WO2020174690A1 - Surface antigen protein of babesia protozoa, method for detecting antibody against babesia protozoa, and diagnostic method for babesiosis - Google Patents

Abstract

The present invention provides: a protein obtained by removing, from the amino acid sequence of the full-length surface antigen protein of a Babesia protozoa, the amino acid sequence of the N-terminal signal peptide thereof and the amino acid sequence of the region from a transmembrane region to the C-terminus thereof; and a detection or diagnostic method for Babesiosis using the protein.

Description

Surface antigen protein of Babesia parasite, method for detecting antibody to Babesia parasite, method for diagnosing babesiosis

The present invention is a surface antigen protein of Babesia parasite, a polynucleotide encoding the protein, a reagent for detecting an antibody against Babesia parasite and a detection method using the reagent, and a reagent for diagnosing Babesiosis, and the reagent. The present invention relates to a method for diagnosing babesiosis.

Babesiosis is a tick-borne infection caused by a parasite (Protozoa genus Babesia). For Babesiosis pathogen (Protozoa of the genus Babesia), Babesia gibsoni, B. canis, B. Vogeli, B.I. Rossi and the like are known, and in Japan, there are many infections mainly caused by Babesia gibsoni. Symptoms are paralysis of red blood cells by protozoa and anemia due to destruction of red blood cells and the associated fever. With respect to treatment, since there are effective drugs, if an accurate diagnosis can be made, it is possible to promptly perform anti-Babesia drug therapy after the diagnosis.

At present, microscopic examination (microscopic examination) is adopted as a main method for definitive diagnosis of babesiosis. At microscopic examination, Babesia protozoa are detected by observing a Giemsa-stained blood smear. However, depending on the protozoa, there are various forms, stains due to staining, and forms that are difficult to distinguish. Therefore, it is essential to have skill and experience to diagnose Babesiosis. In addition, because the parasite parasite rate is not high, the appearance of worms is intermittent and may be overlooked by microscopic examination, making it difficult to distinguish from similar symptoms (misdiagnosis of hemolytic anemia and thrombocytopenia). Furthermore, it is difficult to make a distinctive diagnosis between filariasis and babesiosis.
Even in molecular diagnosis by PCR and serological test by ELISA or indirect fluorescent antibody (IFA) method, there are problems of equipment and sensitivity that require test time, and rapid diagnosis is difficult.

By the way, as a method for detecting Babesiosis, a method of detecting an antibody against Babesia protozoa using a surface protein of Babesia protozoa has been studied (Patent Documents 1 and 2).
Patent Document 1 describes that a dog Babesia antigen protein was produced in Escherichia coli, and Patent Document 2 describes that a truncated dog Babesia surface protein was produced.

Japanese Patent Publication No. 2008-500802 CN 102558332 Publication

When infected with Babesia protozoa, various kinds of antibodies are produced in the body, and thus the presence or absence of infection can be determined by detecting the antibodies. Since the invention described in Patent Document 2 uses a part of the surface antigen protein of canine Babesia protozoa to detect an antibody, it cannot detect an antibody to a region other than that part of the region. For this reason, a false negative will appear as the detection result.
Under the above circumstances, it is desired to develop a method for detecting an antibody against the surface protein of Babesia parasite with high accuracy.

The present inventors have conducted extensive studies in order to solve the above-mentioned problems, and as a result, among the full-length surface antigen proteins of Babesia protozoa, the N-terminal signal peptide and the protein excluding the region from the transmembrane region to the C-terminus have been identified. I succeeded in synthesizing. Then, they have found that the protein can be used for highly accurate detection of an antibody against the Babesia parasite surface antigen protein, and thus for the diagnosis of Babesiosis, and completed the present invention.

