JP6041238B2 - Bacterial hemolytic hemolytic jaundice pathogen antigen polypeptide and marine vaccine containing the same - Google Patents

Description

  The present invention relates to a pathogenic antigen polypeptide of yellowtail hemolytic hemolytic jaundice and a marine vaccine containing the same. Furthermore, the present invention relates to a polynucleotide, an expression vector containing the polynucleotide, a transformant containing the expression vector, and the like useful for the production of the polypeptide and a marine vaccine.

  In order to solve the food shortage accompanying the global population growth, the importance of seafood and fishery resources is attracting attention, and the demand for aquaculture production is increasing year by year. In order to ensure a stable supply of seafood through aquaculture production, it is necessary to reduce the damage caused by infectious diseases that hinders planned production. In the past, treatments using drugs (antibiotics and synthetic antibacterial agents) have been carried out as countermeasures against infectious diseases, but in recent years, prevention using vaccines instead of drugs has become the mainstream from the viewpoint of "food safety and security". It has become.

  All aquatic vaccines currently marketed in Japan are inactivated vaccines using viruses and bacteria inactivated by chemical treatment as antigens. In order to produce an inactivated vaccine, it is essential to culture pathogens in large quantities at low cost. However, many marine microorganisms including fish disease bacteria are difficult to cultivate, and there are many cases where vaccine development does not proceed. Bacterial hemolytic jaundice pathogens are one of the hard-to-cultivate bacteria, and many researchers have tried to improve the culture method so far. Is seen as a barrier to vaccine development.

  The subunit vaccine is a vaccine using a recombinant protein prepared based on the genetic information of the pathogen infection protective antigen, and does not require the pathogen culture. However, there has been no application to bacterial diseases in the fisheries field, and it has been developed only for IPN viruses with a small genome size worldwide.

  Even in yellowtail culture, the developed vaccine has shown tremendous effects against some infectious diseases that have become a problem, and the use of medicines such as antibiotics has been reduced. On the other hand, the damage caused by bacterial hemolytic jaundice, for which no vaccine has been developed, has become prominent. This disease is widespread in cultured yellowtails in western Japan, and also occurs in large fish before shipment, resulting in a large economic loss (estimated damage of about 400 million yen in 2007). Moreover, even if infected fish survive, the effect of bilirubin causes fish meat to suffer for a long period of time, reducing the commercial value, so prevention of this disease is important. In view of the above, manufacturers and contractors are strongly demanded to develop vaccines against yellow hemolytic jaundice.

  Bacterial hemolytic jaundice pathogens have already been isolated and cultured (Non-patent Documents 1 and 2). There is a report that the draft genome analysis of the causative bacterium of the yellowtail hemolytic jaundice has been made (Non-patent Document 3), but no sequence information is disclosed. As a result of searching for an infection protective antigen gene of cultured yellowtail bacteriolytic hemolytic jaundice by expression cloning, it was reported that a protein with a molecular weight of 40 to 60 kDa that reacts with antiserum obtained from infection-resistant hyperbrachi was detected. (Non-Patent Document 4). By immunological screening using mouse antibody against yellowtail hemolytic hemolytic jaundice, three kinds of sequences (possible T4-like proximal tail fiber, phosphodiesterase, DNA topoisomerase IV subunit B) are encoded as novel major antigen genes. It has been reported that a clone was obtained (Non-patent Document 5).

Fish Disease Research (1993) Vol. 28, No. 3, 119-124 Fish Disease Research (1994) Vol. 29, No. 1: 25-28 "Draft Genome Analysis of Cause Bacteria of Bacterial Hemolytic Jaundice", Japanese Society of Fish Diseases (March 26, 2011) Abstract: 17 pages “Search for protective antigen gene of cultured yellowtail bacteriolytic hemolytic jaundice” Japanese Society of Fisheries Science (March 27, 2011) Abstract: 158 "Identification of a novel major antigen gene of the causative agent of yellowtail hemolytic hemolytic jaundice" Japanese Society of Fisheries Science (March 26, 2012) Abstract: p.141

  The present invention identifies an antigenic polypeptide having high vaccine activity against bacterial hemolytic jaundice of a fish of the genus Buri, and provides a subunit vaccine against bacterial hemolytic jaundice of a genus of fish using the antigenic polypeptide. The purpose is to do.

  The present inventors have conducted intensive studies in view of the above object. Until now, there has been little analysis of gene sequence information about pathogens of yellow-bacterial hemolytic jaundice that are difficult to culture. Therefore, first, the present inventors conducted a genome-wide analysis of the pathogen of this yellow bacterial hemolytic jaundice and determined about 1500 protein coding regions. Next, from these, 256 types of antigens that were estimated to be highly likely to be present on the surface of the cells were selected, recombinant proteins were prepared using Escherichia coli, and 145 types expressed in the soluble fraction were selected. Selected. Furthermore, among these soluble antigens, 64 types of antigens that strongly reacted with yellowtail antiserum that had tolerated bacterial hemolytic jaundice were selected as vaccine candidate antigens. As a result of inoculating yellowtails with these antigens and attacking with bacterial hemolytic jaundice pathogens three weeks later, they found four kinds of antigens having excellent vaccine effects and completed the present invention.

