WO2007023628A1

WO2007023628A1 - Secretory antigen 1 protein of babesia gibsoni, dna encoding the same and use thereof

Info

Publication number: WO2007023628A1
Application number: PCT/JP2006/314492
Authority: WO
Inventors: Xuenan Xuan; Honglin Jia; Jinlin Zhou; Hiroshi Suzuki; Kozo Fujisaki; Ikuo Igarashi
Original assignee: National University Corporation Obihiro University Of Agriculture And Veterinary Medicine
Priority date: 2005-08-25
Filing date: 2006-07-21
Publication date: 2007-03-01
Also published as: JP2007053987A; JP4752054B2

Abstract

[PROBLEMS] It is intended to provide a major antigen gene and a major antigen protein of B. gibsoni protozoa. Further, it is intended to provide a diagnostic antigen, a vaccine for prevention and the like for canine B. gibsoni infection using the gene. [MEANS FOR SOLVING PROBLEMS] A DNA encoding a secretory antigen 1 (BgSA1) protein of B gibsoni protozoa; an expression product of a BgSA1 gene (recombinant BgSA1 protein); a synthetic peptide based on a BgSA1 gene sequence; a therapeutic preparation using an antiserum or an antibody against the recombinant BgSA1 protein and the BgSA1 synthetic peptide; a diagnostic preparation using the recombinant BgSA1 protein or the BgSA1 synthetic peptide as an antigen; a DNA diagnosis targeting the BgSA1 gene; a vaccine preparation for prevention using the recombinant BgSA1 protein or the BgSA1 synthetic peptide; a recombinant virus vaccine in which the BgSA1 gene has been integrated; a plasmid vaccine in which the BgSA1 gene has been integrated (DNA vaccine).

Description

Specification

Secreted antigen 1 of Babesia gibsoni, DNA encoding this protein, and use thereof

Technical field

[0001] The present invention relates to a DNA encoding a secretory antigen 1 (BgSAl) of dog Babesia gibsoni (hereinafter referred to as B. gibusoni), a recombinant BgSAl protein, a BgSAl synthetic peptide, and the like. In addition, the present invention relates to a method for diagnosing protozoal infections and a therapeutic / preventive agent for protozoal infections.

Background art

[0002] B. gibsoni is an erythrocytic protozoan parasite that is transmitted by ticks, and canine infectious diseases (B. gibsoni infection) are widely recognized throughout the world including Asia, Africa, Europe and North America. ing. In Japan, the power that has been reported in almost all regions nationwide from Kyushu to Hokkaido, particularly in western Japan.

[0003] Dogs infected with B. gibsoni have anorexia, tendency to sleep, loss of energy, fever, anemia, vomiting, hematuria, hemoglobinuria, splenomegaly, hemoglobininemia, thrombocytopenia, systemic lymphadenopathy Symptoms such as Fever is caused by a pyrogen released during hemolysis or when the monocyte phagocyte system responds to infected erythrocytes or protozoa. Platelet depletion is thought to be caused by suppression of bone marrow production, platelet sequestration in the liver and spleen, increased platelet consumption, and immune-mediated platelet destruction. B. gibsoni-infected dogs can also cause progressive anemia during surgery and immunosuppressive therapy, preventing recovery from the underlying disease and smooth treatment.

[0004] Canine B. gibsoni infection is mainly treated with diminazen preparations, but this product has side effects such as causing neurological symptoms, liver damage, and kidney damage caused by cerebellar hemorrhage. , Its use is limited.

[0005] The diagnosis of this protozoan infection is currently performed mainly by confirming protozoa in blood smears, but this method has a problem of low detection sensitivity for chronically infected dogs. On the other hand, a serological diagnostic method using a fluorescent antibody method is also used, but cross-reaction with other erythrocytic parasites (B. canis) is observed, and its specificity is problematic. [Fukumoto, S., X uan, X., Nishikawa, Y., Inoue, N., Igarashi, I., Nagasawa, H., Fujisaki, K., and Mika mi, T .: Identification and expression of a 50— kilodalton surface antigen of Babesia gi bsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 39: 2603-2609, 2001.]

