JP2008127283A - Vaccine for feline infectious peritonitis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vaccine for prophylaxis of FIP (feline infectious peritonitis) caused by infection of FIPV (feline infectious peritonitis virus), to provide a method for prophylaxis and a method for diagnosis of the FIP using the vaccine and to provide a reagent for testing the infection of the FIP. <P>SOLUTION: The vaccine for the FIP comprises a protein containing the same or substantially the same amino acid sequence as the amino acid described in sequence number 3 and an amino acid sequence in which a polynucleotide of the same or substantially the same base sequence as the polynucleotide described in sequence number 3 is inserted into a plasmid for expressing Escherichia coli and expressed as an active ingredient and contains preferably a solution composed of anhydrous mannitol oleate ester and squalane as an adjuvant, the method for prophylaxis of the FIP comprises administering the vaccine to a cat. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a vaccine used for the prevention of feline infectious peritonitis (FIP) caused by infection with feline infectious peritonitis virus (FIPV), and a method for preventing and diagnosing FIP.

  FIP is a chronic progressive viral infectious disease found in many felines, and when it develops, it exhibits symptoms such as fever, loss of appetite, inactivity, vomiting, diarrhea, and dehydration. Exudate type in which exudate accumulates, or exudative type that forms pyogenic granulomas in abdominal organs such as liver, kidney, spleen, and organs such as eyes, central nerve, lung, etc., or a mixture thereof. In general, the exudative type has an acute course, and the non-exudative type has a chronic course. The route of infection is thought to be oral infection by FIPV contained in the excrement of affected cats. FIPV is a virus belonging to the genus Coronavirus, has a diameter of about 100 to 150 nm, has an envelope, and has a helical capsid structure inside. The main structural proteins are spike (S protein), membrane (M protein), and nucleocapsid (N protein). The genome of the virus is a single-stranded RNA with a positive strand of about 20,000 bases. .

  FIPV is classified into type I (slow growth) and type II (fast growth) because of the difference in growth in cultured cells. There are variations in the gene sequence of type I viruses, but there are no such reports among type II viruses (see, for example, Non-Patent Document 1). Moreover, unlike type I virus, type II feline coronavirus containing FIPV can be propagated in feline kidney passage cells (CRFK) in which the gene sequence of the S protein region cannot be infected by type I by recombination ( For example, see Non-Patent Document 2.) Furthermore, between type I and type II, the M protein and the N protein cross immunologically, but the S protein shows little crossing. 70% of cats actually infected with FIP are type I, and the rest are type II (see, for example, Non-Patent Document 3). Nevertheless, vaccine development so far has been done using type II. The reasons for this are the slow growth rate in type I tissue culture and the difficulty in assembling infection experiments due to the lower pathogenicity to cats than type II.

  FIPV is characteristic among coronaviruses by causing antibody-dependent enhancement (ADE). ADE means that an antibody, which is a biological defense mechanism for excluding foreign substances, promotes cell entry of viruses and worsens symptoms. Cats naturally infected or vaccinated with FIPV and having neutralizing antibodies in the body do not have antibodies, and often exhibit symptoms that are far more severe and faster than cats that have been infected for the first time (for example, Non-Patent Document 1). See). Although all major neutralizing epitopes exist on the S protein of FIPV, it is known that many of them are infection-enhancing epitopes (see, for example, Non-Patent Documents 4 to 5). On the other hand, although N protein does not contain an infection-enhancing epitope, it does not exist on the surface of virus particles or infected cells. Therefore, it is considered difficult to completely prevent infection by immunization with N protein alone. In fact, there has been a report that a recombinant expressed with vaccinia virus in the frame of a gene encoding the N protein of type II FIPV has been used for vaccines, but data supporting the infection prevention effect has not been obtained (Patent Document 1) reference.).

  Among the three major structural proteins of FIPV, S protein having neutralizing epitope and infection-enhancing epitope has been used for vaccine antigens from early on. Venema et al. Produced a recombinant vaccine in which a gene encoding an antigenic determinant (S protein) relating to neutralization of type II virus was incorporated into vaccinia virus. However, infection was not protected and results of earlier onset were obtained ( For example, refer nonpatent literature 6.). For the type I S protein, the entire nucleotide sequence of DNA relative to the gene RNA is determined, and the polypeptide encoded thereby is expressed in E. coli (see Patent Document 2). However, there is no report about the effect of the vaccine. In addition, peptide fragments or fusion proteins containing a specific amino acid sequence of the feline corona S gene have been used in diagnostic and therapeutic vaccines (see Patent Document 3). Furthermore, a vaccine using a chimeric corona S protein as an antigen (see Patent Documents 4 and 5), and a vaccine using an S protein obtained by mutating a major epitope causing an infection enhancing effect by mutagenesis (see Patent Document 6). Etc.

  In addition, there is an inactivated vaccine using an immunogenic FIPV precursor cultured in a Crandall cat kidney cell line, but it has not been put into practical use (see Patent Document 7). Although live vaccines using attenuated temperature-sensitive strains (see Patent Document 8) have been put to practical use, evaluations in Europe and the United States regarding the effectiveness and safety of the vaccines vary. This is probably because temperature sensitive strains that cannot grow at high temperatures have limited sites where they can grow in the body. In addition, there are live vaccines in which FIPV nucleotides are inserted into the ORF of feline herpesvirus (see Patent Document 9), and those using a modified poxvirus-FIPV recombinant as an antigen (see Patent Document 10).

  Among those using N protein, those relating to recombinant raccoon poxvirus expressing N protein and M protein (see Patent Document 11) and those relating to nucleotide sequences encoding N protein and M protein (see Patent Document 12) Although there is a vaccine consisting of DNA encoding N protein and physiological saline (see Patent Document 13), it has not yet provided a safe and effective vaccine. Furthermore, there is a vaccine in which the gene region of the N protein of type I FIPV is expressed by baculovirus and immunized three times with an existing cat triple-type vaccine (see Patent Document 14). ). This is not the result of FIPV alone, and since immunization is performed three times despite the fact that immunization is generally performed twice, it is considered that the immunogenicity is low and it is not practical.

  As described above, at the present time, no vaccine has been developed that is sufficiently satisfactory with respect to the protective effect, safety and practicality of FIPV infection.

