KR20150085984A - Vaccine composition using Streptococcus parauberis cytoplasmic membrance protein in fishes - Google Patents

Abstract

The present invention relates to a vaccine composition prepared by using the cellular membrane protein of Streptococcus parauberis which is a causative agent of the fish streptococcus. More specifically, the present invention selects the protein having an antigenic activity against Streptococcus parauberis which is a causative agent of the fish streptococcus to use the protein as the vaccine composition for the prevention and treatment of streptococcus.

Description

(Vaccine composition using Streptococcus parauberis cytoplasmic membrane protein in fishes) using Streptococcus parauverus cytoplasmic membrane protein

The present invention relates to a method for producing Streptococcus pneumoniae parauberis ) cytoplasmic membrane protein.

Streptococcus parauberis ( Streptococcus parauberis ) has a high segregation rate in domestic flounder farms and is emerging as a major cause of streptococcal infection. In flukes infected with S. parauberis, dark coloration, nocturnal hemorrhage, abnormal swimming and poor feeding, and death due to cachexia.

Flounder is a species that occupies a large part of domestic marine aquaculture. Domestic flounder farms are mostly located in the form of aquaculture tanks in Jeju, South and West coasts, and the damage caused by streptococcal infections is frequent. However, epidemiological studies have been lacking in response to major causative agents Than in those who were treated with antibiotics.

However, recent epidemiological studies have shown that S. parauberis is a major pathogen of streptococcal infection in flounder (Nho SW, Shin GW, Park SB, Jang HB, Cha IS, Ha MA, Kim YR, Park YK , Dalvi RS, Kang BJ, Joh SJ, and Jung TS FEMS Microbiol Lett. 2009 Apr; 293 (1): 20-7, Shin GW, Palaksha KJ, Yang HH, Shin YS, Kim YR, Lee EY, Kim HY. Kim YJ, Oh MJ, Yoshida T. Jung TS, Bull Eur Assoc., Fish Pathol.

Currently, S. parauberis has only been identified, but the research on the prevention and treatment of S. parauberis has been limited. In addition, an antibiotic resistance gene for erythromycin and tetracycline was found in S. parauberis and antibiotic treatment was limited. (Park YK, Nho SW, Shin GW, Park SB, Jang HB, Cha IS, Ha MA, Kim YR, Dalvi RS, Kang BJ, Jung TS, Vet Microbiol 2009 Apr 14; 136 (1-2): 76-81. Therefore, development of a vaccine is urgent as a fundamental preventive measure.

Accordingly, the inventors of the present invention have studied a method of effectively preventing Streptococcus, and as a result, selected proteins having antigenicity against Streptococcus parauberis , and found that excellent vaccine efficacy was confirmed by inoculation with zebrafish Thus completing the present invention.

It is an object of the present invention to provide a vaccine composition for treating or preventing a streptococcal infection of fish comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

Another object of the present invention is to provide an expression vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 2 encoding the polypeptide.

It is still another object of the present invention to provide a host cell transformed with the expression vector.

It is another object of the present invention to provide a feed composition for the treatment or prevention of streptococcal infectious disease of fish comprising the vaccine composition.

It is yet another object of the present invention to provide a method for treating or preventing streptococcal infection of fish by administering the vaccine composition to fish.

In order to accomplish the above object, the present invention provides a vaccine composition for treating or preventing streptococcal infection of fish comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides an expression vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 2 encoding the polypeptide.

The present invention also provides a host cell transformed with the expression vector.

The present invention also provides a feed composition for the treatment or prevention of streptococcal infectious disease of fish comprising the vaccine composition.

In addition, the present invention provides a method for treating or preventing a streptococcal infection of a fish by administering the vaccine composition to a fish.

Use of a protein having excellent antigenicity against Streptococcus parauberus, which is a causative organism of streptococci according to the present invention, can be effectively utilized as a vaccine composition for the prevention and treatment of streptococci.

