Isolation of Genomic DNA of RBIV-KOR-TY1

Genomic DNA of RBIV-KOR-TY1 was isolated as follows to amplify the ORF A to G genes selected in Example 2.

After collecting and dissecting the sea bream infected with RBIV-KOR-TY1, the spleen, kidney and brain were extracted, and EMEM (Eagle's minimum essential medium) medium without bovine placenta serum was added at 10 times the total tissue weight. After grinding with a crusher, Antibiotic-Antimycotic (Invitrogen corporation) was added to 1X and left at 4 ° C. for 10 hours, and then the tissue grinding solution was sterile filtered using a sterile filtration filter having a pore size of 0.22 μm.

The filtrate was diluted 1/500 to GF (Grant fin) cells and incubated for 5 days at 25 ° C. to obtain RBIV-KOR-TY1 genomic DNA from RBIV-KOR-TY1 culture medium and virus-infected GF cells. Isolation was carried out using a PCR Preparation Kit (Roche, Germany). In addition, the genomic DNA of RBIV-KOR-TY1 was isolated from the spleen, kidney, and brain tissues of sea bream infected with RBIV-KOR-TY1.

Example 4

Amplification of ORF A, B, C, D, E, F, and G Genes of RBIV-KOR-TY1

In order to amplify the ORF A, B, C, D, E, F and G genes of selected RBIV-KOR-TY1, PCR primers were prepared for the ORF A, B, C, D, E, F and G genes. .

First, forward primer-5 'GCT AGC ATG GCA CCG TGT GTA CTA CAG TGT 3' (SEQ ID NO: 15) and reverse primer-5 'AAG with Hind III cleavage site to amplify ORF A gene CTT TTA ATA CCC CTG TAA TTG TAT TTT 3 '(SEQ ID NO: 16) was synthesized and used for the PCR reaction.

Subsequently, 0.5 U of EX Taq-polymerase (Takara, Japan) and 10 pmole of the primer (SEQ ID NO: 15 + SEQ ID NO: 16) were added to genomic DNA of RBIV-KOR-TY1 isolated in Example 3, DNA amplification was performed 30 times in a sequence of 45 seconds at 94 ° C., 45 seconds at 60 ° C., and 45 seconds at 72 ° C. using a fast thermal cycler (MJ reacher, USA).

The amplified DNA product was treated with restriction enzymes Nhe I and Hind III, gel electrophoresis and DNA was isolated to obtain ORF A gene of RBIV-KOR-TY1 to be used for cloning to the vector in the next step.

RBIV-KOR-TY1 ORF B, C, D, E, F and G genes were also amplified according to the same experimental procedures as above using the PCR primer pairs as described below, to obtain each gene. The base sequence of the primer pair used for each gene is as follows:

ORF B: forward primer-5 'GCT AGC ATG TCT GCA ATC TCA GGT G 3' (SEQ ID NO: 17)

         Reverse primer-5 'AAG CTT TTA CAG GAT AGG GAA GCC TGC 3' (SEQ ID NO: 18)

ORF C: forward primer-5 'GCT AGC TAC CGT GGG TAA GGC AGG TAA 3' (SEQ ID NO: 19)

        Reverse primer-5 'AAG CTT AGC TCA TCT ACG GCT ACT ATG 3' (SEQ ID NO: 20)

ORF D: forward primer-5 'GCT AGC TCA CCC ACG GTG TCA CCA CCA CCA 3' (SEQ ID NO: 21)

        Reverse primer-5 'AAG CTT TAC TCG TTA CCG TTG GGA CGA 3' (SEQ ID NO: 22)

ORF E: forward primer-5 'GCT AGC ATG AGT GCA ATA AAG GCA TGA TA 3' (SEQ ID NO: 23)

        Reverse primer-5 'AAG CTT GCC TTT GCA GAA TAC CTA TGT GAA 3' (SEQ ID NO: 24)

ORF F: forward primer-5 'GCT AGC TAC TAC CGG GAA GTC AAC ATA 3' (SEQ ID NO: 25)

        Reverse primer-5 'AAG CTT CTG TAG GAT CCG TTT TAT CTG 3' (SEQ ID NO: 26)

ORF G: forward primer-5 'GCT AGC ATG CAT GTA CAT GTG TCT GTT 3' (SEQ ID NO: 27)

        Reverse primer-5 'AAG CTT CTA CGT CTT ATC AGC TCA TCG 3' (SEQ ID NO: 28)

Example 5

Construction of Recombinant Expression Vectors Comprising ORF A, B, C, D, E, F and G Genes of RBIV-KOR-TY1

The ORF A to G genes of RBIV-KOR-TY1 obtained as a result of PCR in Example 4 were cloned into pET28a (Novagen, USA), which are prokaryotic expression vectors, respectively, to prepare a recombinant expression vector. The manufacturing process is as follows.