[0006] An effective vaccine for canine B. babesia infection has not yet been developed. A major factor is that the search for effective antigens related to infection protection is delayed. Against this background, the search for the major antigens of B. gibsoni, the establishment of diagnostic methods with high sensitivity and specificity using recombinant proteins as antigens, and the development of effective recombinant vaccines are strongly desired.

[0007] An object of the present invention is to search and clone a major antigen gene of B. gibsoni protozoa, to provide a major antigen gene and a major antigen protein of B. gibsoni protozoa, and to use this gene for a dog The objective is to provide antigens for diagnosis of B. gibsoni infection and vaccines for prevention.

Disclosure of the invention

[0008] In order to achieve the above object, in the present invention, a cDNA library of B. gibson protozoa is constructed, and a gene encoding the main secretory antigen BgSAl is obtained by immunoscreening using B. gibsoni-infected dog serum. Identification 'clawed'. Furthermore, the BgSAl gene was expressed in Escherichia coli, and the antigenicity and immunogenicity of the expression product were examined. As a result, DNA encoding the BgSAl protein of Babesia gibsoni of the present invention was found.

[0009] The present invention is as follows.

[1] Secreted antigen of Babesia gibsoni-DNA encoding the l (BgSAl) protein ₀

[2] A DNA encoding a signal peptide and a mature protein of the secreted antigen l (BgS A1) protein of Babesia gibsoni shown in the following (A) or (B).

(A) a protein having amino acid sequences 1 to 544 of SEQ ID NO: 2 in the sequence listing

(B) the amino acid sequence of Nos. 1 to 544 of SEQ ID NO: 2 in the Sequence Listing, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions; and Protein that can react with BgSAl protein antibody

[3] Secreted antigen l (BgS) of Babesia gibsoni-(Babesia gibsoni) shown in (C) or (D) below Al) DNA encoding the mature protein of the protein.

(C) a protein having amino acids 24 to 544 of SEQ ID NO: 2 in the sequence listing

(D) It consists of an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence Nos. 24 to 544 of SEQ ID NO: 2 in the sequence listing, and Protein that can react with BgSAl protein antibody

[4] A DNA encoding a signal peptide and a mature protein of the secretory antigen l (BgS Al) protein of Babesia gibsoni shown in the following (a) or (b):

(a) DNA having positions 52 to 1686 of the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing

(b) encodes a protein that is hybridized under stringent conditions with a complementary strand of nucleotide sequence 52 to 1686 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and that can react with an anti-BgSAl protein antibody DNA

[5] A DNA encoding a mature protein of a secreted antigen BgS Al) protein of Babesia gibsoni shown in (c) or (d) below.

(c) a DNA having positions 121 to 1686 of the base sequence set forth in SEQ ID NO: 1 in the sequence listing

(d) a protein that is hybridized under stringent conditions with a complementary strand of nucleotide sequence 121 to 1686 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and that can react with an anti-BgSAl protein antibody DNA to encode

[6] Recombinant DNA comprising the DNA and plasmid DNA of any one of [1] to [5]

[7] A recombinant virus comprising the DNA according to [1] to [5]!

[8] A host comprising the recombinant DNA according to [6] or the recombinant virus according to [7].

[9] The host according to [8], wherein the host is a bacterium.

[10] A secreted antigen l (BgSAl) protein of Babesia gibsoni containing a signal peptide and a mature protein as shown in (A) or (B) below.

(B) the amino acid sequence of Nos. 1 to 544 of SEQ ID NO: 2 in the Sequence Listing, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions; and Protein that can react with BgSAl protein antibody [11] A secreted antigen l (BgSAl) protein of a mature protein, Babesia gibsoni, as shown in (C) or (D) below.

[12] A fusion protein comprising the signal peptide and mature protein of the BgSAl protein according to [10] or the mature protein of the BgSAl protein according to [11] and a dartathione S transferase (GST) leader protein.

[13] A vaccine for treating or preventing protozoan infections containing the recombinant DNA according to [6] or the recombinant virus according to [7] as an active ingredient.

[14] A diagnostic method for a protozoan infection using the signal peptide and mature protein of BgSAl protein according to [10], the mature protein of BgSAl protein according to [11], or the fusion protein according to [12].