1, Streptococcus This is the result of DNA electrophoresis of the candidate antigen gene from E. coli .
FIG. 2 shows electrophoresis of antigen candidate protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) of SEQ ID NO: 1 and protein blotting Western blotting using His probe antibody.
Fig. 3 shows Western blot results using streptococcal antiserum produced in mice. Fig.
FIG. 4 is a graph showing the survival rate after the inoculation of antigenic candidate protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) of SEQ.
FIG. 5 shows that the antigen protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) is allergic to the S. parauberis serotype I, S. parauberis serotype II and S. iniae, .
6 is a diagram showing a process of preparing a zebrafish rearing environment and preparing an experiment.
FIG. 7 is a diagram showing the amino acid sequence of the antigen candidate protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) of SEQ ID NO: 1.

Hereinafter, the present invention will be described in detail.

 The present invention provides a vaccine composition for treating or preventing streptococcal infection of fish comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The streptococcus is preferably Streptococcus parauberis .

The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is an antigen candidate protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) present in the cytoplasmic membrane of Streptococcus parauberis in a specific embodiment of the present invention.

The fish is preferably at least one species selected from the group consisting of flounder, perch, trout, oyster, and dome, but is not limited thereto.

In addition, the present invention provides an expression vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 2 encoding the polypeptide.

The expression vector is preferably pET-30a, but is not limited thereto.

Also, variants of the above sequences are included within the scope of the present invention. The mutant is a nucleotide sequence having a functional characteristic similar to that of the nucleotide sequence of SEQ ID NO: 2, although the nucleotide sequence thereof is changed. Specifically, the sequence comprises a nucleotide sequence having at least 70% homology, more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 2 can do.

 The "% of sequence homology" for the polynucleotide is identified by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., Gap), as compared to < / RTI >

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "expression vector" is often used interchangeably with a "recombinant vector ". The term "recombinant vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The present invention also provides a host cell transformed with the expression vector.

The gene sequence of SEQ ID NO: 2 may be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

The present invention also provides a feed composition for the treatment or prevention of streptococcal infectious disease of fish comprising the vaccine composition.

The streptococcus is preferably Streptococcus parauberis .

In addition, the present invention provides a method for treating or preventing a streptococcal infection of a fish by administering the vaccine composition to a fish.

The streptococcus is preferably Streptococcus parauberis .

< Example  1> In- In-silico  Screening for candidate antigen proteins

Streptococcus the candidate antigen protein were selected by computer simulation through a silico - parauberis) KCTC of using the genomic analysis of the 11537 (STP_) and S. parauberis NCFD 2020 (SPB_) strain. At this time, the transmembrane helix number, subcellular localization, and adhesion of the protein were considered. Extracellular (EC), cell wall (CW) 7 orfs and cytoplasmic membrane (CM) 16 orfs were selected from a total of 1868 proteins of S. parauberis KCTC 11537, and S. parauberis NCFD 2020 Among the total 2146 proteins, extracellular 4 orfs, cell wall 7 orfs, and cytoplasmic membrane 14 orfs were selected. The final 40 proteins were selected as candidate antigen proteins of Streptococcus pauberus strain except for overlapping among them, and are shown in Table 1 below.