First, the PCR products amplified in Example 4 were digested with restriction enzymes Nhe I- Hind III to obtain respective gene products. In addition, in order to prepare an expression vector for cloning each gene of the present invention, pET28a vector was transformed into E. coli JM109 competent cells, and then inoculated into LB broth to which 50 µg / ml of kanamycin was added. After incubation at 37 ° C., the plasmid was extracted in bulk. Nhe I restriction enzyme (invitrogen, USA) and reaction buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl ₂ , 50 mM KCl) were added to 1 mg of the extracted plasmid, followed by reaction at 37 ° C. for 1 hour. Cut. Subsequently, Hind III restriction enzyme and reaction buffer solution (50 mM Tris-HCl (pH 8.0), 10 mM MgCl ₂ , 50 mM NaCl) were added to the pET28a vector digested with NheI, and reacted at 37 ° C. for 1 hour. Cut. Next, pET28a vector digested with restriction enzymes Nhe I and Hind III and each gene were mixed at a ratio of 1: 1, and then 1 unit of T4 DNA ligase (Invitrogen, USA) and reaction buffer solution (66 mM Tris-HCl ( pH 7.6), 6.6 mM MgCl ₂ , 10 mM DTT, 66 μM ATP) were added and reacted at 37 ° C. for 30 minutes to allow the gene to be inserted into the Nhe I- Hind III position of the vector.

Recombinant expression vectors A to G, into which each of the ORF A to G genes of RBIV-KOR-TY1, thus inserted, were inserted by an automated sequencer (Appllied Biosystemics, USA) using a T7 promoter primer. The RBIV-KOR-TY1 ORF A to G gene was confirmed to be properly cloned.

As an example of the recombinant expression vector obtained by this example, a recombinant expression vector pET28a / RBIV-KOR-TY1-B containing the ORF B gene of RBIV-KOR-TY1 is shown in FIG. 1.

Recombinant expression vectors pET28a / RBIV-KOR-TY1-A, pET28a / RBIV-KOR-TY1-C, pET28a / RBIV-KOR-TY1-D, pET28a / RBIV-KOR-TY1-E, pET28a / RBIV-KOR-TY1 -F and pET28a / RBIV-KOR-TY1-G replace the ORF B gene portion in the pET28a / RBIV-KOR-TY1-B of FIG. 1 with ORF A, C, D, E, F, and G genes, respectively.

Example 6

Transformation of Host Cells by Recombinant Expression Vectors

The recombinant expression vector prepared in Example 5 was mixed with E. coli JM109 (TAKARA, Japan) and reacted for 30 minutes on ice (4 ° C), followed by reaction for 1 minute and 30 seconds at 42 ° C, and then again on ice (4 ° C). It was left for 30 minutes.

As a result, 800 μl SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl) was added to stabilize E. coli transformed with the recombinant expression vector, and reacted at 37 ° C. for 1 hour.

The stabilized transformants were spread by spreading 100 μl into 50 μg / ml kanamycin-added LB plates and incubated at 37 ° C. for 15 hours to select 30 transformants for each gene.

Example 7

Expression of recombinant proteins A, B, C, D, E, F and G proteins of RBIV-KOR-TY1

In order to optimize the culture conditions of the transformants for expressing the ORF A to G protein in large quantities from the ORF A to G gene cloned in the transformed host cell obtained in Example 6 The degree of expression was examined at different antibiotic concentrations.

1) Induction of expression by IPTG concentration and treatment time

The E. coli BL 21 (DE3) / RBIV-KOR-TY1-A transformant obtained by the method of Example 6 was incubated for 10 hours in 3 mL LB medium containing 50 mg / mL of kanamycin, and then diluted to 1/100. Was inoculated in 100 mL LB medium containing 50 mg / mL kanamycin and incubated at 37 ℃.