[15] The signal peptide and mature protein of the BgSAl protein according to [10], [11

A therapeutic or preventive agent for a protozoal infection comprising the mature protein of the BgSAl protein according to [12] or the fusion protein according to [12] as an active ingredient.

[16] The signal peptide and mature protein of the BgSAl protein according to [10], [11

A therapeutic or prophylactic agent for a protozoal infection comprising an antibody against the mature protein of the BgSAl protein according to [12] or the fusion protein according to [12].

[17] The signal peptide and mature protein of the BgSAl protein according to [10], [11

A diagnostic method for a protozoan infection using an antiserum or an antibody against the mature protein of the BgSAl protein according to [12] or the fusion protein according to [12].

[0010] According to the present invention, it is possible to provide DNA encoding the BgSAl protein, which is a major antigen gene of B. gibsoni protozoa, and further, using this gene, an antigen for diagnosis of canine B. gibsoni infection And prophylactic vaccines can be provided.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The present invention relates to DN encoding BgSAl protein of Babesia gibsoni. Regarding A. This DNA was constructed by constructing a cDNA library of B. gibsoni protozoa, identified by immunoscreening using B. gibsoni-infected dog sera, cloned, and cleaved to 52 to 52 of the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing. DNA strength No. 1686 We found that it is DNA encoding the BgSAl protein. Of the nucleotide sequences 52 to 1686, the numbers 52 to 120 are the DNA encoding the signal peptide, and the numbers 121 to 1686 are the DNA encoding the mature peptide.

[0012] Furthermore, the present invention relates to a BgSAl protein of Babesia gibsoni. This protein is a protein having amino acids 1 to 544 of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and a protein having 24 to 544 of the amino acid sequences described in SEQ ID NO: 2 of the sequence listing. Among amino acid sequences 1 to 544 of the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of 1 to 23 corresponds to the signal peptide, and the 24 to 544 corresponds to the mature peptide of the BgSAl protein.

[0013] The present invention is a force that is a DNA that codes for the secretory antigen l (BgSAl) protein of Babesia gibsoni. Specifically, this DNA is represented by the following (A) or ( It can be a DNA encoding the protein shown in B).

[0014] Protein (A) is a Babesia gibsoni BgSAl protein containing a signal peptide and a mature protein.

[0015] The protein (B) is substantially the same protein as the protein (A), and the DNA of the present invention has activity as an encoded BgSAl protein, that is, a property capable of reacting with an anti-BgSAl protein antibody. As long as it is not impaired, it may encode a protein containing substitution, deletion, insertion, addition, or inversion of one or several amino acids at one or more positions.

[0016] Furthermore, the DNA encoding the secretory antigen l (BgSAl) protein of Babesia gibsoni of the present invention is specifically a babe shown in (C) or (D) below. Shea Gibso DNA encoding the mature protein of the BgSAl protein of (Babesia gibsoni).

[0017] Protein (C) is a mature protein of the BgSAl protein of Babesia gibsoni that does not contain a signal peptide! /.

[0018] The protein (D) is substantially the same protein as the protein (C), and the DNA of the present invention has an activity as an encoded BgSAl protein, that is, a property capable of reacting with an anti-BgSAl protein antibody. As long as it is not impaired, it may encode a protein containing substitution, deletion, insertion, addition, or inversion of one or several amino acids at one or more positions.

[0019] Furthermore, the DNA encoding the secretory antigen l (BgSAl) protein of Babesia gibsoni of the present invention is specifically the babe shown in the following (a) or (b). It can be a DNA encoding the BgSAl protein of Babesia g¾soni.

[0020] DNA (a) is DNA encoding the BgSAl protein of Babes ia gibsoni containing a signal peptide and a mature protein.

[0021] DNA (b) is substantially the same DNA as DNA (a), and the DNA of the present invention is neutralized with a complementary strand of DNA (a) under stringent conditions and has anti-resistance. It may be DNA encoding a protein that can react with the BgSAl protein antibody.