number Locus tag location gene One STP_0018 Extracellular amidase 2 STP_0130 Extracellular Xaa-Pro dipeptidyl-peptidase 3 STP_0197 Extracellular N-acetyl-muramidase 4 STP_0342 Extracellular alpha-amylase 5 STP_1253 / SPB_1157 Extracellular penicillin-bindingprotein 1A 6 STP_1551 / SPB_1829 Extracellular Alkalinephosphatase 7 STP_1590 Extracellular prophage LambdaSa1, N-acetylmuramoyl-L-alanine amidase, family 4 8 SPB_2015 Extracellular CHAP domain protein 9 STP_0140 Cell wall signal peptidase I 10 STP_0535 / SPB_0281 Cell wall N-acetylmuramoyl-L-alanine amidase 11 STP_1223 Cell wall zinc carboxypeptidase 12 STP_1405 / SPB_1598 Cell wall secretionproteinBug4 13 STP_1447 / SPB_1652 Cell wall lpxtg-motifcellwallanchordomainprotein 14 STP_1567 Cell wall SiM protein 15 SPB_0377 Cell wall AP domain protein 16 SPB_0381 Cell wall LPXTG-motif cell wall anchor domain protein 17 SPB_0674 Cell wall phage minor structural protein, N-terminal domain protein 18 SPB_1927 Cell wall pilin isopeptide linkage domain protein 19 STP_0225 / SPB_1215 Cytoplasmic membrane LytRfamilyregulatoryprotein 20 STP_0274 / SPB_1171 Cytoplasmic membrane penicillinbindingprotein2x 21 STP_0438 / SPB_0179 Cytoplasmic membrane Zinc-bindingproteinAdcAprecursor 22 STP_0503 / SPB_0252 Cytoplasmic membrane competenceassociatedendonuclease 23 STP_0539 / SPB_0285 Cytoplasmic membrane 막 protein 24 STP_0689 / SPB_0517 Cytoplasmic membrane pneumococcal vaccine antigen-like protein 25 STP_0697 Cytoplasmic membrane laminin binding protein 26 STP_0700 Cytoplasmic membrane 심포치 27 STP_0801 / SPB_0626 Cytoplasmic membrane phosphateABCtransporter, extracellularphosphate-binding lipoprotein 28 STP_0845 Cytoplasmic membrane putative truncated prophage LambdaSa1, minor structural protein 29 STP_0956 / SPB_0817 Cytoplasmic membrane protease 30 STP_1063 / SPB_0928 Cytoplasmic membrane foldaseproteinPrsA1precursor 31 STP_1610 Cytoplasmic membrane sporulation protein 32 STP_1616 Cytoplasmic membrane penicillin-binding protein 1B 33 STP_1749 Cytoplasmic membrane penicillin-binding protein 2a 34 STP_1850 / SPB_2075 Cytoplasmic membrane transglycosylase protein 35 SPB _0027 Cytoplasmic membrane metal ABC transporter 기판 - binding lipoprotein 36 SPB_0398 Cytoplasmic membrane 심포치 37 SPB_1817 Cytoplasmic membrane BAAT / acyl-CoA thioester hydrolase C-terminal domain protein 38 SPB_1837 Cytoplasmic membrane ABC transporter, solute-binding protein 39 SPB_1884 Cytoplasmic membrane 과산 40 STP_0585 / SPB_0333 Cytoplasmic membrane mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase family protein / gametolysin

< Example  2> Antigen candidate protein Cloning

In order to obtain an antigen candidate gene of the protein in the above <Example 1> GDNA (genomic DNA) of Streptococcus strain was isolated using DNeasy Blood & Tissue kit (Qiagen, Cat No. 69506) and genes were obtained.

The strains used for the isolation of genomic DNA were S. parauberis serotype I (aquatic animal disease laboratory, S. parauberis P2 strain, Seoul National University), S. parauberis serotype II (two strains of S. parauberis YSFST02-111 strain, Chonnam National University) ).

Specifically, the isolated gDNA was obtained as a primer capable of amplifying the antigen gene, and the introduction into the expression vector was performed using In-fusion ^TM Advantage PCR cloning kit (Clontech, USA). The primers and the amplified genes used for antigen gene acquisition are shown in Table 2 and FIG. The obtained antigen gene was cloned into a pET-30a expression vector linearized with NdeI and XhoI using In-Fusion HD Enzyme Premix (Clontech, Cat No. ST0345). The plasmids of the expression vectors thus prepared were transformed into E. coli BL21 (DE3). The transformants were cultured in LB medium containing 50 μg / ml of antibiotics at 37 ° C. At OD = 600 to 0.5 When it reached, 0.5 mM IPTG (isopropyl- beta -thio-D-galactopyranoside) was added and the culture was further incubated for 3 hours at 37 C. For further analysis, the cells were collected by centrifugation and the pellet was disrupted by sonication The supernatant was then recovered and the recombinant fusion protein was analyzed by SDS-PAGE.