That is, the present invention is as follows.
[1] A protein in which the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminal have been removed from the amino acid sequence of the full-length surface antigen protein of Babesia parasite.
[2] The following protein (a), (b) or (c).
(A) a protein consisting of any amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) (b) represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any amino acid sequence, and which reacts with an antibody against Babesia parasite (c) SEQ ID NO: 2n (n is an integer of 1 to 4) Which has a homology of 80% or more with any of the amino acid sequences represented by the formula (1) and which reacts with an antibody against Babesia protozoa [3] (1) or (2) A polynucleotide.
[4] The polynucleotide of (d), (e) or (f) below.
(D) Polynucleotide consisting of any base sequence represented by SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) (e) SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) (F) SEQ ID NO: which is a protein which hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences shown in (1) to (3) under stringent conditions and which reacts with an antibody against Babesia parasite (f). 2n-1 (n represents an integer of 1 to 4) and a protein comprising a nucleotide sequence having a homology of 80% or more with a polynucleotide comprising any of the nucleotide sequences represented by 2n-1 and reacting with an antibody against a Babesia parasite. A vector containing the polynucleotide described in [5] (3) or (4), which encodes the polynucleotide.
[6] The vector according to (5), which is a viral vector.
[7] A silkworm host containing the polynucleotide according to (3) or (4) or the vector according to (5) or (6).
[8] A method for producing the protein, comprising the step of culturing or breeding the silkworm host according to (7) and recovering the protein according to (1) or (2) from the silkworm host after culturing or breeding.
[9] A protein produced by the method according to (8).
[10] A reagent for detecting an antibody against Babesia parasite, which comprises the protein according to (1), (2) or (9).
[11] A reagent for diagnosing babesiosis containing the protein according to (1), (2) or (9).
[12] The reagent according to (10) or (11) for use in immunochromatography.
[13] Babesia, which comprises reacting a sample collected from a test animal with the protein described in (1), (2) or (9) or the reagent described in (10) or (11). Method for detecting antibody to protozoa.
[14] A method for diagnosing babesiosis using the detection result detected by the method described in (13) as an index.
[15] A method for treating babesiosis, which comprises administering a therapeutic drug for babesiosis to a test animal in which babesiosis is diagnosed by the method according to (14).
[16] A vaccine for treating or preventing babesiosis, which comprises the protein according to (1), (2) or (9).
[17] A kit for detecting an antibody against Babesia parasite, which comprises the protein according to (1), (2), or (9).
[18] A diagnostic kit for babesiosis containing the protein according to (1), (2) or (9).
[19] The kit according to (17) or (18) for use in immunochromatography.
[20] A protein in which the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminus are removed from the amino acid sequence of the full-length surface antigen protein of Babesia parasite for the detection or diagnosis of Babesiosis ..
[21] The following protein (a), (b) or (c) for detecting or diagnosing babesiosis.
(A) a protein consisting of any amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) (b) represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any amino acid sequence, and which reacts with an antibody against Babesia parasite (c) SEQ ID NO: 2n (n is an integer of 1 to 4) A protein having an amino acid sequence showing 80% or more homology with any of the amino acid sequences represented by

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to detect an antibody against Babesia protozoa with high accuracy and to diagnose babesiosis.
In the present invention, by establishing a method for detecting antibodies to Babesia protozoa by immunochromatography, it is possible to easily detect the presence or absence of infection with Babesia protozoa in a veterinary hospital or at home, and in a short period of time, and also a similar symptom (for example, filararia). It is possible to diagnose Babesiosis.

FIG. 1 is a construction diagram of a recombinant donor plasmid for producing a baculovirus using the Gateway LR reaction. It is a figure which shows the expression result in the silkworm of recombinant PChi protein. It is a figure which shows the purification result of vector pDEST8-30K6G-BgTRAP-dH8STREP-1274-8 and BgTRAP. It is a figure which shows the purification result of vector pDEST8-30K6G-dH8STREP-BgTRAP-1381-9 and BgTRAP. It is a figure which shows the purification result of vector pDEST8-30K6G-dH8STREP-BgTRAP-1381-9 and BgTRAP. It is a figure which shows the result of SDS-polyacrylamide gel electrophoresis of BgTRAP. It is a figure which shows the quantitative result of purified BgTRAP. It is a figure which shows the result of having investigated the influence by the temperature of BgTRAP. It is a figure which shows the full length amino acid sequence of BgTRAP. FIG. 3 is a diagram of a plasmid for expressing BgTRAP in E. coli. It is a figure which shows the result of SDS-polyacrylamide gel electrophoresis of the expression product by Escherichia coli. It is a figure which shows the result of the immuno chromatography using a half strip. It is a figure which shows the result of the immuno chromatography using a full strip.