US Patent No. 5811104 JP 7-327683 A JP 2003-93078 A JP 2003-180385 A JP 2004-313196 A Special table 10-505236 JP 7-504897 gazette Japanese Patent No. 2608935 Special Table 2000-501927 Special Table 2000-501930 Japanese Patent Laid-Open No. 7-184661 Japanese Patent No. 2903129 JP 2000-302692 A JP 2004-35494 A Pedersen et al., Am. J. et al. Vet. Res. , 41, 868, (1980) Herreweh et al., J. MoI. Virol. , 72, 4508, (1998) Takara et al., Arch. Virol. , 117, 85, (1991) Corapi et al. Virol. , 66, 6695, (1992) Olsen et al. Virol. , 66, 956, (1992) Vennema et al., J. MoI. Virol. , 64, 1407 (1990)

  The present invention provides a vaccine useful for preventing FIP infection, a method for preventing feline infectious peritonitis using the vaccine, a diagnostic method, and a reagent for testing for co-infectious peritonitis infection.

  The feline infectious peritonitis vaccine according to the present invention contains, as an active ingredient, a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence set forth in SEQ ID NO: 3.

  In addition, the feline infectious peritonitis virus vaccine according to the present invention includes a protein fragment containing an epitope site having the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 3 and / or a fusion protein with the protein fragment Is contained as an active ingredient.

  Furthermore, the feline infectious peritonitis virus vaccine according to the present invention is an amino acid sequence obtained by inserting a polynucleotide having the same or substantially the same base sequence as the polynucleotide of SEQ ID NO: 3 into an E. coli expression plasmid. Contains a protein containing as an active ingredient.

  The feline infectious peritonitis virus vaccine according to the present invention as described above preferably contains an adjuvant, more preferably an oil adjuvant as the adjuvant, and the main component of the oil adjuvant is anhydrous mannitol oleate-added squalane solution. It is preferable that

  The method for preventing feline infectious peritonitis according to the present invention comprises administering the vaccine according to the present invention as described above to a cat, and preferably administering the vaccine to the cat at least twice within 30 days.

  Further, the method for diagnosing feline infectious peritonitis according to the present invention detects or quantifies a FIPV antibody in a biological sample using a protein containing the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 3 as an antigen. Is.

  Furthermore, the test reagent for feline infectious peritonitis infection according to the present invention comprises a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence set forth in SEQ ID NO: 3.

  The present invention provides vaccines and methods for the prevention of feline infectious peritonitis virus infection. The vaccine of the present invention uses a protein having an amino acid sequence encoded by the polynucleotide described in SEQ ID NO: 3 as an antigen. By using these proteins, a vaccine that can be expected to have preventive and / or therapeutic effects on a wide range of strains can be obtained. Moreover, since these proteins do not contain an infection-enhancing epitope, high safety can be expected for the vaccine of the present invention.

  Feline infectious peritonitis virus is a serious infectious disease that often has a lethal course after onset. Various vaccines have been tried so far, but their evaluation has not been determined. On the other hand, the vaccine of the present invention has an excellent preventive effect and high safety compared to known vaccines. Therefore, the vaccine of the present invention is useful for preventing infection with feline infectious peritonitis virus.

  The vaccine of the present invention comprises a protein containing the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 3 or a fragment thereof as an active ingredient. The amino acid sequence and the base sequence encoding the amino acid sequence are obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 derived from the FIPV type I strain Yayoi strain and the base sequence shown in SEQ ID NO: 2. A method for preventing FIP using a protein encoded by this gene is not known. FIPV type I and type II are classified by the difference in antisera against S protein. The homology between the gene and amino acid sequence of the S protein in both is less than 50%. On the other hand, FIPV N protein shows 90% or more homology between type I and type II, and antisera against type I and type II N proteins show cross-reactivity. That is, this type I virus can be used as a common antigen not only for type I but also for type II FIPV. Furthermore, the protein described in SEQ ID NO: 1 induces an immune response that suppresses the growth of FIPV, but does not contain an infection-enhancing epitope. Therefore, a vaccine excellent in safety and preventive effect can be provided. Furthermore, by adding an adjuvant that has been confirmed to be safe to N protein, which is a vaccine antigen, the immune effect is further enhanced, and further, two immunization injections can be used to safely protect cats from highly fatal FIPV infections. It became possible to prevent.

  In the present invention, a desirable amino acid sequence is an amino acid sequence having a homology of 93% or more, more preferably 98% or more, with respect to SEQ ID NO: 3. The number of amino acid residue mutations that can be introduced into the amino acid sequence shown in SEQ ID NO: 3 is usually 26 or less, preferably 7 or less. In order to maintain the three-dimensional structure of the original protein, the amino acid to be substituted is desirably an amino acid having a chemical property close to that of the original amino acid. Specific examples of such conservative substitution include, for example, mutual substitution between nonpolar amino acids of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Asn, Gln. And the substitution between acidic amino acids Asp and Glu, and mutual substitution of basic amino acids with Lys, Arg, and His.

  A protein fragment containing an amino acid sequence constituting an epitope can also be used as the active ingredient of the vaccine of the present invention. The epitope site of a protein can be determined by several known methods. As long as the protein fragment is a fragment immunologically equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 3, it can be used alone or in combination with a plurality of fragments consisting of different amino acid sequences. You can also. Even if a single fragment is not immunologically equivalent, a combination of fragments is included in the present invention as long as it shows immunologically equivalent activity by combining multiple fragments. . In addition, protein fragments that can achieve immunologically equivalent activity by a combination of auxiliary components, for example, immunologically equivalent activity to a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 by blending an immunostimulant The protein fragments shown are also included in the present invention. Furthermore, artificially synthesized protein fragments such as fusion with other proteins can also be used in the present invention.

  The host immune response to the administered protein is indexed by 1) antibody titer against the administered protein, 2) cellular immunity activation, 3) tolerance to attack virus, and differences from controls in clinical findings. Can be compared.

1) Comparison by antibody titer The antibody titer against the administered protein can be measured by various immunoassays. For example, the antibody titer can be measured by adding a serum sample of a test animal to a plate sensitized with a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 and further reacting with a labeled antibody against the immunoglobulin of the animal. it can.

2) Comparison by cellular immunity activation effect The cellular immunity activation effect by the administered protein can be evaluated by CTL assay that measures the degree of activation of T cells in peripheral blood in vitro. . In addition, a method of measuring the activation level of T cells by a technique such as ELISA or real time PCR using perforin, which is a biomolecule involved in cytokines or cytotoxicity, as an index is also used.

3) Comparison of immunological equivalence by an attack test The immunological equivalence of an administered protein can be evaluated by inoculating an individual who has been administered the protein with a pathogen and its protective effect. The protective effect can be compared by clinical findings such as body temperature and body weight after inoculation with a pathogen, or survival rate and survival days.