As a result, # 1 (STP-0018; amidase) and # 8 (SPB-2015; CHAP domain protein) which were not expressed well were used as a negative control group as shown in SDS- 35 protein (SPB_0027, metal ABC transporter substrate-binding lipoprotein) was expressed (Figure 2).

gene name The sequence (5'-3 ') One Forward primer TAAGAAGGAGATATACATATG AAAAAGAGAATTTTATCAG Reverse primer GTGGTGGTGGTGGTGCTCGAG GTTTGGTAAGATGTATAC 2 Forward primer TAAGAAGGAGATATACATATG AAGTATAATCAATTTTCTTATATAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTCGGTATCTTTAGTTTAG 3 Forward primer TAAGAAGGAGATATACATATG AGATCACGTTTAAAATTAA Reverse primer GTGGTGGTGGTGGTGCTCGAG ATCATATTTTGTTAAATC 4 Forward primer TAAGAAGGAGATATACATATG ACAAATGATTTAATTATG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTATTTTGTTCTTAGG 5 Forward primer TAAGAAGGAGATATACATATG ATTAAAATCAAAAATCCAAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTGACCAGCTGTTCCATTAC 6 Forward primer TAAGAAGGAGATATACATATG GCAGTAACTACACATTCG Reverse primer GTGGTGGTGGTGGTGCTCGAG ATTAATTTTCTTTGGTGC 7 Forward primer TAAGAAGGAGATATACATATG CAAAGAGTTTTAGATAGTGAC Reverse primer GTGGTGGTGGTGGTGCTCGAG ACTATTTTTGATTGATCTGATG 8 Forward primer TAAGAAGGAGATATACATATG AAAAAGAGAATTTTATCAG Reverse primer GTGGTGGTGGTGGTGCTCGAG GTTTGGTAAGATGTATAC 9 Forward primer TAAGAAGGAGATATACATATG AAAAATTTTATTAAAGAATG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTAAAAGATCAATGCG 10 Forward primer TAAGAAGGAGATATACATATG CTTTTTGTACAAAAGAATG Reverse primer GTGGTGGTGGTGGTGCTCGAG AATTGTGTAAGTCTTATAGTACC 11 Forward primer TAAGAAGGAGATATACATATG ACTAATGTAAAAACTGGTC Reverse primer GTGGTGGTGGTGGTGCTCGAG ATTTTCTTTACGACGTTTTAC 12 Forward primer TAAGAAGGAGATATACATATG AAAAATGGGAAATGGTTAGC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTCAATATGCTTTTCTAATG 13 Forward primer TAAGAAGGAGATATACATATG TATCATACTAAAGATAGG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTCATGTTTATACTTTTTTG 14 Forward primer TAAGAAGGAGATATACATATG GCAAAGCGTGAAGCTAAAC Reverse primer GTGGTGGTGGTGGTGCTCGAG AGCTTCTTTACGTTTACG 15 Forward primer TAAGAAGGAGATATACATATG TTGTCTAAACAAACAAGACAAG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTGGTGGTATTGCAAACG 16 Forward primer TAAGAAGGAGATATACATATG AAAAAATCATTATTAATTAG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTAGTTCTTTTTTACTT 17 Forward primer TAAGAAGGAGATATACATATG CTTTATTATTATGGAGCAAAAACC Reverse primer GTGGTGGTGGTGGTGCTCGAG AAAAACCTAAATCTTGGAAG 18 Forward primer TAAGAAGGAGATATACATATG AGAAATTTTATAAACTATTTG Reverse primer GTGGTGGTGGTGGTGCTCGAG CTTATTCTTTCTTTGTCTCC 19 Forward primer TAAGAAGGAGATATACATATG AAAATCGGAAAAAAAATACTCC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTGTACAGGCGCAACTGGAG 20 Forward primer TAAGAAGGAGATATACATATG AATAAGTTTATTAAAGGTTTTA Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTTAATTTTATGAAGTGC 21 Forward primer TAAGAAGGAGATATACATATG AAAAAGAAAGTTCTATTGC Reverse primer GTGGTGGTGGTGGTGCTCGAG ATGTGCATTTTATTTCTTGTGC 22 Forward primer TAAGAAGGAGATATACATATG AGAGGTCAAATGTCAAATC Reverse primer GTGGTGGTGGTGGTGCTCGAG GCGTTCTATATCGACCTGACC 23 Forward primer TAAGAAGGAGATATACATATG GCTAAAGAACCATGGG Reverse primer GTGGTGGTGGTGGTGCTCGAG CTTGATAATAACTGCATC 24 Forward primer TAAGAAGGAGATATACATATG TTGAAATTTTTCAAACGTG Reverse primer Gt ; 25 Forward primer TAAGAAGGAGATATACATATG TTACCGATTTGCTTAATAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTAATTCTTGATATAAAAT 26 Forward primer TAAGAAGGAGATATACATATG AAACAAATTCGCAACCGTC Reverse primer GTGGTGGTGGTGGTGCTCGAG GTTTTTCTTGTCTTTAAAA 27 Forward primer TAAGAAGGAGATATACATATG AATATTAAAAAGAAATTC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTCTTTGAAACTTTTCCATC 28 Forward primer TAAGAAGGAGATATACATATG GGAACATCAAATTTTAGTGGC Reverse primer GTGGTGGTGGTGGTGCTCGAG CGCTGTTCTCAACCACATATAAG 29 Forward primer TAAGAAGGAGATATACATATG AAGCTGTTGAATGGCTTTTTC Reverse primer GTGGTGGTGGTGGTGCTCGAG GTTTCCTTGATATAGCTC 30 Forward primer TAAGAAGGAGATATACATATG AAAAAATCTAATAAGTTAATTAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTATCAACTGATGTTTTC 31 Forward primer TAAGAAGGAGATATACATATG AAAAATAAATTTTATATTGTC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTGATAACATAAAAATA 32 Forward primer TAAGAAGGAGATATACATATG CAAGCTTTATCAAAGTTTGG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTTTCTAATATTGAAAC 33 Forward primer TAAGAAGGAGATATACATATG GTTAAAAAAATAGAAGCTA Reverse primer GTGGTGGTGGTGGTGCTCGAG TCTGAAGTAGTCTATAACACC 34 Forward primer TAAGAAGGAGATATACATATG TATAGAATTAAAAATTTG Reverse primer GTGGTGGTGGTGGTGCTCGAG ATAACCCCAAGCAGACATC 35 Forward primer TAAGAAGGAGATATACATATG AAGAAGAAATTTACTTTTGC- SEQ ID NO: 3 Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTCGATAAACCTTCTG-SEQ ID NO: 4 36 Forward primer TAAGAAGGAGATATACATATG AAAACAATTGGAAAATCAG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTTATCTATATTGAC 37 Forward primer TAAGAAGGAGATATACATATG AAAAATTTTATTAAAATAA Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTACCATGTTTTTCCTTATG 38 Forward primer TAAGAAGGAGATATACATATG AAAAAAAATTGGTTAGCAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTCTAAAGCTTTATC 39 Forward primer TAAGAAGGAGATATACATATG AACCAATTAAAGAAGAAAAAAAC Reverse primer GTGGTGGTGGTGGTGCTCGAG TTTTTTTCTAATATTGAAAC 40 Forward primer TAAGAAGGAGATATACATATG ACGCTTCAGAAAGGTAAG Reverse primer GTGGTGGTGGTGGTGCTCGAG TTGATTATTTTTATAATCAC