The present invention artificially removes the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminus, from the amino acid sequence of the full-length surface antigen protein of Babesia parasite (natural surface antigen protein). Surface antigen proteins having different amino acid sequences. In the present specification, a protein obtained by removing the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminal in the amino acid sequence of the full-length surface antigen protein of Babesia parasite is referred to as “the surface of the present invention”. It is called an antigen protein.

The surface antigen protein of the present invention has the N-terminal signal peptide and the region from the transmembrane region to the C-terminal removed, but includes all the regions that appear on the surface of Babesia parasite. When a Babesia parasite is infected, an antibody against the Babesia parasite surface antigen protein is produced in the infected animal.However, since the antibody is any region of the surface antigen protein that appears on the surface of the Babesia protozoa, it becomes an antigen in the body. There will be many types of antibodies to these regions. Therefore, when a truncated antigen protein in which a part of the surface antigen protein is cleaved is used for antibody detection, it is not possible to detect many kinds of antibodies against the region produced on the surface of Babesia parasite produced in the body (false Negative) occurs.

Since the surface antigen protein of the present invention is the entire antigen protein (entire region) that appears on the surface of Babesia parasite, the above problems can be solved. That is, the antibody produced in the infected animal body reacts by using the surface antigen protein of the present invention as an antigen, regardless of which area is present on the surface of the Babesia parasite of the surface antigen protein, Since it can be detected, it becomes possible to detect the infection of Babesia parasite with high accuracy.

Further, in the present invention, the surface antigen protein of the present invention is produced by a protein expression system using silkworm. The surface antigen protein of Babesia parasite has heretofore been difficult to express, and the protein produced in Escherichia coli or the like was a partial fragment protein that does not include a partial region of the surface antigen protein. If the antigenic protein contains all the regions appearing on the surface of Babesia protozoa, it can react with the antibody against all the regions appearing on the surface of Babesia protozoa of the surface antigen protein produced in the infected animal body, so that The diagnosis can be performed accurately.
The present invention diagnoses Babesiosis by detecting an antibody against a surface protein of Babesia protozoa in a biological sample (specimen) by an immunochromatography method. Also, the kit used for the detection was successfully developed.

1. Surface Antigen Protein of the Present Invention The protozoan from which the surface antigen protein of the present invention is derived is a protozoan belonging to the genus Babesia. Examples of animals infected with the protozoa include dogs, sheep, horses, and cows. Among the protozoa belonging to the genus Babesia, the amino acid sequence of the full-length surface antigen protein of the dog Babesia protozoa (Babesia gibbsoni) is shown in SEQ ID NO: 10, and the polynucleotide sequence encoding the protein is shown in SEQ ID NO: 9. In the amino acid sequence of the full-length surface protein (SEQ ID NO: 10 and FIG. 9), the region from the 1st methionine (M) to the 23rd glycine (G) is the N-terminal signal sequence, and the 672nd alanine ( The region from A) to the 694th valine (V) is a transmembrane region. The surface antigen protein of the present invention comprises the amino acid sequence of the region of the N-terminal signal sequence (first to 23rd) and the amino acid sequence of the region from the transmembrane region to the C-terminal (672nd to 735th). Is a protein having a sequence in which is deleted.

In the same manner as described above, the surface antigen protein of the present invention is obtained by removing the amino acid sequence of the N-terminal signal sequence region and the region from the transmembrane region to the C-terminus from the full-length protein of Babesia parasites that infect various animals. It can be prepared. The amino acid sequence of the surface antigen protein of the present invention, and the polynucleotide sequence (base sequence) encoding the antigen protein of the present invention, and the full length, the signal sequence region, and the base sequence and amino acid sequence from the transmembrane region to the C terminus, The results are shown in Table 1 below.

However, in the present invention, the origin of the surface antigen protein of the present invention is not limited to the above-mentioned Babesia protozoa, but it is not limited to Babesia canis (protozoan dog Babesia), Babesia vogeli (protozoan dog Babesia), Babesia rossi (protozoan dog Babesia), and Babesia dia (Babesia disia). It is also possible to use a surface antigen protein derived from bovine Babesia protozoa).
The surface antigen protein of the present invention is the amino acid sequence of the region of the N-terminal signal sequence and the amino acid sequence of the region from the transmembrane region to the C-terminal in the amino acid sequence of the full-length surface protein of various Babesia parasites (which encodes a surface antigen protein). In the case of a gene, it can be obtained by removing the nucleotide sequence encoding the region.