That is, the present invention relates to the following.
[1] A polypeptide comprising any one of the following amino acid sequences selected from (1) to (4) and having vaccine activity against bacterial hemolytic jaundice in a fish of the genus Buri:
(1) an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(2) an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(3) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16; and (4) A partial sequence of any amino acid sequence selected from the above (1) to (3), having a length of 10 amino acids or more.
[2] A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to [1].
[3] An expression vector comprising the polynucleotide according to [2].
[4] A transformant transformed with the expression vector according to [3].
[5] An antibody that specifically recognizes the polypeptide of [1].
[6] A composition comprising the polypeptide according to [1].
[7] A vaccine against bacterial hemolytic jaundice of a fish of the genus Buri, comprising the polypeptide of [1].
[8] A method for preventing bacterial hemolytic jaundice in a fish, which comprises administering an effective amount of the polypeptide according to [1] to the fish.
[9] A method for cultivating a fish of the genus Buri, comprising administering an effective amount of the polypeptide according to [1] to the genus Fish of the genus and rearing the fish.

INDUSTRIAL APPLICABILITY According to the present invention, there are provided an antigen polypeptide having high vaccine activity against bacterial hemolytic jaundice of a genus fish, and a subunit vaccine against bacterial hemolytic jaundice of a genus fish containing the antigen polypeptide.
Since the pathogen of bacterial hemolytic jaundice is difficult to cultivate, vaccine development has not progressed so far, but the antigen polypeptide and vaccine of the present invention are prepared in large quantities at low cost by using genetic recombination technology. Therefore, it contributes to the practical use of a vaccine against bacterial hemolytic jaundice in the fish of the genus Buri.

Mortality after inoculation with bacterial hemolytic jaundice pathogens in yellowtail immunized with various antigens Mortality after inoculation with bacterial hemolytic jaundice pathogens in yellowtail immunized with various antigens

1. Antigenic polypeptide of bacterial hemolytic jaundice pathogen The present invention comprises any amino acid sequence selected from the following (1) to (4), and has vaccine activity against bacterial hemolytic jaundice of a fish A polypeptide (hereinafter sometimes referred to as a polypeptide of the present invention) is provided:
(1) an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(2) an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(3) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16; and (4) A partial sequence of any amino acid sequence selected from the above (1) to (3), having a length of 10 amino acids or more.

  The amino acid sequences represented by SEQ ID NOs: 10, 12, 14, and 16 correspond to partial sequences of the amino acid sequences represented by SEQ ID NOs: 2, 4, 6, and 8, respectively.

  The polypeptide of the present invention has vaccine activity against bacterial hemolytic jaundice in the fish of the genus Buri. Examples of the fish of the genus Buri include yellowtail, Japanese cypress, amberjack, Japanese amberjack, and the like, preferably yellowtail.

  The presence or absence of vaccine activity against bacterial hemolytic jaundice in the genus Buri can be evaluated, for example, by the following method.

E. coli (BL21) killed by a vector expressing the polypeptide to be evaluated (pET30a) is administered intraperitoneally to yellowtail 0-year-old fish (body weight 70-140 g) (dosage 2 mg / tail) . Three weeks after antigen administration, the yellowtail is attacked with bacterial hemolytic jaundice pathogen (JBKA-6 strain) by intravascular tail inoculation (amount of attacking bacteria: 5.0 × 10 ^{1 to} 1.5 × 10 ² MPN / tail). The accumulated mortality rate is calculated 18 to 20 days after the attack, and when the mortality rate of the polypeptide administration group is lower than that of the control group ( E. coli administration), the polypeptide to be evaluated is It is evaluated that it has vaccine activity against bacterial hemolytic jaundice. The breeding water temperature of yellowtail is 17-25 ° C.

  Buri bacterial hemolytic jaundice pathogens are isolated and cultured, and the attack test of yellowtail with bacterial hemolytic jaundice pathogens is the “pathophysiology of cultured yellow jaundice” fish disease research (1995) 30: 1: 7. It can be carried out according to page -14.

  The amino acid sequence of (2) above is 70% or more, preferably 80% or more, more preferably 90% or more, with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. More preferably 95% or more, and still more preferably 98% or more.

  As used herein, “identity” of amino acid sequences refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm is an optimal alignment). Is the ratio of the same amino acid residue to the total overlapping amino acid residues in which one may consider the introduction of a gap into one or both of the sequences.

  The identity of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI blastp / Blast 2 sequences (National Center for Biotechnology Information Basic Local Alignment Search Tool) published on the NCBI Internet homepage under the following conditions ( Short queries = off / Expect threshold = 10 / Matrix = BLOSUM62 / Gap Costs = Existence: 11 Extension: 1 / Compositional adjustments = Conditional compositional score matrix adjustment / filter = off / Mask = off).