< Example  3> Identified  Protein metal ABC transporter 기판 - binding  Expression of lipoprotein

The recombinant fusion protein transformed in Example 2 was expressed and the Western blot assay was performed to confirm the antigenicity.

Specifically, the recombinant fusion protein transformed in Example 2 was subjected to SDS-PAGE, and the resulting gel was transferred to a nitrocellulose membrane (Nitrocellulose (NC) membrane). The nitrocellulose membrane was blocked with 5% skim milk in PBS for 1 hour at room temperature to remove nonspecific reaction and washed with PBST (PBS, 0.05% Tween 20) three times for 15 minutes. Then, mouse-produced His-probe antibody (H8; Santa Cruz Biotechnology, Cat SC-57598) and mouse streptococcal antiserum prepared in 0.5% skim milk melted PBST were added, Respectively. The nitrocellulose membrane was washed with PBST three times for 15 minutes each time, followed by secondary reaction with goat anti-mouse IgG and Alkaline-phosphatase-linked antibody (Sigma) for 1 hour at room temperature. The nitrocellulose membrane was washed with PBST three times for 15 minutes and the antigen protein was identified using the AP conjugate substrate kit (Bio-Rad; Cat 170-6432).

As a result, His expression band was not observed in # 1 (STP-0018; amidase), # 8 (SPB-2015; CHAP domain protein) SPB_0027, metal ABC transporter substrate-binding lipoprotein) (Fig. 2).

< Example  4> Zebra fish ( Zebrafish ) metal ABC transporter 기판 -binding lipoprotein  Analysis of protein vaccine efficacy

Zebrafish has been shown to be susceptible to both streptococcal infections and inoculation and intramuscular inoculation, and it has been shown in studies that the LD50 for Streptococcus strains and their mortality typically take 48 to 72 hours (Streptococcus- zebrafish model of bacterial pathogenesis / Neely MN, Pfeifer JD, Caparon M. / Infect Immun 2002 Jul; 70 (7): 3904-14). Based on the results, the sensitivity test and LD50 of Streptococcus parauberus against zebrafish were confirmed. Based on the results, it was confirmed that the immune protein immune protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) .

<4-1> Preparation of zebrafish breeding environment and experiment preparation

As shown in Fig. 6, a large amount of zebrafish was raised in a large water tank, and the water was transferred into a small water tank and separated into experimental groups.

<4-2> Attack inoculation strain History

The inoculated strains were named S. parauberis P2, and the sale agency is a serotype I, which is a school of aquatic animal disease at Seoul National University College of Veterinary Medicine.

<4-3> Zebra fish  Model-based Streptococcus  Parauberus vaccine efficacy assessment confirmation test

① Experimental method

The three groups of zebrafish were anesthetized with Tris-buffered tricaine (3-aminobenzoic acid ethylester, pH 7.0; Sigma) and the antigen candidate protein # 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) 100 ug / ml, and S. parauberis S1 whole bacterin at a concentration of 5 mg / ml in 10 ul. After 3 weeks of immunization, the number of Streptococcus parauberus serotypes for attack was inoculated twice per 10 days at 10-day intervals to induce an attack of inoculation (number of first attacked cells: 4.3 X 10 ⁹ CFU / ml second attacked number of cells inoculated 1.4 X 10 ¹⁰ CFU / ml). As shown in the following Table 3, PBS was inoculated in place of the antigen candidate protein, and a group subjected to an attack inoculation induction test was used as a control group.

group challenge Inoculation number Sp2 (antigen protein 35)

1.410 ¹⁰ CFU / ml 9 Sp3 ( S. parauberis S1 whole bacterin) 5 Sp4 (PBS) 10

② Experimental judgment

The daily survival rate should be recorded and the relative survival rate of the vaccine group should be 50% according to the following formula when the cumulative mortality rate of the control group is 60% or more. The relative percent survival (RPS) calculation equation is shown in Equation (1).

③ Experimental results

As shown in the following Table 4, the result of analysis of the vaccine efficacy of the antigen candidate protein using zebrafish is summarized. (SPB_0027, metal ABC transporter substrate-binding lipoprotein) antigen protein and attacked with S. parauberis type 1 live organisms, two mice died after 1 day of inoculation with the inoculum, but 7 mice survived to finally survive And the survival rate was 100%. In the Sp3 group, inoculated with S. parauberis type 1 whole bacterin and inoculated live cells, 3 out of 5 died and 2 survived and survival rate was 40%. In the Sp4 group, inoculated with control PBS and inoculated with viable cell count, one mouse was killed and eight mice out of 9 mice died, showing a survival rate of 11%. FIG. 4 is a graph showing the following Table 4.