In Table 1, the base sequences shown in SEQ ID NOs: 1, 3, 5 and 7 are represented by the general formula 2n-1 (n represents an integer of 1 to 4), and the amino acids shown in SEQ ID NOs: 2, 4, 6 and 8 are shown. When the sequence is represented by the general formula 2n (n represents an integer of 1 to 4), the surface antigen protein of the present invention is the following protein (a), (b) or (c).

(A) a protein consisting of any amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) (b) represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any amino acid sequence, and which reacts with an antibody against Babesia parasite (c) SEQ ID NO: 2n (n is an integer of 1 to 4) A protein consisting of an amino acid sequence having a homology of 80% or more with any of the amino acid sequences shown in (3) and reacting with an antibody against Babesia parasite

Here, the above-mentioned “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11 pieces, 1-10 pieces, 1-9 pieces, 1-8 pieces, 1-7 pieces, 1-6 pieces, 1-5 pieces, 1-4 pieces, 1-3 pieces, 1-2 pieces or An amino acid sequence in which one amino acid is deleted, substituted or added is included, but not limited to.
In the case of introducing a mutation such as the deletion, substitution or addition, in the case of producing a protein by genetic engineering, a mutation introduction kit utilizing a site-directed mutagenesis method, for example, GeneTailor ^™ Site-Directed Mutagenesis System is used. (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: manufactured by Takara Bio Inc.) and the like.

The surface antigen protein of the present invention is represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) in addition to the amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4). An antibody having an amino acid sequence having 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more homology (identity) with the amino acid sequence, and an antibody against Babesia parasite Also included are proteins that react with. The homology can be searched by using BLAST on a homology search site using the Internet, for example, National Center for Biotechnology Information (NCBI) (for example, refer to the following URL: https: //blast.ncbi. .Nlm.nih.gov/Blast.cgi).

“80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more of the amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4) The present invention having an amino acid sequence having homology (identity) and reacting with an antibody against Babesia protozoa" has an amino acid sequence represented by SEQ ID NO: 2n (n represents an integer of 1 to 4). Surface antigen protein, compared to the binding amount or binding activity of binding to an antibody against Babesia parasite, that is, substantially equivalent activity, that is, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more Alternatively, it is a protein having a binding amount or binding activity of 95% or more.

2. Polynucleotide encoding surface antigen protein of the present invention Examples of the polynucleotide encoding surface antigen protein of the present invention include the following polynucleotides (d), (e) or (f).
(D) Polynucleotide consisting of any of the base sequences represented by SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) (e) SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) Polynucleotide (f) SEQ ID NO: which encodes a protein which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences shown in FIG. 2n-1 (n represents an integer of 1 to 4) and a protein comprising a nucleotide sequence having a homology of 80% or more with a polynucleotide comprising any of the nucleotide sequences represented by 2n-1 and reacting with an antibody against a Babesia parasite. Encoding polynucleotide

Here, the “polynucleotide” means a polymer composed of bases or base pairs such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and includes DNA, cDNA, genomic DNA, chemically synthesized DNA and RNA. It also includes polynucleotides containing non-natural bases as necessary. Examples of non-natural bases include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-0-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, Dihydrouridine, 2'-0-methylpseudouridine, 2'-0-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine , 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl Examples include 2-thiouridine, β-D-mannosylkyueosin, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine and the like.

The polynucleotide encoding the surface antigen protein of the present invention may be the polynucleotide of (d), (e) or (f) above, or may be required for gene expression in addition to the nucleotide sequence. It may be a polynucleotide containing a known base sequence (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) and is not limited.

Here, the “stringent conditions” may be any of low stringent conditions, medium stringent conditions and high stringent conditions, and the “low stringent conditions” include, for example, 5×SSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 32°C. The "moderate stringent conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. The “highly stringent conditions” are, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 50° C. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, factors that affect the stringency of hybridization include multiple factors such as temperature, concentration of polynucleotide, length of polynucleotide, ionic strength, time, and salt concentration, and those skilled in the art can use these factors. It is possible to realize the same stringency by appropriately selecting.