  The amino acid sequence of (3) above is an amino acid in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. Sequence, for example, (1) one or more (preferably 1 to 300, more preferably 1 to 100) in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 More preferably 1-30, even more preferably 1-10, most preferably 1, 2, 3, 4 or 5) amino acid sequences deleted, (2) SEQ ID NO: 2, 4, One or more amino acid sequences represented by 6, 8, 10, 12, 14 or 16 (preferably 1 to 300, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 -10, most preferably 1 2, 3, 4, or 5) amino acid sequence added, (3) one or more amino acid sequences represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 ( Preferably 1 to 300, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1, 2, 3, 4 or 5 amino acids) (4) one or more (preferably 1 to 300, more preferably 1 to 100) in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 Amino acid sequence in which one, more preferably 1 to 30, even more preferably 1 to 10, most preferably 1, 2, 3, 4 or 5 amino acids are substituted with other amino acids, or (5) Amino acid sequence in which the mutations (1) to (4) above are combined (The total number of amino acids deleted, substituted, inserted or added is preferably 1 to 300, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 290 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, for example, (1) 1 to 290 (preferably 1 to 193, more preferably 1 to 96, still more preferably 1 to 48, still more preferably 1 to 19) in the amino acid sequence represented by SEQ ID NO: 2 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 290 (preferably 1 to 193, more) in the amino acid sequence represented by SEQ ID NO: 2 Preferably 1 to 96, more preferably 1 to 48, even more preferably 1 to 19, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Represented by SEQ ID NO: 2 1 to 290 amino acids in the amino acid sequence (preferably 1 to 193, more preferably 1 to 96, still more preferably 1 to 48, still more preferably 1 to 19, most preferably 1, 2, 3, 4 Or an amino acid sequence in which 5 amino acids are inserted, (4) 1 to 290 (preferably 1 to 193, more preferably 1 to 96, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 2. 1 to 48, even more preferably 1 to 19, most preferably 1, 2, 3, 4 or 5 amino acid sequences substituted with other amino acids, or (5) the above (1) to The amino acid sequence in which the mutations of (4) are combined (the total number of amino acids deleted, substituted, inserted or added is 1 to 290, preferably 1 to 193, more preferably 1 to 96, still more preferably 1-48, even more preferred Ku is 1 to 19, and most preferably at 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 124 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4, for example, (1) 1-124 (preferably 1-83, more preferably 1-41, more preferably 1-20, still more preferably 1-8) in the amino acid sequence represented by SEQ ID NO: 4 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 124 (preferably 1 to 83, more) in the amino acid sequence represented by SEQ ID NO: 4 Preferably 1 to 41, more preferably 1 to 20, even more preferably 1 to 8, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Amino represented by SEQ ID NO: 4 1 to 124 (preferably 1 to 83, more preferably 1 to 41, still more preferably 1 to 20, even more preferably 1 to 8, most preferably 1, 2, 3, 4 or (4) 1 to 124 (preferably 1 to 83, more preferably 1 to 41, more preferably 1) of the amino acid sequence represented by SEQ ID NO: 4; Amino acid sequence in which 20 amino acids, even more preferably 1-8, most preferably 1, 2, 3, 4 or 5) amino acids are substituted with other amino acids, or (5) above (1)-( 4) combined amino acid sequence (total number of deleted, substituted, inserted or added amino acids is 1 to 124, preferably 1 to 83, more preferably 1 to 41, more preferably 1 To 20, more preferably 1 to 8, most preferably Preferably 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 108 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 6, for example, (1) 1 to 108 (preferably 1 to 72, more preferably 1 to 36, still more preferably 1 to 18, even more preferably 1 to 7) in the amino acid sequence represented by SEQ ID NO: 6 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 108 (preferably 1 to 72, more) amino acid sequence represented by SEQ ID NO: 6 Preferably 1 to 36, more preferably 1 to 18, even more preferably 1 to 7, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Amino represented by SEQ ID NO: 6 1 to 108 (preferably 1 to 72, more preferably 1 to 36, even more preferably 1 to 18, even more preferably 1 to 7, most preferably 1, 2, 3, 4 or An amino acid sequence in which 5 amino acids are inserted; (4) 1 to 108 (preferably 1 to 72, more preferably 1 to 36, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 6; Amino acid sequence wherein 18 amino acids, even more preferably 1-7, most preferably 1, 2, 3, 4 or 5) amino acids are substituted with other amino acids, or (5) above (1)-( 4) combined amino acid sequence (total number of deleted, substituted, inserted or added amino acids is 1 to 108, preferably 1 to 72, more preferably 1 to 36, still more preferably 1 To 18, more preferably 1 to 7, most preferably Preferably 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 294 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 8, for example, (1) 1 to 294 (preferably 1 to 196, more preferably 1 to 98, still more preferably 1 to 49, still more preferably 1 to 19) in the amino acid sequence represented by SEQ ID NO: 8 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 294 (preferably 1 to 196, more) in the amino acid sequence represented by SEQ ID NO: 8 Preferably 1 to 98, more preferably 1 to 49, still more preferably 1 to 19, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Represented by SEQ ID NO: 8 1 to 294 amino acid sequences (preferably 1 to 196, more preferably 1 to 98, still more preferably 1 to 49, still more preferably 1 to 19, most preferably 1, 2, 3, 4 Or an amino acid sequence in which 5 amino acids are inserted, (4) 1 to 294 (preferably 1 to 196, more preferably 1 to 98, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 8 1 to 49, even more preferably 1 to 19, most preferably 1, 2, 3, 4 or 5 amino acid sequences substituted with other amino acids, or (5) the above (1) to Amino acid sequence in which mutations of (4) are combined (the total number of amino acids deleted, substituted, inserted or added is 1 to 294, preferably 1 to 196, more preferably 1 to 98, still more preferably 1-49, even more preferred Ku is 1 to 19, and most preferably at 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 282 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 10, for example, (1) 1 to 282 in the amino acid sequence represented by SEQ ID NO: 10 (preferably 1 to 188, more preferably 1 to 94, still more preferably 1 to 47, still more preferably 1 to 18) , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 282 (preferably 1 to 188, more) in the amino acid sequence represented by SEQ ID NO: 10 Preferably 1 to 94, more preferably 1 to 47, still more preferably 1 to 18, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) SEQ ID NO: 10 1 to 282 (preferably 1 to 188, more preferably 1 to 94, still more preferably 1 to 47, still more preferably 1 to 18, most preferably 1, 2 to the amino acid sequence represented. (4) 1 to 282 (preferably 1 to 188, more preferably 1 to 94) of the amino acid sequence represented by SEQ ID NO: 10, More preferably 1 to 47, still more preferably 1 to 18, most preferably 1, 2, 3, 4 or 5 amino acid sequences substituted with other amino acids, or (5) the above ( 1) to (4) combined amino acid sequence (total number of deleted, substituted, inserted or added amino acids is 1 to 282, preferably 1 to 188, more preferably 1 to 94, More preferably, 1 to 47, More preferably 1 to 18, most preferably 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1-114 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 12, for example, (1) 1 to 114 (preferably 1 to 76, more preferably 1 to 38, still more preferably 1 to 19, even more preferably 1 to 7) in the amino acid sequence represented by SEQ ID NO: 12 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1-114 (preferably 1-76, more preferably) in the amino acid sequence represented by SEQ ID NO: 12 Preferably 1 to 38, more preferably 1 to 19, still more preferably 1 to 7, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Represented by SEQ ID NO: 12 1 to 114 amino acids in the amino acid sequence (preferably 1 to 76, more preferably 1 to 38, still more preferably 1 to 19, even more preferably 1 to 7, most preferably 1, 2, 3, 4 Or an amino acid sequence into which 5 amino acids have been inserted, (4) 1-114 (preferably 1-76, more preferably 1-38, and even more preferably in the amino acid sequence represented by SEQ ID NO: 12 An amino acid sequence in which 1 to 19, even more preferably 1 to 7, most preferably 1, 2, 3, 4 or 5) amino acids are substituted with other amino acids, or (5) the above (1) to Amino acid sequence in which the mutations of (4) are combined (the total number of amino acids deleted, substituted, inserted or added is 1-114, preferably 1-76, more preferably 1-38, still more preferably 1 to 19, even more preferably 1 7, and most preferably at 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 98 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 14, for example, (1) 1 to 98 (preferably 1 to 65, more preferably 1 to 32, still more preferably 1 to 16, even more preferably 1 to 6) in the amino acid sequence represented by SEQ ID NO: 14 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1-98 (preferably 1-65, more) in the amino acid sequence represented by SEQ ID NO: 14 Preferably 1 to 32, more preferably 1 to 16, even more preferably 1 to 6, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) Amy represented by SEQ ID NO: 14 1 to 98 in the acid sequence (preferably 1 to 65, more preferably 1 to 32, still more preferably 1 to 16, even more preferably 1 to 6, most preferably 1, 2, 3, 4 Or an amino acid sequence in which 5 amino acids are inserted, (4) 1 to 98 (preferably 1 to 65, more preferably 1 to 32, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 14. An amino acid sequence in which 1 to 16, even more preferably 1 to 6, most preferably 1, 2, 3, 4 or 5 amino acids are substituted with other amino acids, or (5) the above (1) to Amino acid sequence in which the mutations of (4) are combined (the total number of deleted, substituted, inserted or added amino acids is 1 to 98, preferably 1 to 65, more preferably 1 to 32, still more preferably 1-16, even more preferably 1-6, most Is Mashiku is 1, 2, 3, 4 or 5).