  Although the polynucleotide described in SEQ ID NO: 2 is derived from the type I FIPV Yayoi strain, the origin of the polynucleotide used as the active ingredient of the vaccine of the present invention is not particularly limited. The polynucleotide may be a natural polynucleotide, an artificially introduced polynucleotide, or a polynucleotide composed of an artificially synthesized sequence. For example, a polynucleotide derived from a mutant strain derived from a Yayoi strain, or a polynucleotide derived from FIPV having a base sequence having a high homology with the Yayoi strain can also be used. Moreover, the polynucleotide which introduce | transduced the artificial variation | mutation into the base sequence of Yayoi strain | stump | stock can also be used.

  Preparation and isolation of the polynucleotide described in SEQ ID NO: 2 can be achieved by utilizing known hybridization techniques and techniques such as polymerase chain reaction (PCR). The base sequence of the probe necessary for the hybridization method and the primer necessary for the PCR method can be designed from the cDNA sequence of the N protein described in SEQ ID NO: 2, for example.

  N protein used as an active ingredient in the vaccine of the present invention can be obtained by a known method. For example, the polynucleotide represented by SEQ ID NO: 2 is inserted into an appropriate expression vector and introduced into a host cell for expression. Examples of the host include bacteria, yeast, insect cells, mammalian cells and the like. More specifically, Escherichia coli for bacteria, Schizosaccharomyces pombe for yeast, and CHO cells and COS cells for mammalian cells. Known vectors can be used as vectors that can be transformed into these hosts. Among them, an expression system using E. coli and a plasmid vector is useful for producing the vaccine of the present invention. As an expression vector, a polynucleotide which is a target gene is incorporated into a replication gene (ColE1 ori) necessary for replication in E. coli and cloned in E. coli. The cloned expression vector can be recovered and transformed into competent cells to add IPTG to induce N protein expression in E. coli. The N protein obtained by the expression in this manner is purified if necessary after confirming the specificity using a monoclonal antibody against the N protein. As a purification method, a known method such as affinity chromatography or ion exchange chromatography is used.

  In the present invention, an arginine-treated protein containing an amino acid sequence expressed in Escherichia coli is preferable from the viewpoint of excellent action and effect as a vaccine.

  In the present invention, the effect of the vaccine can be remarkably improved by treating the protein containing the amino acid sequence expressed in Escherichia coli with arginine as described above. Here, although it is not clear as a reason why the action effect of the vaccine as described above is improved by the arginine treatment, it is considered that the expressed protein is reshaped (refolded) into a structure preferable as a vaccine. Arginine has the effect of increasing the solubility of proteins, and may contribute to the stabilization of protein preparations. Therefore, it is considered that these effects synergistically act by arginine treatment, and the action effect of the vaccine is remarkably improved.

The arginine treatment method is not particularly limited as long as it is a method in which arginine and the protein are brought into contact with each other, but a method in which the protein is dialyzed in a Tris-arginine buffer for a predetermined time is preferable. L-arginine is mentioned as an arginine used for a tris-arginine buffer solution. Moreover, there are no particular limitations on the amount of arginine and tris (tris (hydroxymethyl) aminomethane) in the buffer solution.
The buffer medium is water, but the addition of oxidized glutathione or the like as another component has the advantage that the treatment effect is improved.
In the present invention, for confirmation of the arginine-treated N protein, for example, the absorbance (320 nm) of a solution containing the N protein is measured, and the measured value after treatment is higher than that before treatment. This can be done by checking.

  The N protein of the present invention can be obtained from cultured cells infected with FIPV or chemically synthesized. For the culture of FIPV, primary cells or cell lines derived from mammals are used. In particular, cells derived from cats are suitable for FIPV culture. Specifically, fcwf4 cells and CRFK cells can be used. These cells can be obtained from a cell bank. As the chemical synthesis, a synthesis method is known in which amino acids are sequentially bonded to form an oligopeptide having a target amino acid sequence. A protein consisting of a long amino acid sequence that is difficult to synthesize can also be synthesized by linking protein fragments consisting of a relatively short amino acid sequence that is easy to synthesize.

  When the obtained N protein is used as it is as a vaccine, it cannot be expected to produce antibodies or to prevent infection. Therefore, for example, by formulating a suitable combination with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, etc. Therefore, it is necessary to enhance the production of antibodies against N protein and the protective effect against infection. Addition of various immunostimulants is also effective.

Examples of adjuvants included in vaccines include inorganic substances such as aluminum salts, microorganisms or microorganism-derived substances (BCG, muramyl dipeptide, pertussis, pertussis toxin, cholera toxin, etc.), surfactant substances (saponins) , Deoxycholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil), etc., and these can be used alone or in combination. As an adjuvant blended with the vaccine of the present invention, an oil adjuvant is particularly preferable. More preferred is an oil adjuvant mainly composed of squalane. Specifically, an anhydrous mannitol oleate-added squalane solution is preferable from the viewpoint of safety. The effect of this adjuvant is more remarkable than expected, and the vaccine of the present invention can acquire an excellent protective effect against FIP and high safety by blending this adjuvant.
The anhydrous mannitol oleate-added squalane liquid refers to a solution comprising anhydrous mannitol oleate and squalane.

  The vaccine of the present invention can be administered to animals by, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, or oral methods known to those skilled in the art. Yes. Although the dosage varies depending on the body weight and age of the animal, the administration method, the purpose of use, etc., those skilled in the art can appropriately select an appropriate dosage. In general, feline vaccines are inoculated twice at 2-3 week intervals after 8 weeks of age. Also in the vaccine of the present invention, feline FIP prophylaxis can be achieved by inoculating at least once after 8 weeks of birth, more preferably twice at 2-3 week intervals.

  The vaccine of the present invention is useful for the prevention of FIPV in cats. In the present invention, the prevention of FIPV refers to the action of inhibiting the onset by prophylactic administration to animals that have not been infected with FIPV, or to animals that are in the pre-onset state after infection. Inhibition of onset is 1) the action to prevent the onset itself, 2) the action to delay the onset, 3) the action to reduce the symptoms after the onset, 4) the action to accelerate the healing after the onset, 5) the survival rate after the onset The action of improving, 6) the action of inhibiting the infectivity from an individual who has developed to other individuals is included, and among these actions, the vaccine of the present invention is particularly effective in preventing the onset itself.

  The present invention also relates to a method for examining feline infectious peritonitis virus infection, which detects and / or quantifies antibodies in feline serum using a protein comprising the amino acid sequence shown in SEQ ID NO: 3 as an antigen. As shown in the Examples, N protein derived from the Yayoi strain reacts strongly with antisera from a wide range of FIPV strains. This indicates that the N protein of the Yayoi strain is highly useful as a FIPV vaccine, and at the same time, is particularly useful as a screening antigen for FIPV.