Inoculation number No. of daily dead fish of Challenge Survival rate (%) Relative survival rate
(%) 0 One 2 3 4 5 6 7 Sp2 (antigen protein 35) 9 - 2** - - - - - - 7/7 (100%) 100% Sp3 ( S. parauberis S1 whole bacterin) 5 - 0 One One One - - - 2/5 (40%) 33% Sp4 (PBS) 10 - One** 2 3 3 - - 1/9 (11%) -

< Example  5> Crossover antigen response to causative bacteria causing streptococcal streptococcal infection

( S. iniae , YSFST01-82), S. parauberis serotype I (Department of Aquatic Animal Disease, College of Veterinary Medicine, Seoul National University), which is a recombinant fusion protein showing antigenicity in Example 3 above, S. parauberis P2) and S. parauberis serotype II ( S. parauberis strain YSFST02-111 , Chonnam National University) were identified.

Specifically, 2 g of a metal ABC transporter substrate-binding lipoprotein having antigenicity shown in Example 3 was subjected to SDS-PAGE, and the obtained gel was transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skim milk dissolved in PBS for 1 hour at room temperature to remove the nonspecific reaction and washed three times with PBST for 15 minutes. Subsequently, S. paraaferis serotype I (SP1) and S. parauberis serotype II (SP2) produced in S. cerevisiae (SI) -infected streptococcal antiserum and mouse produced in rabbit in 0.5% skim milk melted PBST Streptococcal streptococcal antiserum was added to each, and a first reaction was carried out at room temperature for 1 hour. The nitrocellulose membrane phosphorus was washed with PBST three times for 15 minutes each, and then subjected to secondary reaction with anti-mouse IgG and HRP-linked antibody (KOMABiotech) at room temperature for 1 hour. The nitrocellulose membrane was washed with PBST three times for 15 minutes each, and the antigen protein was identified using a Western bright ^™ Quantum Western blotting detection kit (Advansta).

As a result, the antigen protein 35 (SPB_0027, metal ABC transporter substrate-binding lipoprotein) selected in the above Example 3 was identified as S. parasuberis serotype I, S. parauberis serotype II, S. iniae (Fig. 5).