  In one embodiment, the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which 1 to 150 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 16, for example, (1) 1 to 150 (preferably 1 to 100, more preferably 1 to 50, still more preferably 1 to 25, still more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 16 , Most preferably 1, 2, 3, 4 or 5 amino acid sequences deleted, (2) 1 to 150 (preferably 1 to 100) in the amino acid sequence represented by SEQ ID NO: 16 Preferably 1 to 50, more preferably 1 to 25, still more preferably 1 to 10, most preferably 1, 2, 3, 4 or 5 amino acid sequences, (3) SEQ ID NO: 16 1 to 150 (preferably 1 to 100, more preferably 1 to 50, still more preferably 1 to 25, still more preferably 1 to 10, most preferably 1, 2 to the amino acid sequence represented. (4) 1 to 150 (preferably 1 to 100, more preferably 1 to 50) of the amino acid sequence represented by SEQ ID NO: 16, More preferably 1-25, even more preferably 1-10, most preferably 1, 2, 3, 4 or 5) amino acid sequences substituted with other amino acids, or (5) above ( 1) to (4) combined amino acid sequence (total number of deleted, substituted, inserted or added amino acids is 1 to 150, preferably 1 to 100, more preferably 1 to 50, More preferably, 1-25, More preferably 1-10, most preferably 1, 2, 3, 4 or 5).

  In one preferred embodiment, the amino acid sequences of (2) and (3) above are natural animal mysterin polypeptides. “Natural” means that the amino acid sequence constituting the polypeptide is encoded in the genome of the natural pathogen of the bacterial hemolytic jaundice of the fish of the genus Buri.

  It is known that there are usually polymorphisms (individual differences) in the nucleotide sequence constituting a gene. The amino acid sequences of (2) and (3) include amino acid sequences different from the amino acid sequence of (1) generated by polymorphism.

  The length of the amino acid sequence of the above (4) is not limited as long as the polypeptide of the present invention has vaccine activity against bacterial hemolytic jaundice in the fish of the genus Brassica, but usually 10 amino acids or more, preferably 15 amino acids or more, more preferably Is 20 amino acids or more, more preferably 50 amino acids or more, and still more preferably 100 amino acids or more.

  The length of the polypeptide of the present invention is not limited as long as it has vaccine activity against bacterial hemolytic jaundice of the fish of the genus Buri, and a polypeptide having a desired length can be selected according to the purpose of use. For example, the polypeptide of the present invention has a length of 3000 amino acids or less, 2500 amino acids or less, 2000 amino acids or less, 1500 amino acids or less, 1000 amino acids or less, 500 amino acids or less, 400 amino acids The following, 300 amino acids or less, etc. can be selected as appropriate.

  In the polypeptide of the present invention, the amino acid sequence of a portion other than any one of the amino acid sequences selected from (1) to (4) above indicates that the polypeptide of the present invention is a vaccine activity against bacterial hemolytic jaundice As long as it has, it will not specifically limit.

  In one embodiment, the N-terminus and / or C-terminus of the polypeptide of the present invention includes at least one tag polypeptide or signal sequence.