  A known technique can be used as a method for detecting and / or quantifying the antibody. For example, an antigen is immobilized on a solid phase, and an antibody that binds to the antigen can be detected by an antibody that recognizes the antibody (second antibody). Alternatively, the presence of an antigen-specific antibody can be detected by capturing an antibody contained in a sample on a solid phase and reacting the antibody with an antigen. In either method, the antibody or antigen can be labeled for easy detection. For labeling antigens and antibodies, enzymes, fluorescent substances, luminescent substances, colored particles, or radioisotopes are used. In addition, a method of detecting an antibody by using aggregation of an antibody on a particle carrier such as latex or gold colloid sensitized with an antigen as an index can be used. In addition to simple particle aggregation, immunochromatography is also available in which the particles bound to the antigen are moved by capillary flow and trapped in a detection zone where the second antibody is immobilized.

  An individual in which an antibody that binds to a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 is detected is diagnosed as having experience with FIPV infection. Alternatively, the degree of symptoms can be diagnosed by following changes in antibody titer. For example, an increase in antibody titer occurs following virus growth. Therefore, an individual whose antibody titer is increasing is likely to be accompanied by virus growth. If the antibody titer decreases after the symptoms are resolved, the individual is diagnosed as likely to have recovered.

  The present invention also relates to a reagent for testing feline infectious peritonitis virus infection, comprising a protein comprising the amino acid sequence set forth in SEQ ID NO: 3. The protein containing the amino acid sequence set forth in SEQ ID NO: 3 in the reagent of the present invention can be immobilized on a solid phase or particles according to the various immunoassay formats described above. Further, according to the various immunoassay formats described above, a diagnostic kit can be obtained by combining with a labeled antibody, additional reagents necessary for detecting the label, a positive control, a negative control, or the like.

  Hereinafter, the present invention will be described more specifically based on examples.

Purification of viral protein FIPV type II 79-1146 was purchased from ATCC and cultured in feline kidney-derived cell line (CRFK). As a culture solution, a solution obtained by adding 10% fetal calf serum (FCS) to Eagles minimum essential medium (E-MEM) was used.

Purification of virus CRFK cells were inoculated with 100 TCID ₅₀ virus, adsorbed at 37 ° C. for 1 hour, and then added with a culture medium and cultured. The culture supernatant was collected on days 2 to 3 where CPE was observed. The collected supernatant was concentrated, layered on a 20% to 60% sucrose density gradient, and centrifuged at 27,000 rpm for 2 hours to collect virus particle bands. The collected virus solution is dissolved in NTE buffer (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA), and then centrifuged at 100,000 g for 1 hour to dissolve the precipitate in NTE buffer. A virus solution was used for Western blotting.

Protein fraction:
Sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.1% BPB) was added to the purified virus, heat-treated at 100 ° C. for 5 minutes, and electrophoresed on 10% polyacrylamide gel. Electrophoresis (SDS-PAGE) was performed. The gel after electrophoresis was taken out, the position corresponding to the molecular weight of the N protein was judged from the position of the marker protein, and the gel at the position containing the N protein was cut out. The protein contained in the gel was collected to obtain a purified N protein, and the obtained purified N protein was used as an ELISA antigen.

Base sequence of FIPV type I Yayoi strain FIPV type I Yayoi strain is a virus (Hayashi, T., Yanai, T., Tsurudome, M., Nakayama, H., Watabe, 15 passages in the milk-only mouse brain). Y., and Fujiwara, K.,: 1982: Jpn. J. Vet Sci .; 43: 669-676.), A fetal fetus-derived cell line feline catus whole fetus. Incubated with (fcwf-4). As the culture solution, a solution obtained by adding 10% FCS to a culture solution in which E-MEM and L-15 medium were mixed in equal amounts was used.

Preparation of viral RNA:
After fcwf-4 cells inoculated with FIPV type I Yayoi strain were adsorbed at 37 ° C. for 1 hour, the culture solution was added and the cells were cultured in a CO ₂ incubator. Cells exhibiting CPE were detached and recovered with a cell scraper. For the infected cells, total RNA was extracted using ISOGEN (Nippon Gene), and finally the ethanol-precipitated and dried pellet was dissolved in RNase free water to obtain an RNA solution. From the RNA solution, mRNA was extracted with an mRNA purification kit (Pharmacia) using an Oligo-dT column.

RT-PCR:
P1 (sense side) 5′-CTGACAATTTGAGGTGAACATG-3 ′ (SEQ ID NO: 4)
P2 (antisense side) 5′-ACAGCATTGTAGGAAAACGAGC-3 ′ (SEQ ID NO: 5)
For RT-PCR, One Step RNA-PCR kit (TaKaRa) was used. Add 5 mM MgCl ₂ , 1 mM dNTP, 0.4 μM sense and antisense primer, 0.1 U / μL Taq polymerase, 0.1 U / μL reverse transcriptase and mRNA to the reverse transcriptase reaction buffer and reverse at 50 ° C. for 30 minutes. After inactivation of RTase at 94 ° C. for 2 minutes, the gene was amplified by PCR reaction of 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 2 minutes. The specificity of the PCR product was confirmed by 1% agarose gel electrophoresis.

Cloning:
The PCR product was precipitated with ethanol, dissolved in sterilized distilled water, the purified PCR product was mixed with the ligation buffer, and the plasmid vector pT7BlueVector (Novagen) was mixed. DNA ligase was added and a ligation reaction was performed at 16 ° C. for 1 hour. . The circularized DNA was transformed into competent cells of E. coli NovaBlue strain, plated on carbenicillin-added agar plate, and cultured overnight at 37 ° C. After colonizing the colony formed on the medium and culturing overnight with carbenicillin-added L-Broth, the plasmid was extracted and cut with a restriction enzyme, and the size of the DNA fragment was confirmed by 1% agarose gel electrophoresis. The success or failure of cloning was confirmed. Escherichia coli confirmed to be cloned was cultured overnight in carbenicillin-added L-Broth, and the recombinant plasmid DNA was extracted and purified.

Base sequence:
From the purified recombinant plasmid DNA, the base sequence of the N protein gene based on the dideoxy method was determined using the vector base sequence upstream and downstream of the DNA containing the N protein gene region as a primer. Of the total length of about 20 kb of the FIPV gene RNA, the base sequence and the deduced amino acid sequence of the N protein cDNA of Yayoi strain were analyzed and shown in SEQ ID NOs: 1 and 2. The N protein gene consists of an ORF of 1,134 bases from the initiation codon ATG to the termination codon TAA, which encodes 378 amino acids, and is calculated to express an initial protein of 43.47 kDa.