[0022] Furthermore, the DNA encoding the secretory antigen l (BgSAl) protein of Babesia gibsoni of the present invention is specifically a basociabium- (BgSAl) protein represented by the following (c) or (d): Bessiah Gibso

-It can also be DNA encoding the BgSAl protein of (Babesia gibsoni). (c) a DNA having positions 121 to 1686 of the base sequence set forth in SEQ ID NO: 1 in the sequence listing

(d) encodes a protein that is hybridized under stringent conditions with the 121 to 1686th nucleotide sequence of the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing and that can react with the anti-BgSAl protein antibody DNA

[0023] DNA (c) is a DNA encoding the mature protein of the BgSAl protein of Babesia gibsoni.

[0024] DNA (d) is substantially the same DNA as DNA (c), and the DNA of the present invention is neutralized with a complementary strand of DNA (c) under stringent conditions and has anti-resistance. It may be DNA encoding a protein that can react with the BgSAl protein antibody.

[0025] The BgSAl gene of the present invention can be used for the preparation of a recombinant BgSAl protein or a BgSAl synthetic peptide, and can also be used for DNA diagnosis targeting this gene.

[0026] The present invention relates to a BgSAl protein of Babesia gibsoni shown in the following (A) or (B).

[0027] As described above, protein (A) is a BgSAl protein of Babesia gibsoni containing a signal peptide and a mature protein. Furthermore, protein (B) is substantially the same protein as protein (A), and the BgSAl protein of the present invention has impaired activity as a Bg SAl protein, that is, a property capable of reacting with an anti-BgSAl protein antibody. Unless otherwise indicated, it may be a protein containing substitution, deletion, insertion, addition, or inversion of one or several amino acids at one or more positions.

[0028] The present invention further relates to a mature protein of BgSAl protein of Babesia gibsoni shown in the following (C) or (D).

(D) 1 to several amino acids in Nos. 24 to 544 of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing A protein comprising an amino acid sequence containing amino acid substitutions, deletions, insertions, additions, or inversions, and capable of reacting with an anti-BgSAl protein antibody

[0029] As mentioned above, protein (C) is a mature protein of BgSAl protein of Babesia gibsoni. Furthermore, protein (D) is substantially the same protein as protein (C), and the BgSAl protein of the present invention does not impair the activity as a BgSAl protein, that is, the ability to react with an anti-BgSAl protein antibody. As long as it is a protein containing one or several amino acid substitutions, deletions, insertions, additions or inversions at one or more positions! /.

[0030] The present invention further includes a fusion protein comprising the signal peptide of the BgSAl protein and the mature protein or the mature protein of the BgSAl protein and the glutathione S-transferase (GST) leader protein. The dartathione S transferase (GST) leader protein allows stable and high expression to express the BgSAl protein and makes the expressed protein soluble.

[0031] Since the base sequence of the DNA having the base sequence shown in SEQ ID NO: 1 was clarified by the present invention, PCR was carried out by PCR using an oligonucleotide prepared based on the base sequence as a primer. It can be obtained by amplification from a gibsoni protozoan cDNA library or by conventional solid phase DNA synthesis techniques. The primer used for PCR is not particularly limited.

[0032] The DNA of the present invention has one or several amino acids at one or more positions as long as the activity as the encoded BgSAl protein, ie, the property of reacting with an anti-BgSAl protein antibody is not impaired. It may encode a protein containing substitutions, deletions, insertions, additions, or inversions. Here, the term “several” differs depending on the position and type of the protein in the three-dimensional structure of amino acid residues. This is because amino acids with high affinity exist depending on amino acids, such as isoleucine and parin, and such amino acid differences do not significantly affect the three-dimensional structure of proteins.

[0033] DNA encoding a protein substantially identical to the BgSAl protein as described above is substituted, deleted, inserted, added, or inverted by substitution of an amino acid residue at a specific site, for example, by site-directed mutagenesis. It is obtained by modifying the base sequence to include Also, above Such modified DNA can also be obtained by a conventionally known mutation treatment. Mutation treatment includes in vitro treatment of DNA encoding BgSAl protein with hydroxylamine or the like, and microorganisms that retain DNA encoding BgSAl protein, such as bacteria belonging to the genus Escherichia, by ultraviolet irradiation or N-methyl— N '-tro-N--trosoguanidine (NTG) or a method of treating with a mutagen that is usually used for mutagenesis such as nitrous acid.