  A tag polypeptide refers to a polypeptide that is added to facilitate the detection and purification of the polypeptide. Examples of tag polypeptides include, but are not limited to, epitope tags, fluorescent polypeptides, immunoglobulin Fc regions, and the like. An epitope tag refers to a peptide that is specifically recognized by an antibody or other binding partner, specifically, a Flag tag, a polyhistidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, or an MBP tag. Etc. can be mentioned. Examples of the fluorescent polypeptide include GFP, YFP, RFP, CFP, BFP, EGFP and the like. Such tag polypeptides are well known to those skilled in the art, and various antibodies that specifically recognize the tag polypeptides are commercially available.

  The signal sequence refers to a polypeptide sequence that directs the transport and localization of the polypeptide from the synthesis site to a specific site inside the cell or outside the cell simultaneously with or after translation of the polypeptide. Examples of the signal sequence include a leader sequence that induces secretion of the polypeptide, a nuclear translocation signal sequence (for example, a nuclear translocation signal sequence of SV40 T antigen), a nuclear translocation signal sequence, a nucleolus localization signal, and the like. However, it is not limited to this. Such signal sequences are well known to those skilled in the art, and can be appropriately selected according to the purpose.

  In a preferred embodiment, the polypeptide of the present invention consists of any amino acid sequence selected from the above (1) to (4).

  The polypeptide of the present invention may be modified. Examples of such modifications include lipid chain addition (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.

In addition, the polypeptide of the present invention contains a suitable labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase). , Peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin) Etc.).

  In this specification, the term “polypeptide” is used to mean a salt thereof. Polypeptide salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The polypeptide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation to remove factors other than the target component has been performed, and the state existing in nature has been removed. The purity of the isolated or purified polypeptide of the present invention (ratio of the weight of the polypeptide of the present invention to the total polypeptide weight) is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, 100%).

  There is no restriction | limiting in particular about the manufacturing method of polypeptide of this invention, You may manufacture according to a well-known peptide synthesis method, and you may manufacture using a well-known gene recombination technique. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. If the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining portion, and the product has a protecting group, the protecting polypeptide can be eliminated to produce the desired polypeptide.

  When the polypeptide of the present invention is produced using genetic recombination technology, first, a polynucleotide encoding the polypeptide of the present invention as described later is obtained, and the host cell is expressed using an expression vector capable of expressing the polypeptide. The polypeptide can be produced by transforming and culturing the resulting transformant. A method for producing the polypeptide of the present invention using the polynucleotide and gene recombination techniques will be described later.

  The polypeptide of the present invention is useful as an active ingredient of a vaccine against bacterial hemolytic jaundice of a fish of the genus Buri.

2. Polynucleotide Encoding Bacterial Hemolytic Jaundice Pathogen Antigen Polypeptide The present invention provides a polynucleotide comprising a nucleotide sequence encoding the polypeptide of the present invention.

  The polynucleotide of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.

  Examples of the polynucleotide of the present invention include, but are not limited to, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. The nucleotide sequence represented by SEQ ID NO: 1 is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, the nucleotide sequence represented by SEQ ID NO: 3 is the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, The nucleotide sequence represented by SEQ ID NO: 5 is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6, the nucleotide sequence represented by SEQ ID NO: 7 is the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8, The nucleotide sequence represented by SEQ ID NO: 9 is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10, the nucleotide sequence represented by SEQ ID NO: 11 is the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 12, The nucleotide sequence represented by SEQ ID NO: 13 is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 14. The nucleotide sequence represented by SEQ ID NO: 15 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, encoding respectively.

  The polynucleotide of the present invention can be easily produced by utilizing a known gene recombination technique based on the sequence information described in the sequence listing of the present specification. For example, the polynucleotide of the present invention can be produced by designing an appropriate primer based on sequence information and performing RT-PCR using cDNA prepared from total RNA isolated from a bacterial hemolytic jaundice pathogen of yellowtail as a template. I can do it. Alternatively, the polynucleotide of the present invention may be synthesized by a polynucleotide synthesizer based on the sequence information described in the sequence listing of the present specification.

  The obtained polynucleotide encoding the polypeptide of the present invention can be used as it is or after digestion with a restriction enzyme or adding a linker, if desired. The polynucleotide may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  The polynucleotide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation to remove factors other than the target component has been performed, and the state existing in nature has been removed. The purity of the isolated or purified polynucleotide of the present invention (ratio of the weight of the polynucleotide of the present invention to the total weight of the polynucleotide) is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, 100%).

  The polynucleotide of the present invention is useful for producing the polypeptide of the present invention.

3. Expression vector and transformant The present invention provides an expression vector containing the polynucleotide of the present invention and a transformant containing the expression vector.

  The expression vector can be produced by operably linking the polynucleotide of the present invention to a promoter in an appropriate expression vector. Examples of the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host cell to be used.

  Host cells include prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.) and the like. Examples of eukaryotic cells include yeast (Saccharomyces cerevisiae, etc.), insect cells (night stealing larva-derived cell lines (Spodoptera frugiperda cell; Sf cells), etc.), mammalian cells (human cells (293, etc.)). Monkey cells (such as COS-7), Chinese hamster cells (such as CHO cells)), and the like.

  Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.

  Plasmid vectors that can be amplified in E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmid vectors that can be amplified in Bacillus subtilis (eg, pUB110, pTP5, pC194), and can be amplified in yeast Plasmid vectors (eg, pSH19, pSH15) and the like can be mentioned, and can be appropriately selected according to the type of host used and the purpose of use.

  The type of viral vector can be appropriately selected according to the type of host cell used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.