Homology:
Table 1 shows the N genes of the Yayoi strain, KU-2 strain, Black strain, UCD1 strain, 79-1146 strain belonging to FIPV type II, feline intestinal coronavirus type II (FECV) 79-1683 strain belonging to FIPV type I. Amino acid sequence homology was shown. By comparing this amino acid sequence, the gene of the Yayoi strain showed high homology of 90% or more with the other 5 strains. The homology in the table is calculated using the maximum matching of the genetic information processing software GENETYX, and the virus name indicates the virus described in the margin of Table 1, respectively. The numerical values in the table indicate the homology of the base sequence in the upper right of 100% and the amino acid sequence in the lower left as described on the diagonal line.

Recombinant N protein expression PCR:
Based on the obtained gene sequence, primers added with a restriction enzyme recognition sequence (Nde I, Xho I) were prepared so that an ORF (Open reading frame) of the N protein gene could be expressed. In the antisense primer, the underlined base was changed from cytosine to guanine to prevent the translated codon from becoming methionine.
P3 (sense side) 5′-TTGCATATGGCCACACAGAGAGACAACGCGTC-3 ′ (SEQ ID NO: 6)
P4 (antisense side) 5′-GGCTCGAGGTTCGTAACCCTCATCAAT G ATCTC-3 ′ (SEQ ID NO: 7)
Recombinant purified plasmid DNA into which 5 mM MgCl ₂ , 1 mM dNTP, 0.4 μM sense and antisense primers, 0.1 U / μL Taq polymerase, and N protein gene were inserted and cloned into the PCR reaction buffer was added, After 2 minutes at 94 ° C, the gene was amplified by a PCR reaction of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes, and 72 ° C for 5 minutes. The specificity of the PCR product was confirmed by 1% agarose gel electrophoresis.

Cloning:
Plasmid vector pT7BlueVector (Novagen) was used for cloning. The PCR product was precipitated with ethanol, dissolved in sterilized distilled water, the purified PCR product and the vector DNA fragment were mixed in a ligation buffer, DNA ligase was added, and a ligation reaction was performed at 16 ° C. for 1 hour. The circularized DNA was transformed into competent cells of E. coli NovaBlue strain, plated on carbenicillin-added agar plate, and cultured overnight at 37 ° C. The colonies formed on the medium were picked and cultured overnight in L-Broth supplemented with carbenicillin, and then the plasmid was extracted to reconfirm the nucleotide sequence and amino acid sequence (SEQ ID NO: 3). Moreover, it cut | disconnected with the restriction enzyme, the size of the DNA fragment was confirmed by 1% agarose gel electrophoresis, and the success or failure of cloning was confirmed. Escherichia coli confirmed to be cloned was cultured overnight in carbenicillin-added L-Broth, and the recombinant plasmid DNA was extracted and purified.

Construction of recombinant plasmid DNA Purified plasmid DNA was digested with restriction enzymes at the Nde I and Xho I sites located on the primers. In addition, pET16b (Novagen) was used as an expression plasmid vector, which was also digested with Nde I and Xho I restriction enzymes. Both were electrophoresed on a 1% agarose gel, the gel containing the DNA was cut out, and the DNA was extracted and purified. DNA ligase was added to both purified fragments, and a ligation reaction was performed at 16 ° C. for 1 hour to form a circular plasmid, which was transformed into competent cells and cloned.

Recombinant protein expression The purified cDNA was further transformed into Escherichia coli BL21 (DE3) strain, plated on chloramphenicol and carbenicillin agar plates, and cultured at 37 ° C overnight. The antibiotic-resistant colonies were fished, and 40 mL of carbenicillin-added L-Broth was cultured at 37 ° C. for 3 hours, and then the expression of the fusion N protein to which histidine was added was induced with 0.1 mM IPTG. Three hours after induction, the proliferated Escherichia coli was collected, solubilized using sonication and 0.5% Triton X-100, and then the ultracentrifugated supernatant at 100,000 g for 1 hour was collected. From this soluble fraction, the fusion N protein bound to nickel resin was eluted with imidazole and purified using nickel column affinity chromatography. The eluted purified protein was dialyzed against 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM EDTA, 200 mM NaCl and used in the experiment.

  Further, as another purification method, it can be purified by the following method. The FIPV N protein gene was inserted into pET16b at the Nde I-Xho I site, and a fusion N protein containing Hist-Tag was expressed in BL21 (DE3). After sonication, Escherichia coli was pulverized using a high-pressure homogenizer, and the supernatant centrifuged at 7000 rpm for 40 minutes was salted out with 50% saturated ammonium sulfate. The salted-out pellet was solubilized with 350 mL of 50 mM Tris-HCl (pH 9.0), 8 M urea, 2.5% glycerin, filtered through a 5,1,0.22 filter, made 1 L with the same buffer, Purification by exchange chromatography. Ion exchange chromatography was performed using Q-Sepharose FF, which is an anion exchanger. The equilibration solution was 50 mM Tris-HCl (pH 9.0), 8M urea, 2.5% glycerin, and elution was performed by a salt concentration gradient in which 1M NaCl was added to the equilibration buffer (FIG. 5). N protein elution fractions were prepared in two types: a sample dialyzed overnight at 4 ° C. with PBS (−) and a sample dialyzed according to the following buffer and procedure. Base buffer (50 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM EDTA, 200 mM NaCl) +4 M urea overnight at 4 ° C. Next, 4 ° C. for 4 hours with basic buffer + 2M urea + 375 μM GSSG (Oxidized glutathione) +400 mM L-arginine. Finally, dialysis was performed overnight at 4 ° C. against the basic buffer (hereinafter referred to as “Tris-arginine dialysis”).

Specificity Western blot:
A sample buffer was added to the prepared antigen and heat-treated at 100 ° C. for 5 minutes, followed by development with SDS-PAGE of 10% gel. The gel was removed and transferred to a PVDF membrane using a transfer device. The transfer film was immersed and blocked with 5% skim milk.
As primary sera, FIPV (79-1146 strain) -infected cat serum (type II antiserum) and anti-serum (type I antiserum) immunized with a purified virus of FIPV Yayoi strain were used. The transfer film was reacted at 37 ° C. for 1 hour, and then washed three times with PBS (−) containing 0.05% Tween20.
POD-labeled goat anti-cat IgG or POD-labeled goat anti-rabbit IgG was used as secondary serum. After immersing in a serum diluted solution to which the labeled antibody was added and reacting at 37 ° C. for 1 hour, the plate was washed 3 times with 0.05% Tween 20-added PBS (−). The transfer film is dipped in a substrate solution (0.02% DAB, 0.006% H ₂ O ₂ , 0.05M Tris-HCl (pH 7.5)) for about 5 to 20 minutes, and then developed with distilled water. The reaction was stopped by washing.