"A nucleotide sequence having a homology (homology) of 80% or more with a polynucleotide comprising any of the nucleotide sequences represented by SEQ ID NO: 2n-1 (n is an integer of 1 to 4), and a Babesia parasite "A polynucleotide encoding a protein that reacts with an antibody against" is a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2n-1 (n represents an integer of 1 to 4), and 80% or more, 90% or more, 95 %, 96% or more, 97% or more, 98% or more or 99% or more of a nucleotide sequence having a homology (identity), wherein the protein encoded by the nucleotide sequence is SEQ ID NO: 2n. -1 (n represents an integer of 1 to 4), the protein encoded by the nucleotide sequence represented by -1 (n is an integer of 1 to 4) is substantially equivalent to the binding amount or binding activity of binding to the Babesia antibody, ie, 50% or more A polynucleotide encoding a protein having a binding amount or binding activity of 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

3. Vector The method for producing the vector of the present invention is not particularly limited, but, for example, different restriction enzymes on the 5′ side of the start codon and the 3′ side of the stop codon of the nucleotide encoding the surface antigen protein of the present invention, respectively. A primer having a sequence is set, and this coding region is gene-amplified. The vector of the present invention can be obtained by cutting out the amplified region with a restriction enzyme and incorporating it into a plasmid vector or a viral vector.

Specifically, a plasmid containing a polynucleotide encoding the surface antigen protein of the present invention cloned from a PCR product is cleaved with a restriction enzyme. The vector fragment of the present invention can be obtained by purifying the cleaved polynucleotide fragment by agarose gel electrophoresis or the like and incorporating this polynucleotide fragment into an expression vector using a known method (Sambrook J. et al. , Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)).

4. Silkworm host In the present invention, a silkworm individual, cell or tissue can be used as a silkworm host for producing the surface antigen protein of the present invention. In one embodiment of the present invention, the silkworm host refers to an individual, cell or tissue of Bombyx mori, and the individual includes not only larvae but also eggs, pupae, cocoons or adults. In the present invention, the silkworm host may be a cell, a cell mass or a tissue, and the cell may be single or plural.
Strains can be distinguished from other strain sets by all or part of their traits (eggs, larvae, pupae, cocoons, adult traits, genotypes) and origins, and retain all of their traits. A single set of silkworms that can be propagated while being reproduced.

The systematic classification in this specification follows the method of the Genetic Resource Development Research Center. Strains are first classified alphabetically by their main purpose trait, with a 2-position attached to them, and a branch of the same origin with a 3-position to represent a strain.
In the present invention, in order to introduce the vector of the present invention into the silkworm host, a known method such as an injection method or a baculovirus expression system can be used (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition). (Cold Spring Harbor Laboratory Press (2012)) For example, when a baculovirus expression system is used, a silkworm host sensitive to the virus can be used.

“The silkworm host is “susceptible” to a virus refers to the ability to infect the virus and to propagate it. Whether or not it is susceptible is determined, for example, by preparing a recombinant virus into which a gene for a marker protein (eg, luciferase, GFP) that allows easy evaluation of growth is introduced, and infecting a target silkworm, It can be judged by evaluating the amount of the marker protein in the silkworm at various times.

The silkworm host used in the present invention is, for example, BmNPV-sensitive or AcNPV-sensitive. In one of the preferable embodiments of the present invention, a BmNPV-sensitive strain, more preferably a BmNPV-sensitive strain, is used as the silkworm. Examples of silkworm strains that can be preferably used include a49, c11, c51, c60, d17, e15, f10, f38, g05, g30, g32, 131, 1311, 1312, n41, r02, r21, t70, w601 and fylu, as well as variants thereof having the same biological properties as those of the c11, d17, f10 or f38 lines. The breeding and breeding of these strains can be performed under the conditions commonly used by those skilled in the art. Regarding the biological characteristics, the description in JP2007-159406A can be referred to.

The silkworm strain is a core institution of the National BioResource Project (NBRP), Kyushu University Genetic Resource Development and Research Center (Kyushu University Graduate School of Agriculture Research; Kyushu University Graduate School of Agricultural Research; (Fax) 092-802-4820) can be sold by the applicant in accordance with the provisions of Article 27-3 of the Patent Law Enforcement Regulations or in accordance with the 11th Regulation of the Budapest Treaty Regulations ( http://www.nbrp.jp/report/reportProject.jsp; jsessionid=BE73451C6E54680014762FDD194C0F721?project=silkworm).