  In addition, a promoter that can initiate transcription in the host cell can be selected according to the type of host cell to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SRα promoter and the like are preferable.

The expression vector of the present invention may contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like in a functional manner, if desired. Good. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (sometimes abbreviated as Amp ^r ), neomycin resistance gene (Neo). G418 resistance) which may be abbreviated as ^r ).

  The expression vector of the present invention is preferably isolated or purified.

  Since the expression vector of the present invention can express the polypeptide of the present invention in an appropriate host cell, it is useful for producing the polypeptide of the present invention.

  The expression vector of the present invention is described above according to a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, protoplast fusion method, electroporation method, DEAE dextran method, Gene Gun gene transfer method, etc.) By introducing it into a host cell, a transformant into which the expression vector has been introduced (the transformant of the present invention) can be produced. The transformant can express the polypeptide of the present invention. The transformant of the present invention is useful for producing the polypeptide of the present invention.

  The polypeptide of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the type of host and isolating the polypeptide of the present invention from the culture. The transformant whose host is Escherichia is cultured in an appropriate medium such as LB medium or M9 medium, usually at about 15 to 43 ° C. for about 3 to 24 hours. The transformant whose host is Bacillus is cultured in a suitable medium, usually at about 30 to 40 ° C. for about 6 to 24 hours. The transformant whose host is yeast is cultured in a suitable medium such as a Burkholder medium, usually at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Culturing of a transformant whose host is an insect cell or an insect is carried out in an appropriate medium such as Grace's Insect medium supplemented with about 10% bovine serum, usually at about 27 ° C. for about 3 to 5 days. The transformant whose host is an animal cell is cultured in an appropriate medium such as MEM medium supplemented with about 10% bovine serum, usually at about 30 ° C. to 40 ° C. for about 15 to 60 hours. In any culture, aeration and agitation may be performed as necessary. For isolation or purification of the polypeptide of the present invention from the culture, for example, the cell lysate or the culture supernatant is subjected to a plurality of chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. Can be achieved.

4). Antibody specifically recognizing bacterial hemolytic jaundice pathogen The present invention provides an antibody specifically recognizing the polypeptide of the present invention (hereinafter sometimes referred to as the antibody of the present invention).

  “Specific recognition” of the antigen X by the antibody means that the affinity of the antibody for the antigen X in the antigen-antibody reaction is stronger than the affinity for an antigen other than the antigen X.

The antibody of the present invention can be produced by an existing general production method using the polypeptide of the present invention or a partial peptide having antigenicity as an immunogen. In this specification, the antibodies include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAbs), chimeric antibodies that can be produced using gene recombination techniques, humanized antibodies, single chain antibodies, human antibodies, and these Binding fragments of, but not limited to. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.

  The antibody of the present invention is useful as a reagent for detecting the polypeptide of the present invention and the bacterial hemolytic jaundice pathogen of the fish of the genus Buri.

5. Vaccine against bacterial hemolytic jaundice in a fish of the genus Buri The immune reaction against the polypeptide of the present invention is induced by immunizing a fish of the genus Bacilli with the polypeptide of the present invention, and bacterial hemolysis comprising the polypeptide of the present invention or an antigenic epitope thereof The jaundice pathogen is also eliminated by this acquired immune response. Thus, yellowtails immunized with the polypeptides of the present invention acquire resistance to infection with bacterial hemolytic jaundice pathogens. Accordingly, the present invention provides a vaccine against bacterial hemolytic jaundice in the fish of the genus Buri (including the polypeptide of the present invention) (hereinafter sometimes referred to as the vaccine of the present invention). By administering an effective amount of the polypeptide of the present invention to the fish of the genus Buri, bacterial hemolytic jaundice in the fish of the genus Buri can be prevented.

  The fish to be administered with the vaccine of the present invention is a fish belonging to the genus Buri (Buri, Hiramasa, Kampati, Hironaga Kampati, etc.), which is an infection target of bacterial hemolytic jaundice pathogen, and preferably a yellowtail.

  The vaccine of the present invention is not limited to the one containing only the polypeptide of the present invention, and may further contain a pharmaceutically acceptable liquid or solid carrier. The present invention also provides such a composition (a pharmaceutical composition for fisheries). Examples of the liquid carrier include water, phosphate buffer (PBS), and physiological saline. Examples of the solid carrier include excipients such as talc and sucrose. The form of the vaccine of the present invention is not particularly limited and may be any of an injection, an oral preparation, and an immersion agent. However, an injection form that has a long-lasting effect with a small amount of administration should be adopted. Is preferred. In the case of an oral preparation, the polypeptide of the present invention may be mixed with normal fish feed.

  The vaccine of the present invention is administered to an administration subject by a technique usually used in aquatic vaccines, for example, an injection method, an immersion method, an oral method, or the like. In the injection method, the vaccine of the present invention is inoculated intraperitoneally, intramuscularly, subcutaneously, intradermally, intravenously, etc. (preferably intraperitoneally) into an injectable fish. In the immersion method, the fish is immersed for about 0.05 to 24 hours in a liquid containing the components of the vaccine of the present invention. Since the immersion method may reduce the vaccine effect as compared with the injection method, booster immunization may be performed as necessary. In the oral method, a feed containing the components of the vaccine of the present invention is freely fed. When the oral method is adopted, continuous administration for 5 to 14 days is desirable.

  The vaccine for injection can be prepared by suspending the polypeptide of the present invention in a sterilized fish physiological saline or the like. In addition to the polypeptide of the present invention and physiological saline, the injectable vaccine includes a suspending agent, a stabilizer, an emulsifier, a buffer, a preservative, a solubilizing agent, Other suitable additives can also be blended.