[0034] By expressing the DNA having the mutation as described above in an appropriate cell and examining the reactivity with the anti-BgSAl protein antibody, DNA encoding a protein substantially identical to the BgSAl protein is obtained. It is done. Further, from a DNA encoding a BgSAl protein having a mutation or a cell having the same, for example, complementation of a DNA having a base sequence consisting of base numbers 121 to 1686 among the base sequences described in SEQ ID NO: 1 in the sequence listing Isolation of a DNA encoding a protein that reacts with the chain under stringent conditions and reacts with an anti-BgSAl protein antibody is substantially identical to the BgSAl protein. DNA encoding the protein is obtained.

[0035] Here, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to express numerical values for this condition clearly, for example, DNAs with high homology, for example, DNAs with 90% or more homology, hybridize and have lower homology. DNA each other high Buridizu Shinano, condition or 60 ° C, 1 X SSC is a typical Southern hybrida I See Chillon wash, ^{_{conditions,, 0. 1 0/0 SDS}} , or preferably ί, 0. 1 X SSC, Haiburidizu conditions are exemplified by a salt concentration you Ne th person in 0. 1 ^_0/0 SDS.

[0036] The genes that are nobbridized under these conditions include those in which a stop codon has occurred in the middle or those that have lost their activity. These may be tested for reactivity with anti-BgSAl protein antibodies. Can be easily removed. The modification of the BgSAl protein as described above may not impair the activity as a BgSAl protein, but may be more reactive with an anti-BgSAl protein antibody that recognizes the protein. preferable.

[0037] The present invention relates to a recombinant DNA comprising the DNA of the present invention and a plasmid DNA. The Examples of rasmid DNA include recombinant plasmids in which the DNA of the present invention is introduced into known plasmids pUC, pTV, pGEX, pK :, pTrcHis, and the like.

[0038] Furthermore, the present invention relates to a recombinant virus containing the DNA of the present invention. The virus that can contain the DNA of the present invention is not particularly limited, and examples thereof include known vaccinia virus vectors, adenovirus vectors, Sendai virus vectors, herpes virus vectors, baculoinores vectors, and the like. Can do.

[0039] Furthermore, the present invention relates to a host comprising the above recombinant DNA of the present invention or the recombinant virus of the present invention. The host is not particularly limited, but can be, for example, a bacterium. Examples of bacteria include Escherichia coli, BCG bacteria, and lactic acid bacteria. Examples of cells for recombinant virus infection include known Vero cells, MDCK cells, CA1 cells, COS cells, and SF9 cells.

[0040] The BgSAl protein can be produced by connecting the DNA of the present invention to a suitable expression vector and culturing cells such as BCG bacteria belonging to the genus B. cerevisiae transformed by this. If a DNA having the base sequence shown in SEQ ID NO: 1 is used as the DNA of the present invention, a BgSAl protein having the amino acid sequence shown in SEQ ID NO: 2 can be obtained. Further, if the above-described modified DNA of the present invention is used, a modified BgSAl protein can be obtained.

[0041] The present invention relates to a vaccine for the treatment or prevention of protozoal infections containing the recombinant DNA of the present invention or the recombinant virus of the present invention as an active ingredient. The present invention includes a recombinant BgSAl protein or a preventive vaccine preparation using a BgSAl synthetic peptide; a recombinant virus vaccine incorporating the BgSAl gene; and a plasmid vaccine (DNA vaccine) incorporating the BgSAl gene. The dosage and administration method can be the same as those for normal vaginal and BCG vaccines.

[0042] Furthermore, the present invention relates to a therapeutic or preventive agent for protozoan infections containing the BgSAl protein signal peptide and mature protein, mature protein of BgSAl protein, or fusion protein of the present invention as an active ingredient. That is, a therapeutic or prophylactic preparation using the recombinant BgSA 1 protein or BgSAl synthetic peptide of the present invention as an antigen can be provided. When the protein or peptide of the present invention is used as a therapeutic or prophylactic agent, the The protein can be administered 1) as is, or 2) with a pharmaceutically acceptable carrier and Z or diluent, and 3) if necessary, with the aid of Alternatively, it may be administered by transdermal absorption from the mucosa by a method such as spraying. The carrier referred to here is, for example, human serum albumin, and the diluent is, for example, PBS, distilled water, or the like.