When the OD600 value was 0.6, IPTG (isopropylthio-β-D-galactoside) was added at various concentrations to induce protein expression. The concentration of added IPTG was 1 mM, 5 mM, 10 mM, 20 mM, and the expression level of recombinant protein A was measured by SDS-PAGE (polyacrylamide gel electrophoresis) analysis 4 hours after IPTG treatment.

On the other hand, in order to investigate the expression pattern of recombinant protein according to IPTG treatment time, in cultures with and without 1 mM IPTG, immediately before addition (0 hours), 2 hours, 4 hours, 6 hours after addition The expression profile of the recombinant protein at time, 8 hours was examined by SDS-PAGE analysis.

SDS-PAGE analysis method used to investigate the expression level of the recombinant protein induced is as follows. 1 mL of each sample was collected, centrifuged at 14,000 rpm for 30 seconds, the supernatant was discarded, and the pellet was resuspended in 500 µl sterile PBS. Cells were crushed by sonication while keeping a 1.5 mL tube containing suspension samples on ice. At this time, the process was sonicated for 15 seconds and cooled three times for 10 seconds. The crushed sample was centrifuged at 14,000 rpm for 5 minutes, 100 μl of the supernatant was mixed with 100 μl of 2X SDS-sample buffer and boiled at 100 ° C. for 10 minutes. This was again centrifuged at 14,000 rpm for 5 minutes and 20 μl was loaded onto a 10% SDS-PAGE gel. After separation for about 2 hours at 50 ~ 150 V and stained with Coomassie staining solution (Coomassie staining solution; 0.1% Coomassie blue R250, 42% acetic acid, 42% methyl alcohol) for 30 minutes, destaining solution I (5) % Acetic acid, 7.5% methyl alcohol) for 1 hour. The resulting polyacrylamide gel photographs are shown in FIGS. 2 and 3.

As shown in FIG. 2, lanes 2 to 5 treated with 1 mM of IPTG were expressed in a large amount of recombinant protein, and the longer the treatment time, the greater the expression amount. On the other hand, it can be seen that the recombinant protein is not expressed in a large amount in lanes 7-10 not treated with IPTG.

In addition, as shown in Figure 3, a large amount of recombinant protein is expressed in the IPTG treatment of 1 mM to 20 mM, especially in IPTG 10 mM treatment (lane 5) or 100 μg treatment of IPTG 1 mM and kanamycin (lane 7) It can be confirmed that the expression in a greater amount than.

Example 8

Preparation of Recombinant Inactivated Vaccines

The transformant was inactivated to prepare a vaccine using the transformant obtained in Example 6. The deactivation process is as follows.

Transgenic strains A to G containing ORF A, B, C, D, E, F and G genes of RBIV-KOR-TY1, respectively, were incubated at 37 ° C. for 10 hours in LB medium containing 50 mg / mL of kanamycin. After diluting to 1/100, the cells were inoculated in 1,000 mL of LB medium containing 50 mg / mL kanamycin and incubated at 37 ° C. When the OD600 value was 0.6, IPTG was added at a concentration of 1 mM, incubated at 37 ° C. for 5 hours, centrifuged at 5,000 rpm for 30 minutes, the supernatant was discarded, and the pellet was resuspended in 100 mL sterile PBS. Formalin (37% Formaldehyde solution) was added to the suspension sample in 0.5% of the total volume to inactivate transformants A, B, C, D, E, F and G for 3 days at 4 ℃.

The inactivated suspension sample was centrifuged at 5,000 rpm for 30 minutes, the supernatant was discarded, and the pellet was resuspended in 100 mL of sterile PBS and centrifuged three times. Sterile PBS was added so that the protein concentration was 20 μg / mL, and 100 unit / mL penicillin G, 100 μg / mL streptomycin was added thereto and stored at 4 ° C.

Example 9

Safety test of recombinant inactivated vaccine

For the safety experiment of the recombinant inactivated vaccine prepared in Example 8, 20 sea bream fish (weight; 9 ± 1 g) were placed in a 300L water bath, and each recombinant vaccine was prepared to have a concentration of 40 μg / mL. Vaccine mortality was observed for 15 days while inoculated at the back muscles of dolphin fry at twice the concentration of the vaccine. As a result, death by the recombinant vaccine treatment of the present invention did not occur at all, ensuring the safety of the recombinant vaccine.