<110> Korea Research Institute of Bioscience & Biotechnology (KRIBB) <120> Vaccine composition using Streptococcus parauberis cytoplasmic          membrance protein in fishes <130> P14R16D0008 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 310 <212> PRT <213> Streptococcus parauberis cytoplasmic membrane protein <400> 1 Met Lys Lys Lys Phe Thr Phe Ala Leu Thr Ala Ala Leu Ser Leu Phe   1 5 10 15 Met Leu Ser Ala Cys Ser Thr Ser Gly Gln Gln Ala Lys Lys Glu Glu              20 25 30 Lys Leu Asn Val Val Ala Thr Asn Ser Val Ile Ala Asp Met Thr Lys          35 40 45 Asn Ile Ala Gly Asn Lys Val Asn Leu His Ser Ile Val Pro Val Gly      50 55 60 Gln Asp Pro His Glu Tyr Glu Pro Leu Pro Glu Asp Val Glu Lys Thr  65 70 75 80 Ser Ser Ala Asp Leu Ile Phe Tyr Asn Gly Ile Asn Leu Glu Asp Gly                  85 90 95 Gly His Ala Trp Phe Thr Lys Leu Ile Lys Asn Ala Lys Lys Glu Lys             100 105 110 Asn Lys Asp Tyr Phe Ala Val Ser Asp Gly Ile Asp Val Ile Tyr Leu         115 120 125 Glu Gly Gln Asn Glu Lys Gly Lys Glu Asp Pro His Ala Trp Leu Asn     130 135 140 Leu Glu Asn Gly Met Ile Tyr Ser Lys Asn Ile Ala Lys Gln Leu Ile 145 150 155 160 Lys Lys Asp Pro Lys Asn Lys Thr Tyr Tyr Gln Ser His Leu Lys Ser                 165 170 175 Tyr Leu Ala Lys Leu Asp Asn Leu Asp Lys Glu Ala Lys Ser Lys Phe             180 185 190 Asp Lys Ile Pro Ala Asp Lys Lys Met Ile Val Thr Ser Glu Gly Cys         195 200 205 Phe Lys Tyr Phe Ser Lys Ala Tyr Asp Val Ser Ser Ala Tyr Ile Trp     210 215 220 Glu Ile Asn Thr Glu Glu Glu Gly Thr Pro Ser Gln Ile Ser Thr Leu 225 230 235 240 Ile Glu Lys Leu Lys Lys Lys Lys Lys Leu Ser Ala Ile Phe Val Glu Ser                 245 250 255 Ser Val Asp Ser Arg Pro Met Lys Ser Val Ser Lys Asp Ser Gly Ile             260 265 270 Pro Ile Tyr Ser Glu Ile Phe Thr Asp Ser Val Ala Lys Lys Gly Lys         275 280 285 Pro Gly Asp Ser Tyr Tyr Ala Met Met Lys Trp Asn Leu Asp Lys Ile     290 295 300 Ser Glu Gly Leu Ser Lys 305 310 <210> 2 <211> 930 <212> DNA <213> Streptococcus parauberis <400> 2 atgaagaaga aatttacttt tgctttgact gctgctttgt cattattcat gctatctgct 60 tgctcgacta gtggtcaaca ggcaaaaaaa gaagaaaaat taaatgttgt tgcaactaat 120 tctgttattg cagatatgac aaaaaatata gccggtaata aggttaattt gcatagtatt 180 gttccagttg gtcaagaccc ccatgagtat gaaccattac ctgaagatgt tgaaaaaaca 240 agttcggcgg atttgatttt ttacaatgga attaacttag aagatggtgg acatgcttgg 300 tttactaaat tgattaagaa tgccaaaaaa gaaaaaaata aagattattt tgcggtatca 360 gatggtattg atgttatcta tttagagggg caaaatgaaa aaggtaaaga agatcctcat 420 gcttggttaa atcttgaaaa tggtatgatt tattcaaaaa atattgctaa gcaattaatc 480 aagaaagatc ctaaaaataa aacttactat caatctcatt taaaaagtta tttggcaaaa 540 ctagataatt tagataaaga agctaaatct aagtttgata agataccagc agataaaaaa 600 atgattgtaa caagtgaagg ctgcttcaaa tatttctcta aagcctatga tgttccatca 660 gcatatattt gggaaattaa tacagaagaa gaaggaacac cgagtcaaat ttcaactctc 720 attgagaaat taaaaaagaa aaaattatca gctatatttg ttgaatctag tgttgatagt 780 cgacctatga aatctgtttc taaggatagt ggaattccaa tttattctga aatttttaca 840 gattctgttg ccaaaaaagg gaaacctggt gatagttact atgccatgat gaaatggaat 900 ttagataaga tttcagaagg tttatcgaaa 930 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> metal ABC transporter substrate-binding lipoprotein forward          primer <400> 3 taagaaggag atatacatat gaagaagaaa tttacttttg c 41 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> metal ABC transporter substrate-binding lipoprotein reverse          primer <400> 4 gtggtggtgg tggtgctcga gtttcgataa accttctg 38

Claims (10)

1. A vaccine composition for treating or preventing a streptococcal infection of a fish comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
The vaccine composition for the prevention of streptococcal infection according to claim 1, wherein the fish is at least one species selected from the group consisting of flounder, perch, trout, oyster, and dome.
The vaccine composition for preventing streptococcal infection according to claim 1, wherein the streptococcal is Streptococcus parauberis .
An expression vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 2 encoding the polypeptide according to claim 1.
A host cell transformed with an expression vector according to claim 4.
6. The host cell according to claim 5, wherein the host cell is Escherichia coli.
A feed composition for the treatment or prevention of a streptococcal infection of a fish comprising the vaccine composition according to claim 1.
8. The feed composition according to claim 7, wherein the streptococcus is Streptococcus parauberis .
A method for treating or preventing a streptococcal infection of a fish by administering the vaccine composition according to claim 1 to the fish.
10. The method according to claim 9, wherein the streptococcal is Streptococcus parauberis .

Images