[0043] The present invention relates to a method for diagnosing a protozoal infection using the signal peptide and mature protein of the BgSAl protein of the present invention, the mature protein of the Bg SAl protein, or a fusion protein. A diagnostic method for protozoal infections using the recombinant BgSAl protein or BgSAl synthetic peptide of the present invention as an antigen can be provided. Diagnosis of protozoal infection can be carried out by examining the reactivity of a sample, for example, serum with the above protein

[0044] Further, the present invention provides a therapeutic and prophylactic agent for protozoal infections comprising an antibody against the signal peptide and mature protein of the BgSAl protein, mature protein of BgSAl protein, or fusion protein of the present invention as an active ingredient. Can be provided. Furthermore, according to the present invention, it is possible to provide a method for diagnosing a protozoal infection using an antiserum or an antibody against the signal peptide and mature protein of the BgSAl protein, the mature protein of BgSAl protein, or the fusion protein of the present invention. That is, a therapeutic or prophylactic agent and diagnostic method using antiserum or antibody against the recombinant BgSAl protein and BgS A1 synthetic peptide of the present invention. Antiserum against the recombinant BgSAl protein and BgSAl synthetic peptide of the present invention is obtained by administering the recombinant BgSAl protein or BgSAl synthetic peptide of the present invention to Freund's complete adjuvant (Difco) multiple times to animals such as mice, rabbits, etc. Obtained by collecting blood. Furthermore, the antibody is obtained by appropriately purifying the obtained antiserum.

[0045] When the antibody is used as a therapeutic and prophylactic agent for protozoal infections, an IgG fraction is purified from antiserum obtained by immunizing animals with BgSAl frequently (using physiological saline as a solvent), Treatment and prevention can be performed by administration to dogs infected with B. gibso ni. In addition, after preparation of a monoclonal antibody against BgSAl, treatment and prevention can be performed by purifying the IgG fraction (using physiological saline as a solvent) and administering it to dogs infected with B. gibsoni.

[0046] For diagnostic methods using antisera or antibodies, see Usagi or Makoto against BgSAl. A mouse polyclonal antibody or monoclonal antibody can be produced, and BgS Al secreted in the blood of B. gibsoni-infected dogs can be detected by sandwich ELISA or immunochromatographic method.

Example

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

In the invention, various molecular biology, protozoology, immunology and biochemical techniques were used. These techniques were referred to Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harber Laboratory Press and other related documents. Mac vector (Oxford Molecular) was used as DNA analysis software.

[0048] Example 1

The identification 'cloning' base sequence decoding of the B. gibsoni BgSAl gene in the present invention is described.

B. gibsoni NRCPD strain [Fukumoto et al., J Parasitol 84: 954-959 (2000)] From infected dog red blood cells Acid Guanidinium-Pheno-Chloroform method [Chomczynski et al., Anal Biochem 162: 156-159 (1987) ] Extracted total RNA. Poly A + RNA was purified from Total RNA using mRNA Isolation Kit (Oligotex-dT30, Takara) according to the manufacturer's recommended method. Construction of the cDNA library and immunoscreening and plasmid cloning (in vivo Excision) of cDNA clones described below were all performed using Stratagen's reagent kit according to our recommended method. About 5 g of B. gibsoni mRNA was used as a saddle, and cDNA was synthesized using ZAP-cDNA Synthesis Kit. The obtained cDNA was size-fractionated with a Sepharose CL-2B gel column, inserted into Uni-ZAP XR Vector arms, and packaged with Gigapacklll Gold packaging extraction. The packaging product was infected with E. coli XL1-Blue M RF 'strain to obtain a library containing about 500,000 cDNA clones. B. gibsoni infection Canine serum (contained secreted antigen derived from worms! /) Is given to normal dogs frequently (5 times at 2 week intervals, 5 ml / dose). Using the obtained serum, the cDNA library was immunoscreened to obtain about several tens of overlapping positive clones. These positive clones were converted into plasmids (converted to pBluescript) by the in vivo Excision method. After purifying the plasmid containing the cDNA fragment with a plasmid purification kit (Qiagen), Dye Primer Cy PCR was performed using cle Sequencing Kit (Perkin Elmer) according to the method recommended by the manufacturer. PCR products were analyzed using ABI PRISM 377 DNA sequencer (Perkin Elmer) to determine the nucleotide sequence of the cDNA fragment. As a result, it was found that all these clones were derived from the same gene. Among them, clones having long fragments were used for further analysis. The total length of the cDNA was 1880 bp, and it was confirmed that it had an open reading frame (ORF) of 1635 bp (SEQ ID NO: 1). The predicted protein had an amino acid strength of 544 residues and an estimated molecular weight of 62 kDa (SEQ ID NO: 2). When a signal peptide search (http://www.cbs.dtu.dk/services/SignalP/) was performed using the presumed amino acid sequence, the N-terminal 23-residue amino acid was a signal peptide. It was assumed. Hereinafter, this gene is referred to as a BgSAl gene. A homology search of the amino acid sequence predicted from the BgSAl gene was performed using the BLAST method of the National Center for Biotechnology Information, and it was found that there was significant homology with any of the genes reported to date for protozoa and other species. It did not show sex.