Death of sea bream by treatment with recombinant vaccines A, B, C, D, E, F and G of the present invention         Recombinant vaccine type      Mortality rate (mortality number / experimental number of animals) A 0 (0/20) B 0 (0/20) C 0 (0/20) D 0 (0/20) E 0 (0/20) F 0 (0/20) G 0 (0/20)

Example 10

Validation of Recombinant Inactivated Vaccines

In order to confirm the efficacy of the recombinant inactivated vaccine of the present invention, the recombinant inactivated vaccines A, B, C, D, E, F and G prepared in Example 8 were mixed and tested in a combination of four.

Test 1 mixed recombinant inactivated vaccines A, B and E, test 2 mixed B and C, test 3 mixed B and D, test 4 mixed B, F and G In addition, a test group treated with a mixture of sterile PBS and antibiotics was prepared as a control. The four combinations described above were combined in a manner based on the B protein and adding a protein having a similar function thereto.

Experiments to confirm the efficacy of the vaccine was carried out as follows.

RDV-large juvenile fish were found to have been infected with 300 L FRP tank, and four test vaccines prepared above were injected into the back muscle area by 0.1 ml each and were bred at natural water temperature for 15 days. Vaccines treated with RBIV-KOR-TY1 strains cultured in GF cells were infected with a concentration of 10 ⁴ TCID ₅₀ / mL. While breeding sea bream infected with RBIV-KOR-TY1, mortality by RBIV-KOR-TY1 was investigated and analyzed.

As a result, as shown in Figure 4, compared to the control vaccine vaccines 1 to 4 had a protective effect against RBIV-KOR-TY1 delayed mortality of sea bream, especially in the case of test 4 accumulated mortality rate One month later, it was confirmed that it provides excellent vaccine effect of less than 30%.

Example 11

Analysis and Synthesis of A, B, C, D, F and G Peptides of RBIV-KOR-TY1

Analyzing amino acid sequences encoded by the six ORF A, C to G genes of the present invention; High antigenicity, second; Exposed to the surface, third; Portions satisfying three such hydrophilic properties were selected and synthesized as vaccine peptides.

The peptide sequence synthesized for each ORF protein is as follows, and the synthesis of the peptide was made by Peptron (PEPTRON, Korea).

ORF A: AWPLFDDRMRVDQCRBI (SEQ ID NO: 29)

ORF C: TLQSHKQLYSDYRTEC (SEQ ID NO: 30)

        APPDRDDDRWTVPTGCRBIV (SEQ ID NO: 31)

ORF D: PTEPEPEEPEEEEDEC (SEQ ID NO: 32)

        TEPEPEEPEEEEDEYC (SEQ ID NO: 33)

ORF E: QVDRSGAKVPSEAPC (SEQ ID NO: 34)

        CAMRSRDPLYDKVK (SEQ ID NO: 35)

ORF F: YARARGSTHIRRQKVCRBIV (SEQ ID NO: 36)

ORF G: CSPETRNSNFKHSVTY (SEQ ID NO: 37)

        DMSHITKEMALNTKQHTCRBIV (SEQ ID NO: 38)

The present invention is a gene encoding an immunogenic protein of RBIV-KOR-TY1 that can be used in the production of a vaccine against doldom iridovirus, immunogenic proteins encoded by it, peptides derived therefrom, recombinant comprising the gene By providing a vector, a transformant comprising the recombinant vector, and an inactivated vaccine prepared by using the transformant, an inexpensive iridovirus vaccine having an excellent vaccine effect against the iridovirus is provided.

Attach sequence list electronic file  

Claims (55)