Purification and detection of expressed N protein From the results of Western blotting, the recombinant N protein having the amino acid sequence of SEQ ID NO: 3 was not eluted with 50 mM imidazole in nickel column affinity chromatography, but could be eluted with 500 mM imidazole.
The N protein of FIPV Yayoi strain has a calculated molecular weight of about 43 kDa, but is expressed as a fusion protein of about 48.4 kDa by adding the fusion protein on the vector. An estimated 48.4 kDa band was detected from SDS-PAGE CBB staining and immunoblot images using anti-FIPV Yayoi rabbit serum or FIPV (79-1146 strain) -infected cat serum. Furthermore, since it reacted with both anti-sera of FIPV type I and type II, it was confirmed that the expressed protein was N protein (FIGS. 2, 3, and 4). On the other hand, fractions 1 and 2 that were purified and eluted with an ion exchanger were analyzed by SDS-PAGE and Western blot. As a result, the elution fraction purified by ion exchanger chromatography from an immunoblot image using infected cat serum. Since the peak 1 was detected as a molecular weight of 48.5 kDa in calculation, it was confirmed that the N protein of FIPV was contained in a high concentration in the eluted fraction of peak 1 (FIGS. 5 and 6).

Examination of adjuvants O included in four types of mixed inactivated vaccines [Kyoto Microken Filine-4 and Kyoto Microken Filine-7 (trade name, Microbial Chemistry Laboratories, Inc.)] marketed in Japan as adjuvants / W (oil-in-water) type oil adjuvant, anhydrous mannitol oleate (AMOE) added squalane solution (anhydrous mannitol oleate 6 vol%, makeup base squalane 40 vol%), ISA which is also an O / W type oil adjuvant Using ELISA-25 (Sepic) and aluminum hydroxide gel (Reheis), the ELISA antibody titer against the expressed N protein was compared, and the optimal adjuvant was selected. N protein antigen and each adjuvant were mixed so that 3 mg of coronavirus antibody-negative SPF cats of 3 to 3.5 months of age were used per group, and 0.1 mg per animal was mixed to make 1 mL. The anti-N-ELISA antibody titer was measured for the sera obtained by immunizing the cat neck subcutaneously twice at intervals of 3 weeks.
As a result, no increase in antibody was observed in the aluminum hydroxide gel in the second immunization. AMOE-added squalane showed a higher antibody titer than ISA-25. Therefore, in subsequent infection protection tests, AMOE-added squalane solution was used as an adjuvant (FIGS. 7, 8, and 9).

Preparation of vaccine Antigen preparation:
The purified expressed N protein (N protein consisting of the amino acid of SEQ ID NO: 3) was measured for the absorbance of the antigen (wavelength 562 nm) with a spectrophotometer by the BCA method (Pierce), and the amount of the antigen protein was determined from the BSA calibration curve. Calculated and adjusted to 0.1 mg per 0.5 mL.

Adjuvant:
From the above examination results, 0.5 mL of AMOE-added squalane as an adjuvant was mixed with 0.5 mL of antigen and mixed well to obtain a vaccine. As a control, 0.5 mL of PBS was mixed with 0.5 mL of PBS and used. One dose of the above vaccine was 1 mL.

Immunity test (1)
animal:
As experimental animals, 3-3.5 month-old coronavirus antibody-negative SPF cats were used. Four (Cat No. 1, 2, 3, 4) were used for the N protein vaccine immunization group adjusted to 100 μg per head, and 2 (Cat No. 5, 6) were used for the control group (Cat No. 5, 6). Table 2).

Vaccination:
One dose of vaccine was inoculated subcutaneously in the neck twice at 3-week intervals (FIG. 1). Blood was collected immediately before each immunization, and the antibody titer of the serum was measured.

Attack method:
An attack test was performed on the cats of the immunization test 2 weeks after the second immunization (FIG. 1). As an attacking virus, FIPV type II strain 79-1146 was used and orally inoculated with 10 ^3.0 TCID ₅₀ (1 mL) (Table 2). Clinical symptoms were observed daily from the day of virus inoculation until the end of the experiment. Body temperature and body weight were measured every 1 to 3 days. Rectal and oral wipes (swabs) were collected at appropriate times and used after virus isolation. In addition, blood was collected in a timely manner for antibody measurement.

Changes in body temperature:
Four immunized cats and two control cats did not show fever due to two immunizations at 3 week intervals. Two control cats showed transient fever exceeding 40 ° C. on the second day and third day after challenge, and the immunized cat (No. 4) also on the third day. Two control cats showed persistent fever for about 10 days from the 13th and 15th day after challenge until death. Fever immunized cat no. No. 4 showed a heat generation exceeding 40 ° C. twice after a transient heat generation. Other immunized cats maintained a normal temperature of about 38 ° C. to 39 ° C. (FIG. 10).

Change in weight:
During the immunization period (5 weeks after the first immunization), all cats showed a steady increase in body weight. Control cat no. No. 5 shows the control cat No. 3 from the third day after the first attack. No. 6 showed a decrease in body weight from the second day after the first attack, and thereafter a temporary recovery of the body weight was observed, but a decrease in body weight was shown from the 17th day after the attack until death. The immunization cat No. 4 also showed a decrease in body weight from the third day after the attack, but then decreased and died from the 29th day after repeated weight increase and decrease. Other immunized cats showed a steady increase in body weight even after challenge (FIG. 11).

Blood count:
The white blood cell count was determined in the control cat no. With 5 and No6, it fell to about 50 × 10 ² / μL and died. In other immunized cats, no extreme increase or decrease was observed (FIG. 10). The red blood cell count diminished to about 500 × 10 ⁴ / μL in the control cat after the attack and died. In addition, the dead immunized cat no. 4 also fell to the same level. There was no extreme increase or decrease in other immunized cats. The hematocrit value also changed almost the same as the number of red blood cells (FIGS. 12, 13, and 14).