[0049] Example 2

The construction of a recombinant vector plasmid for expressing B. gibsoni BgSAl protein in E. coli is described.

B. gbsIlc DNA fragment (sequence from which signal peptide of 23 residues is removed) pBluescript plasmid inserted with digestion with restriction enzymes EcoRI and Xhol to excise BgSAlc DNA fragment, and E. coli expression vector pGEX-4T-3 (Amersham Biosciences) EcoRI and Xhol sites. The recombinant plasmid pGEX / BgSAl was purified with a plasmid purification kit (Qiagen).

[0050] Example 3

Describe the production of B. gibsoni BgSAl protein in E. coli and the properties of the produced protein.

After transforming the E. coli DH5 strain with the recombinant plasmid obtained in Example 2, the cells were cultured in LB medium containing ampicillin at 37 ° C, and when the OD600nm force of the culture reached 0.3-0.5, IPTG Was added to a final concentration of 0.5 mM, and the culture was further continued at 37 ° C for 4 hours. Recombinant protein expression was performed on a 10% SDS-polyacrylamide gel [Laemmli et al, Nature 227: 680-685 (1970)], and then confirmed by Coomassie staining. As a result, the expression of a recombinant protein of about 85 kDa was confirmed, and it was confirmed that it was a fusion protein of GST leader protein (26 kDa) and B. gibsoni BgSAl protein (59 kDa) (Fig. 1).

[0051] Example 4

The purification of recombinant BgSAl protein from E. coli in the present invention and the production of antibodies against the purified recombinant protein are described.

Recombinant BgSAl protein expressed in E. coli by the method described in Example 3 was purified according to the method recommended by Amersham Biosciences. Fig. 1 shows the electrophoresis image of the recombinant BgSAl fusion protein after purification. After mixing 200 μl of a solution containing 100 g of recombinant BgSAl fusion protein and 200 μ1 of Freund's complete adjuvant (Difco), BALB / c mice (8 weeks old, female) were inoculated intraperitoneally. Two weeks and four weeks later, 100 g of recombinant BgSAl fusion protein was mixed with Freund's incomplete adjuvant (Difco) and boosted. Blood was collected 2 weeks after the final inoculation, and the serum was stored at -20 ° C.

[0052] Example 5

The identification of native (native) BgSAl protein by immunoblotting is described. Using the anti-recombinant BgSAl fusion protein mouse serum obtained in Example 4, immunoblotting [Towbin et al., Proc Natl Acad Sci USA 76: 4350-4354 (1979)] was used to identify the natural BgSAl protein. went. As shown in FIG. 2, a specific band of 59 kDa was detected in B. gibsoni-infected erythrocyte lysate. The molecular weight of the native BgSAl protein was similar to the estimated theoretical value (59 kDa).