A gene having the nucleotide sequence of any one of SEQ ID NOs: 1 to 7, and encoding the immunogenic protein of the Korean sea-bream idovirus RBIV-KOR-TY1.  The gene according to claim 1, which is an ORF A gene having the nucleotide sequence of SEQ ID NO: 1. The gene according to claim 1, which is an ORF B gene having a nucleotide sequence of SEQ ID NO: 2. 8. The gene according to claim 1, which is an ORF C gene having the nucleotide sequence of SEQ ID NO: 3. 8. The gene according to claim 1, which is an ORF D gene having a nucleotide sequence of SEQ ID NO: 4. 8. The gene according to claim 1, which is an ORF E gene having a nucleotide sequence of SEQ ID NO: 5. The gene according to claim 1, which is an ORF F gene having the nucleotide sequence of SEQ ID NO: 6. 6. The gene according to claim 1, which is an ORF G gene having the nucleotide sequence of SEQ ID NO. delete delete delete delete delete delete delete delete Oligonucleotide primer pairs represented by SEQ ID NOs: 15 and 16. Oligonucleotide primer pairs represented by SEQ ID NOs: 17 and 18. Oligonucleotide primer pairs represented by SEQ ID NOs: 19 and 20. Oligonucleotide primer pairs represented by SEQ ID NOs: 21 and 22. Oligonucleotide primer pairs represented by SEQ ID NOs: 23 and 24. Oligonucleotide primer pairs represented by SEQ ID NOs: 25 and 26. Oligonucleotide primer pairs represented by SEQ ID NOs: 27 and 28. Amplifying the gene of claim 1 by PCR using the oligonucleotide primer pair of any one of claims 17 to 23 as a template using the whole genomic DNA of Korean sea-bream iridovirus RBIV-KOR-TY1 as a template, and A method of amplifying and obtaining the gene of claim 1, comprising the step of isolating the amplified gene. A recombinant expression vector comprising the gene of claim 1 inserted into an expression vector for a prokaryotic cell. The recombinant expression vector according to claim 25, wherein the expression vector for prokaryotic cell is pET28a. 28. A prokaryotic host cell transformed with the recombinant expression vector of claim 25 or 26. 28. The transformed prokaryotic host cell of claim 27, wherein the host cell is E. coli. 29. A method for mass production of recombinant protein from said transformed host cell, comprising culturing the transformed host cell of claim 27 or 28 in the presence of IPTG in a suitable nutrient medium. The method of claim 29, wherein the concentration of IPTG is in the range of 1 mM to 10 mM. 31. The method of claim 29 or 30, wherein the processing time of the IPTG is at least 2 hours. 32. The method of claim 31, wherein the processing time of the IPTG is 4-6 hours. A recombinant protein produced by culturing the transgenic host cell of claim 27. 29. A method of making a recombinant inactivated vaccine comprising treating the transgenic host cell of claim 27 with an inactivating agent. 35. The method of claim 34, wherein the inactivating agent is formalin. 36. The method of claim 35, wherein the concentration of formalin is 0.1% to 0.7% (v / v). The method of claim 36, wherein the concentration of formalin is 0.5% (v / v). A recombinant inactivated vaccine prepared by inactivating the transformed host cell of claim 27. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine A prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF A gene having the nucleotide sequence of SEQ ID NO: 1. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine B prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF B gene having the nucleotide sequence of SEQ ID NO: 2. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine C prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF C gene having the nucleotide sequence of SEQ ID NO: 3. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine D prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF D gene having the nucleotide sequence of SEQ ID NO: 4. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine E prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF E gene having the nucleotide sequence of SEQ ID NO: 5. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine F prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF F gene having the nucleotide sequence of SEQ ID NO: 6. Activating vaccine. 39. The recombinant fire of claim 38, wherein said vaccine is an inactivated vaccine G prepared by inactivation of a host cell transformed with a recombinant vector comprising an ORF G gene having the nucleotide sequence of SEQ ID NO: 7. Activating vaccine. A combined recombinant inactivated vaccine comprising at least two of the recombinant inactivated vaccines of claims 39 to 45. 47. The combined recombinant inactivation according to claim 46, comprising inactivated vaccines B and C, inactivated vaccines B and D, inactivated vaccines A, B and E, or inactivated vaccines B, F and G. vaccine. 48. The combined recombinant inactivation vaccine according to claim 47, comprising inactivation vaccines B, F and G. 46. A method of immunizing fish comprising administering to the fish the recombinant inactivated vaccine of any one of claims 38-45 or the combined recombinant inactivated vaccine of any one of claims 46-48. 50. The method of claim 49, wherein said inactivated vaccine is administered to the back muscles of the fish. 51. The method of claim 49 or 50, wherein the dose of the vaccine is 200 μg recombinant protein / kg body weight. 46. A kit comprising at least one of the recombinant inactivated vaccines of claims 39-45. The kit of claim 52 comprising inactivated vaccines B and C, inactivated vaccines B and D, inactivated vaccines A, B and E, or inactivated vaccines B, F and G. 54. The kit of claim 53, wherein the kit comprises inactivated vaccines B, F and G. delete

Images