5. Method for Producing Protein In order to produce the surface antigen protein of the present invention, a polynucleotide encoding the protein or a vector containing the polynucleotide is inoculated into or infected with a silkworm host, and the silkworm host (for example, silkworm individual In vivo, in cells, in tissues, etc.).
The polynucleotide encoding the surface antigen protein of the present invention can be directly inoculated into a silkworm host, inoculated as a plasmid vector, or inoculated as a viral vector.

When using a virus as a vector, prepare a recombinant virus that incorporates the polynucleotide into the virus and infect the silkworm host with this recombinant virus. Conventional techniques can be used in the step of producing a recombinant viral vector (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory virus (2012)). Since its genome is usually a circular DNA of 130 kbp, it is impossible to directly introduce the target gene.Therefore, the target gene is once integrated into a transfer plasmid and introduced by homologous recombination or transfer reaction. Regarding the point, various techniques have been developed, and a commercially available construction system (for example, Bac-to-Bac system) may be used in the present invention.

For example, a recombinant viral vector can be obtained by incorporating a polynucleotide encoding the surface antigen protein of the present invention into BmNPV or AcNPV, but not only BmNPV or AcNPV, but also mutants thereof, for example, for suppressing disease expression. Those lacking a specific gene, those overexpressing a specific gene for easy entry into cells, and the like can be used in the present invention.
The recombinant virus can be used in the subsequent infection step after being propagated using cultured cells, if necessary. Further, the obtained virus may be in the form of suspension, freeze-dried powder or the like.

Inoculation or infection of a silkworm individual with a recombinant vector (virus vector, plasmid vector, etc.) into which a polynucleotide encoding the surface antigen protein of the present invention has been introduced can be carried out, for example, to approximately 5th instar larvae. Those skilled in the art can appropriately set the amount of inoculation, the route of infection, and the like. For example, 10 μL of recombinant virus solution prepared to 1×10 ⁶ pfu/mL can be subcutaneously injected into the chest using an injection needle. A method in which it is mixed with feed and orally inoculated is also possible (see Japanese Patent No. 3030430).

When a silkworm individual is used, the time for collecting the surface antigen protein of the present invention from the silkworm host can be determined by the recombinant protein concentration in the silkworm body fluid, the absolute amount of the target protein per silkworm individual, etc. .. Normally, the absolute amount of protein production per head increases as the larval body grows over time. On the other hand, however, some individuals may die after the 7th day. The state of the silkworms at the time of recovery is almost the same as that when they were not infected with the virus, but they may be slightly different depending on the silkworm strains. Virus-infected silkworms usually do not become pupae, even after long term breeding.

The surface antigen protein of the present invention can be recovered from all tissues of the silkworm host (eg, individual). Collection is performed by a method of directly obtaining a body fluid from an individual by using an injection needle or the like, a method of grinding an individual by adding an appropriate solution as necessary, and a body fluid utilizing a contraction phenomenon by freezing and thawing the individual. A method of extracting or the like can be used. In the secretory system, the body fluid containing the secreted surface antigen protein of the present invention can also be collected.

The recovered liquid containing the surface antigen protein of the present invention may be subjected to steps such as separation, purification, lyophilization, and crystallization, if necessary.
Whether the recovered protein is the target surface antigen protein of the present invention can be confirmed by, for example, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and the like.

6. Vaccine In the present invention, an immunologically effective amount of the surface antigen protein of the present invention, or a vector incorporating a polynucleotide encoding the surface antigen protein of the present invention so as to express the protein in vivo is used. A vaccine preparation for treating or preventing Babesiosis (infection against Babesia parasite) can be prepared.
The vaccine may include a pharmaceutically acceptable carrier.
An "immunologically effective amount" means an amount that is effective in the treatment or prevention of babesiosis, as administered in a single dose or as part of a continuous dose, which amount is the health condition of the individual, It can be adjusted according to the vaccine prescription and the like.

Examples of the pharmaceutically acceptable carrier include aluminum compounds such as aluminum hydroxide and aluminum phosphate, Freund's adjuvant, muramyl peptide and the like.
Vaccine preparations further include sodium phosphate, potassium phosphate, sodium hydroxide, pH adjusting agents such as hydrochloric acid, kanamycin sulfate, antibiotics such as erythromycin, lactose, potassium glutamate, D-sorbitol and aminoacetic acid. It is possible to add stabilizers, tonicity agents such as sodium chloride and the like.