  Conventionally, various adjuvants have been used to improve vaccine effects and the like. In one embodiment, the vaccine of the present invention can exert a sufficient vaccine effect without using an adjuvant. However, the present invention does not limit the use of an adjuvant, and an adjuvant can be blended in addition to the above components as desired.

  Examples of adjuvants include, but are not limited to: oily adjuvants (mineral, plant and animal fats, oil soluble vitamins such as vitamin E), surfactants for adding these, alum , Aluminum compounds, bentonite, muramyl dipeptide derivatives, interleukins, endotoxins.

  In one embodiment, the vaccine of the present invention is substantially free of oily adjuvants. Since the vaccine of the present invention can exert a sufficient vaccine effect, it is not necessary to add an oil-based adjuvant. When using a vaccine containing an oil-based adjuvant in aquaculture of edible fish, it is necessary to provide a landing period that is sufficient to remove the oil-based adjuvant remaining in the recipient's body, but a vaccine that does not substantially contain an oil-based adjuvant. By using it, this landing prohibition period becomes unnecessary. In addition, “substantially does not contain” means that the content of the oil-based adjuvant is less than the vaccine effect enhancing amount. In a preferred embodiment, the vaccine of the present invention does not contain an oily adjuvant.

  The amount of the polypeptide of the present invention contained in the vaccine of the present invention is not particularly limited as long as it can achieve prevention of bacterial hemolytic jaundice pathogen infection in the fish of the genus Buri when used as the vaccine of the present invention. It can be appropriately selected depending on the form of antigen, administration route, administration subject and the like. For example, when E. coli dead bacteria transformed with a vector expressing this polypeptide are administered by injection, the amount is 0.5 to 5 mg / 100 μL (E. coli dead bacteria protein amount / volume).

  The dose of the vaccine of the present invention is not particularly limited as long as it can achieve prevention of bacterial hemolytic jaundice pathogen infection in the fish of the genus Buri, and can be appropriately selected depending on the mode of antigen, administration route, administration subject, etc. . For example, when E. coli killed bacteria transformed with a vector expressing this polypeptide are administered by injection, the E. coli killed protein is usually 0.5 to 5 mg / tail.

  In addition, since the effective amount can be appropriately adjusted by increasing or decreasing the volume of the vaccine administered to fish, the content of the polypeptide of the present invention in the vaccine of the present invention is limited to the above. There is no.

  The desired dose when intraperitoneally injecting the vaccine of the present invention into a fish varies depending on various factors such as the amount of active ingredient contained in the vaccine to be administered, the type of fish, age, and body weight, and should be specified in general. I can't. However, if the dose is too large, the administration work becomes complicated, and if the dose is too small, there is a concern that an error in the dose for each administration may increase. About 0.025 to 0.5 mL is preferably injected intraperitoneally according to the body weight.

  The vaccine of the present invention can be administered without being particularly limited by the weight, age, etc. of the fish if it is a fish of the genus Buri. In order to use the vaccine more effectively, it is preferable to administer the vaccine before infection with a bacterial hemolytic jaundice pathogen, for example, at the stage of fry.

  The vaccine of the present invention may be administered once as long as the vaccine effect (bacterial hemolytic jaundice pathogen infection prevention effect) continues, but may be administered multiple times. Multiple doses can be expected to enhance the vaccine effect. The administration interval in the case of multiple administration is usually 1 to 30 days. Moreover, the frequency | count of administration is 2-5 times normally. The frequency of administration of the vaccine of the present invention is preferably 1 to 3, most preferably 1.

  The present invention also provides a method for cultivating the fish of the genus Buri, which comprises administering an effective amount of the vaccine of the polypeptide of the present invention to the fish of the genus Buri, and rearing the fish of the genus Buri.

  Breeding fish are known in the art and are raised until they are large enough for shipment to the market while feeding under appropriate artificial conditions.

  In one embodiment, a vaccine of the invention that is substantially free of oily adjuvant is administered to fish. By using such a vaccine, it is possible to land the product before the landing prohibition period (for example, 49 weeks after the administration of the vaccine), which must be provided when using an oil-based adjuvant, and ship it to the market.

  All references cited in this specification, including publications, patent documents, etc., are cited by reference as if they were individually incorporated by reference specifically and in their entirety. To the same extent, it is incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

[Test Example 1]
(Selection of antigen candidates)
Bacterial cell hemolytic hemolytic jaundice pathogen JKBA-6 strain isolated in Kagawa Prefecture in 1991 was cultured in L-15 medium supplemented with 10% calf serum, and the cells were collected and genomic DNA Extracted. Next, the extracted genomic DNA was fragmented to prepare a library, and genomic sequencing was performed using a plurality of next-generation sequencers.

  Specifically, 187,150 reads were analyzed using Roche 454 GS FLX Titanium and approximately 65 million base pairs were decoded. Of these, 173,826 reads (93%) were assembled, resulting in 215 contigs and 2,345 singletons.

  Next, genome analysis was performed by the paired end method (method for arbitrarily determining the base sequence present at a position of 0.2 kb or more) using the next-generation sequencer of Illumina. The amount of data acquired was 43,005,291 sequences (78 bases per sequence), and a total base sequence of 3.4 Gbp was determined.

  Furthermore, genome analysis was also performed by the mate pair method (a method for arbitrarily determining a base sequence present at a position separated by 3.0 kb or more). The amount of data acquired was 354,647,712 sequences (100 bases per sequence), and a total of 35 Gbp (35 billion base pairs) was determined.

  As a result of assembling these analysis results, the entire genome of this pathogen was summarized as seven large sequence information (scaffold). About 1,500 ORFs were predicted, and antigen candidate genes were predicted using PSORTb and LipoP 1.0 server based on this sequence information, and 427 were selected as candidates.