Virus detection:
Rectal swabs collected after challenge were serially diluted 10-fold, inoculated into CRFK cells, and observed at 37 ° C. for 1 week using CPE (cytopathic effect) as an index. Further, RNA was extracted from rectal swabs, and viral genes were detected by RT-PCR, thereby confirming virus growth in vivo. In virus isolation from rectal swabs after challenge, no virus that infects CRFK cells was observed in any cat. In addition, in virus detection by RT-PCR method, no. On the third day at 1.2,6, no. No. 2, 3, 4 on the 14th day. 2 at 49 days. The relationship between the presence of virus in the rectum and the onset of this disease could not be derived (Table 3). However, for oral swabs, the control cat no. 5 is the third day after the attack, and also the control cat no. FIPV was detected by RT-PCR on day 3 and day 7. On the other hand, in the immunized cat, no virus was detected by either virus isolation or RT-PCR method (Table 4).

Measurement of neutralizing antibody titer:
An equal amount of 0.025 mL of feline serum diluted 4-fold in a 96-well plate and a virus solution adjusted to 200 TCID ₅₀ were mixed and reacted at 37 ° C. for 1 hour. CRFK cells were added thereto at 1 × 10 ⁶ / well and cultured for 1 week. The reciprocal of the highest dilution of feline serum that completely suppressed CPE (cytopathic effect) was taken as the neutralizing antibody titer.
Neutralizing antibody was detected from 2 weeks after challenge in 1 N protein immunized cat (No. 4) and 2 control cats (No. 5, 6), and from 0 to 12 weeks for other immunized cats , Both were less than 4 times. As a result of preventing the invasion of the attacking virus by the initial infection protection, the immunized cat did not produce neutralizing antibodies and ADE did not occur. However, it was speculated that neutralizing antibodies were produced in individuals that allowed virus invasion, and the viral load was increased by ADE (Table 5).

Measurement of anti-N antibody titer by ELISA:
The N protein extracted from the virus-infected cell culture supernatant was used as an antigen, diluted with an immobilization buffer, dispensed to an ELISA plate, and immobilized at 4 ° C. overnight. After washing with PBS (−) containing 0.05% Tween 20 and blocking with PBS (−) using 0.3% bovine serum albumin and 5% saccharose, the mixture was allowed to stand at 37 ° C. for 30 minutes. After washing, the feline serum collected as the primary serum was diluted 100 times with a diluent and allowed to react at 37 ° C. for 1 hour. After washing, the diluted POD-labeled goat anti-cat IgG antibody was reacted at 37 ° C. for 1 hour. After washing, the substrate solution was added to develop color at room temperature for 30 minutes, the reaction stop solution was added, and the absorbance was measured at 492 and 630 nm.
From the second week after two immunizations at intervals of 3 weeks, the immunized cat no. In 1, 2, and 4, an increase in the anti-N-ELISA antibody titer was confirmed. In contrast, control cat No. No increase in anti-N antibody was observed in 5 and 6; 3 showed a slight antibody response compared to the control cat. Immunization cat no. No. 4 showed a high antibody titer from the second week after the challenge, and maintained a high antibody titer for 4 weeks after death. Control cat no. In 5 and 6, the antibody titer increased from 1 to 2 weeks after death from the 3rd week after the attack. Immunization cat no. 1 and 2 did not change the antibody titer against virus attack and maintained a constant antibody level. On the other hand, the immunized cat no. 3 gradually increased the antibody titer from the third week after the attack. An extreme increase in anti-N antibody after the attack causes neutralization antibodies against the attacking strain to be produced in cats that could not protect against the initial infection, which ultimately causes an antibody-dependent promoting action (ADE) against the attacking strain, resulting in・ Death with pleural effusion. However, immune cat no. For 1, 2, and 3, it was speculated that neutralizing antibodies were not produced by protecting against the initial infection of the virus, and ADE was avoided (FIG. 15).

Survival rate:
Control cat no. No. 6 is No. 26 on the 26th day after the attack. 5 died on the 29th day and the lethality of the control cat was 100%. Both cats were confirmed to have ascites by necropsy. On the other hand, among N protein immunized cats, no. 4 died with ascites retention 48 days after challenge, but survived longer than control cats. This means that the resistance to the virus in the initial infection was stronger than that of the control cat, and although it would die from ADE, the neutralizing antibody-bearing cat was not prematurely killed by ADE, but a vaccine. The life-prolonging effect by is shown. The immunized cat that survived the first challenge survived for about 2 months after the first challenge, and thus the survival rate of N protein immunized cats was 75% (3 out of 4) (FIG. 16).
On the other hand, each sample obtained by dialysis of N protein purified by ion exchanger with PBS and Tris-arginine was mixed with AMOE-added squalane oil adjuvant so as to be 100 μg / mL. As a result of an infection protection test using orally infected with 10 ^3.0 TCID ₅₀ (1 mL), the non-inoculated control group died of pooled ascites or pleural effusion, In PBS dialysis, 3 out of 5 animals died with ascites or pleural effusion, whereas in Tris-arginine dialysis, 4 out of 5 animals survived. (FIG. 17). From this result, the N protein expressed in Escherichia coli was solubilized by urea and its three-dimensional structure was lost, but the natural type or a similar three-dimensional structure was reconstructed by Tris-arginine dialysis rather than PBS dialysis. It seems to have induced the function.

Reinfection protection test attack method:
Three immunized cats (No. 1, 2, 3) that did not show infection or symptoms of FIPV to the first virus attack were re-attacked 9 weeks after the first attack. Attack, using 79-1146 strain similar to the first, the 10 ^3.0 TCID ₅₀ a (1 mL) were orally inoculated and observed 8 weeks (Fig. 1).

Changes in body temperature:
No. No. 2 showed a transient fever exceeding 40 ° C. from the 4th day after the re-attack, and then a fever around 40 ° C. continued again from the 37th day after the re-attack until the end of the test. No. 1 and 3 maintained a normal temperature around 38.5 ° C. for 8 weeks and showed no signs of infection (FIG. 8).

Weight gain:
No. In 1 and 3, no increase or decrease in body weight was observed for 8 weeks after re-attack, and the weight of the adult cat was maintained. No. No. 2 gradually decreased in weight from the 28th day after the re-attack (FIG. 9).

Blood count:
The white blood cell count is no. In 3, it increased gradually after re-attacking, but was within the normal range. Other cats also maintained normal values (FIG. 12). As for erythrocytes, all cats are within the normal range. 2 showed a gradual decreasing trend after re-attack (FIG. 13). The hematocrit value is No. 2 showed a downward trend after re-attack compared to other cats (FIG. 14).

Virus detection:
From 0, 3, 7, 10, 14, 28, and 42 days after reinfection, virus could not be detected by virus isolation using CRFK cells and detection by RT-PCR method. It was shown that virus growth due to reinfection was suppressed compared to the first attack (Tables 3 and 4).