[0053] Example 6

The reactivity of immune serum to the recombinant BgSAl protein in the present invention is described. The reactivity of B. gibsoni-infected dog sera to thread-replaced BgSAl protein was examined using immunoblotting. As shown in Fig. 3, it was confirmed that the recombinant BgSAl protein reacted strongly with the blood of B. gibsoni-infected dogs. This indicates that the recombinant BgSAl protein is one of the diagnostic and vaccine candidate molecules for canine B. gib soni infection. No reaction between normal dog serum and recombinant BgSAl protein was observed.

Industrial applicability [0054] The present invention is useful in the field of canine B. gibsoni infection diagnostic antigens and preventive vaccines.

Brief Description of Drawings

[0055] FIG. 1 is an electrophoretogram showing a recombinant BgSAl fusion protein which is an expression product of the present invention.

Lane 1: Molecular weight marker. Lane 2: purified recombinant BgSAl fusion protein. Lane 3: Purified GST protein.

[FIG. 2] Immunoblot image showing detection of native BgSAl protein by recombinant BgSAl fusion protein immune serum. Lane 1: Red blood cells infected with B. gibsoni. Lane 2: Normal dog red blood cells.

[FIG. 3] Imunolot image showing the reactivity of recombinant BgSAl fusion protein with B. gibsoni-infected dog serum. Lane 1: purified recombinant BgSAl fusion protein. Lane 2: purified BgSAl protein.

Claims

The scope of the claims

[1] Secretory antigen of Babesia gibsoni-DN A encoding the protein (BgSAl).

[2] A DNA encoding a signal peptide and a mature protein of the secretory antigen l (BgSAl) protein of Babesia gibsoni shown in the following (A) or (B).

[3] A DNA encoding a mature protein of a secreted antigen l (BgSAl) protein of Babesia gibsoni shown in (C) or (D) below.

[4] A DNA encoding a signal peptide and a mature protein of the secretory antigen 1 (BgSAl) protein of Babesia gibsoni shown in the following (a) or (b):

(b) encodes a protein that is hybridized under stringent conditions with a complementary strand of the nucleotide sequence 52 to 1686 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and can react with an anti-BgSAl protein antibody DNA

[5] A DNA encoding a mature protein of a secreted antigen l (BgSAl) protein of Babesia gibsoni shown in (c) or (d) below.

[6] Recombinant DN comprising the DNA of claim 1 to 5 and the DNA of claim 1 and plasmid DNA A.

[7] A recombinant virus comprising the DNA according to claim 1 to 5!

[8] A host comprising the recombinant DNA according to claim 6 or the recombinant virus according to claim 7.

9. The host according to claim 8, wherein the host is a bacterium.

[10] A secreted antigen 1 (BgSAl) protein of Babesia gibsoni containing a signal peptide and a mature protein as shown in (A) or (B) below.

(A) a protein having the amino acid sequences 1 to 544 of SEQ ID NO: 2 in the sequence listing

[11] A secreted antigen l (BgSAl) protein of the mature protein Babesia gibso ni shown in (C) or (D) below.

[12] A fusion protein comprising the signal peptide and mature protein of the BgSAl protein according to claim 10 or the mature protein of the BgSAl protein according to claim 11 and a dartathione S transferase (GST) leader protein.

[13] A vaccine for treating or preventing protozoal infections comprising the recombinant DNA according to claim 6 or the recombinant virus according to claim 7 as an active ingredient.

[14] A diagnostic method for a protozoan infection using the signal peptide and mature protein of the BgSAl protein according to claim 10, the mature protein of the BgSAl protein according to claim 11, or the fusion protein according to claim 12. .

[15] A protozoal infection containing the signal peptide and mature protein of the BgSAl protein according to claim 10, the mature protein of the BgSAl protein according to claim 11, or the fusion protein according to claim 12 as an active ingredient A therapeutic or prophylactic agent.

[16] A protozoan infection comprising as an active ingredient the signal peptide and mature protein of the BgSAl protein according to claim 10, the mature protein of the BgSAl protein according to claim 11, or the fusion protein according to claim 12. Treatment or prevention agent.

[17] A signal peptide and mature protein of the BgSAl protein according to claim 10, an antiserum against the mature protein of the BgSAl protein according to claim 11, or the fusion protein according to claim 12, wherein an antibody is used. Diagnosis method of protozoan infection.