  Of the 427 candidates, 256 types of antigens likely to be present on the surface of the cells were prepared using Escherichia coli to produce recombinant proteins, and 145 types expressed in the soluble fraction were selected. In addition, pET30a (manufactured by Novagen) was used as an expression vector for producing a recombinant protein, and BL21 was used as Escherichia coli.

  Next, 64 types of antigens were selected from 145 types of antigens, which strongly reacted with yellowtail antiserum that had tolerated pathogenic infection of cytobacterial hemolytic jaundice.

(Vaccine efficacy evaluation)
64 types of killed Escherichia coli each expressing the above 64 types of antigens were used. By combining 2 types of each of these E. coli killed bacteria, 32 sets of prototype vaccines were prepared and immunized by intraperitoneal administration to yellowtail 0 year old fish (body weight 136.5 g) (dosage 2 mg as E. coli killed protein) /tail). As a control, JBKA-6 strain formalin killed (FKC), PBS and E. coli were similarly inoculated intraperitoneally. The breeding water temperature throughout the test was 17.7 to 19.9 ° C.

After 3 weeks of immunization, the yellowtail was attacked with the JBKA-6 strain by intravascular tail inoculation (1.4 × 10 ² MPN / tail of attacking bacteria), and the mortality rate was evaluated over time. (Including one type of antigen candidate) (Table 1), a protective effect equivalent to or better than the positive control JBKA-6 strain formalin killed vaccine was obtained (FIG. 1).

The same protective effect was confirmed in a test conducted separately under conditions of a water temperature of 22.2 to 23.0 ° C., an average fish weight of 77.7 g, and an amount of attacking bacteria of 5.6 × 10 ¹ MPN / tail.

In order to identify protective antigens from 4 groups (8 types) of mixed antigens that showed a protective effect, 10 antigens were individually inoculated intraperitoneally with 8 antigens in the same manner as described above. Four weeks after inoculation, they were attacked with bacterial hemolytic jaundice and observed for 18 days of mortality. As a result, the mortality rate was 40% in the PBS group as the control group (no vaccine) and 60% in the E. coli group, while 20% and 1-5 in the 3A2 antigen in vaccine set 1-2. The death rate was 10% for 2B7, 20% for 1C10 in 1-8, and 0% for 3G1 in 3-1. That is, vaccine efficacy was recognized with one antigen each from four vaccine sets (FIG. 2).
At the same time as this test, the above four sets of mixed antigens were also tested. The 18-day mortality rate was 20% for 1-2, 0% for 1-5, 10% for 1-8, and 3-1. 10%.

  The full-length amino acid sequences of 3A2, 2B7, 1C10, and 3G1 are shown in SEQ ID NOs: 2, 4, 6, and 8, respectively. In addition, the corresponding cDNA sequences of 3A2, 2B7, 1C10, and 3G1 are shown in SEQ ID NOs: 1, 3, 5, and 7, respectively. In the vaccine efficacy confirmation test, a polypeptide comprising a partial sequence of each antigen was used. The partial sequences used are as follows.

  When analyzed by BlastP, 2B7 and 1C10 were no-hit. For 3A2, a hypothetical protein derived from Dysgonomonas mossii and a unknown protein derived from Grouper iridovirus were hit for 3G1, respectively. Max-identity was 37% and 35%, respectively. Therefore, all antigens for which the vaccine effect was confirmed were considered to be novel genes.

INDUSTRIAL APPLICABILITY According to the present invention, there are provided an antigen polypeptide having high vaccine activity against bacterial hemolytic jaundice of a fish of the genus Brix, and a subunit vaccine against bacterial hemolytic jaundice of yellowtail containing the antigen polypeptide.
Since the pathogen of bacterial hemolytic jaundice is difficult to cultivate, vaccine development has not progressed so far, but the antigen polypeptide and vaccine of the present invention are prepared in large quantities at low cost by using genetic recombination technology. Therefore, it contributes to the practical use of a vaccine against bacterial hemolytic jaundice of the genus Buri.

Claims (9)

A polypeptide comprising any one of the following amino acid sequences selected from (1) to ( 3 ) and having vaccine activity against bacterial hemolytic jaundice in a fish of the genus Buri:
(1) an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
Table in and (3) (a) SEQ ID NO: 2; (2) SEQ ID NO: 2,4,6,8,10,12,14 or 16 amino acid sequence having the amino acid sequence 95% or more identity represented by 1-48 amino acids are deleted in the amino acid sequence, substitution, insertion or addition in the amino acid sequence;
(B) an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 4;
(C) an amino acid sequence in which 1 to 18 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 6;
(D) an amino acid sequence in which 1 to 49 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 8;
(E) an amino acid sequence in which 1 to 18 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 10;
(F) an amino acid sequence in which 1 to 19 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 12;
(G) an amino acid sequence in which 1 to 16 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 14; or
(H) An amino acid sequence in which 1 to 25 amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by the amino acid represented by SEQ ID NO: 16 .   A polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1.   An expression vector comprising the polynucleotide according to claim 2.   A transformant transformed with the expression vector according to claim 3.   An antibody that specifically recognizes the polypeptide according to claim 1.   A composition comprising the polypeptide of claim 1.   A vaccine against bacterial hemolytic jaundice of a fish of the genus Buri comprising the polypeptide of claim 1.   A method for preventing bacterial hemolytic jaundice in a fish, comprising administering an effective amount of the polypeptide according to claim 1 to the fish.   A method for cultivating a fish of the genus Buri, comprising administering an effective amount of the polypeptide according to claim 1 to the fish of the genus Buri, and rearing the fish.