Neutralizing antibody titer:
Immunization cat no. In No. 2, the neutralizing antibody increased from the second week after the re-challenge, and after 6 weeks the antibody titer was 65,536 times higher. In contrast, immune cat no. 1 and 3 showed no increase in neutralizing antibody against re-attack, indicating infection protection against virus re-attack (Table 6).

Measurement of anti-N antibody titer by ELISA:
The immunized cat No. whose neutralizing antibody titer was negative. 1 and 3 show an O.D. of about 1.0 of the anti-N antibody that had been maintained until the re-attack. D. Values continued to carry the same level of anti-N antibody until week 22 when the study was terminated. In contrast, the immunized cat No. 2 is the O.O. D. A value of 3.5 or more was shown in the second week after the re-attack from a value of 1.7. It was surmised that ADE increased the number of re-attacked viruses, but survived for 8 weeks until the end of the study (Figure 15).

Autopsy findings and survival:
After the test, No. As a result of necropsy of 2, a large amount of ascites had accumulated and may have died after the end of the study, but the control cat in the first challenge died within 4 weeks after the challenge, compared to 8 weeks. Surviving the above shows that the immune effect of this vaccine has also shown a life-prolonging effect against re-attack. In addition, other immune cat No. As a result of necropsy of 1 and 3, no ascites or pleural effusion was retained, and no pathological changes such as nodules in major organs were observed. Therefore, the survival rate in re-attack was 100% (FIG. 16).

Utility:
In general, vaccinations for animals expect a booster effect for unvaccinated animals and maximize their protective effects by two or more vaccinations. In this vaccine, the production of anti-N protein antibody was confirmed by two immunizations, and two immunized cats produced neutralizing antibodies that induce ADE against two virus attacks with a lethality of 100%. First, it achieved protection against this disease. In addition, the other two immunized cats showed a life-prolonging effect despite the production of neutralizing antibodies. This seems to be mainly due to the enhancement of immunity by adjuvants, which strengthened cellular immunity and shows significance compared to vaccines against FIP viruses so far. On the other hand, as a more practical production method than affinity chromatography purification, the superiority of the method of tris-arginine dialysis of N protein purified by an ion exchanger in the presence of urea was confirmed by an infection protection test. Due to the effect of arginine on protein remodeling, the survival rate of the uninoculated control group in the infection protection test was 0%, whereas the survival rate of the group inoculated with Tris-arginine dialyzed purified FIPV was 80%. (FIG. 17).

Infection prevention test settings and schedule. SDS-PAGE image (CBB staining) of affinity chromatography purification of expressed N protein. Lane 1: Before purification. Lane 2: Nickel resin non-adsorbed solution. Lanes 3, 4, 5, 6: 50 mM imidazole eluate. Lanes 7-12: 500 mM imidazole eluate. Western blotting image of affinity chromatography purification of expressed N protein (detected using FIPV79-1146 strain-infected cat serum and POD-labeled goat anti-cat IgG antibody). Lane 1: Before purification. Lane 2: Nickel resin non-adsorbed solution. Lanes 3, 4, 5, 6: 50 mM imidazole eluate. Lanes 7-12: 500 mM imidazole eluate. Western blotting image of affinity chromatography purification of expressed N protein (detected using FIPV Yayoi strain purified virus immunized rabbit serum and POD-labeled goat anti-rabbit IgG antibody). Lane 1: Before purification. Lane 2: Nickel resin non-adsorbed solution. Lanes 3, 4, 5, 6: 50 mM imidazole eluate. Lanes 7-12: 500 mM imidazole eluate. Ion exchange chromatography purification of expressed N protein 1M NaCl concentration gradient and elution peak at 280 nm SDS-PAGE (CBB staining) of the expressed protein purified by ion exchange chromatography (left) and Western blotting image (detected using FIPV Yayoi strain purified virus immunized rabbit serum and POD-labeled goat IgG antibody) (right) Lane 1: Before purification Lane 2: Peak 1 Lane 3: Peak 2 Anti-N-ELISA antibody titer in cats immunized with expressed N protein and aluminum hydroxide adjuvant (Al ₂ O ₃ ). Anti-N-ELISA antibody titer in cats immunized with expressed N protein and ISA-25 adjuvant. Anti-N-ELISA antibody titer of cats immunized with expressed N protein and anhydrous mannitol oleate added squalane (AMOE-S). Changes in the body temperature of the cat during the test period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Changes in the weight of cats during the study period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Changes in the white blood cell count of cats during the study period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Changes in the number of red blood cells in cats during the study period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Changes in cat hematocrit during the study period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Transition of anti-N protein antibody titer during the test period. Cat No. 1,2,3,4 are expressed N protein immunized cats. No. 5 and 6 are control cats. The black arrow is vaccinated. The white arrow is attack virus inoculation. Survival rate after challenge test (1). The white arrow is attack virus inoculation. Survival rate of challenge test of affinity chromatography purified N protein immunized cat and control cat Survival rate after challenge test (2). The white arrow is attack virus inoculation. Ion exchange chromatography purified N protein immunized cat and control cat survival rate

Claims (11)

  A feline infectious peritonitis virus vaccine containing, as an active ingredient, a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 3.   A feline infectious peritonitis virus vaccine containing, as an active ingredient, a protein fragment containing an epitope site of the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 3 and / or a fusion protein with the protein fragment.   The vaccine according to claim 1, comprising as an active ingredient a protein comprising an amino acid sequence expressed by inserting a polynucleotide identical or substantially identical to the polynucleotide of SEQ ID NO: 3 into an E. coli expression plasmid.   The vaccine according to claim 3, wherein a protein containing an amino acid sequence expressed in Escherichia coli is treated with arginine.   Furthermore, the vaccine in any one of Claims 1-4 containing an adjuvant.   The vaccine according to claim 5, wherein the adjuvant is an oil adjuvant.   The vaccine according to claim 6, wherein the main component of the oil adjuvant is an anhydrous mannitol oleate-added squalane solution.   The prevention method of the feline infectious peritonitis which administers the vaccine of any one of Claims 1-7 to a cat.   The method for preventing feline infectious peritonitis according to claim 8, wherein the vaccine is administered to the cat at least twice within 30 days.   A method for diagnosing feline infectious peritonitis, wherein a FIPV antibody in a biological sample is detected or quantified using a protein comprising the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 3 as an antigen.   A reagent for testing feline infectious peritonitis infection, comprising a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence set forth in SEQ ID NO: 3.

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