7. Detection of antibodies to Babesia protozoa, diagnosis of babesiosis Individuals infected with Babesia protozoa produce antibodies specific to the surface antigen protein of the present invention, and by detecting the antibodies, Babesia protozoa-infected animals can be detected. It is possible to identify with high reliability.

In the anti-Babesia protozoan antibody to be detected, or the Babesiosis or Babesia infection to be diagnosed, the Babesia protozoa is, for example, Babesia gibbsoni (Babesia gibbsoni), Babesia canis (Babesia canis), Babesia vogelige (Babesia variae), rossi (Babesia Rossi), Babesia divergens (Babesia divergence), Babesia bovis (Babesia bovis), Babesia motasi (Babesia motashi), and Babesia equii (Babesia equi). Further, the antibody to be detected is of the merozoite stage in the Schizogony stage of the life cycle of Babesia parasite. In some cases, the body cannot be detected in the merozoite stage, but by using the surface antigen protein of the present invention, an antibody against the protein can be detected.

The method for detecting anti-Babesia protozoa antibody is to contact a biological sample (eg, blood, serum, plasma) obtained from a subject with the purified surface antigen protein of the present invention, and detect the antigen-antibody reaction as an index. .. The antibody can be detected by a known immunological test method such as an ELISA method and an indirect fluorescent antibody method (IFA), but it is advantageous in that immunochromatography is simple and convenient.

Immunochromatography is an immunoassay measurement method that utilizes an antigen-antibody reaction. The surface antigen protein of the present invention, which is an antigen, is applied to the determination part. When the antibody against the Babesia parasite contained in the specimen (biological sample) binds to the surface antigen protein of the present invention in the determination part, it is visualized by the coloring component of the reagent.
The method for developing the color is not particularly limited, and examples thereof include a gold colloid method (a method in which gold colloid is bonded as colored particles).
An antibody against the Babesia parasite is detected by the color development in the determination part. Then, when the determination part develops color, it can be diagnosed as being positive, that is, having Babesiosis or being infected with the dog Babesia protozoa.

In the present invention, the "diagnosis", in addition to a definitive diagnosis that it is Babesiosis, or infected with Babesia protozoa, there is a possibility or risk of Babesiosis, or it can be infected with Babesia protozoa Includes preliminary diagnosis of sex or risk.

8. Kit The surface antigen protein of the present invention is used as a reagent for detecting an antibody against Babesia protozoa or as a diagnostic reagent for Babesia disease. The present invention also provides a kit containing the protein.
Examples of the kit of the present invention include a kit for immunochromatography. Further, the kit of the present invention contains, in addition to the above-mentioned surface antigen protein of the present invention, a diluent, a color former, etc., but is not limited thereto.
In the kit of the present invention, the immunochromatographic test strip is composed of, for example, a sample pad, a coating membrane, a conjugate pad, a sample absorption pad, and the like.

In one aspect of the present invention, the sample pad is, for example, a member where a sample (including a test substance) is dropped. The characteristics of the sample pad control the flow rate of the spread of the dropped sample. Further, the conjugate pad is a member for binding the target antibody contained in the sample solution and its antigen by an immunochemical reaction. The conjugate pad carries an antigen (labeled surface antigen protein of the present invention) labeled by an appropriate detection means. The coating membrane is a member for detecting a target antibody, and includes a determination unit in which the labeled surface antigen protein of the present invention for the target antibody is fixed, and a control unit in which the antibody for the surface antigen protein of the present invention is fixed. It is provided. The sample absorption pad is a member for absorbing unreacted labeled antigen, developing solvent and the like.

Examples of the labeling substance for detection include colored particles, metal colloids (gold colloid, platinum colloid, etc.), colored latex, dyes, and fluorescent dyes. Any one of them may be used alone, or two or more of them may be appropriately combined. Can be used. In the present invention, for example, gold colloid, platinum colloid and the like can be mentioned.
In addition to the above, the kit of the present invention can include, for example, a container (housing case) for an immunochromatographic test strip used for installing an immunochromatographic test strip, an instruction manual, and the like.

9. Therapeutic method, etc. The present invention administers a therapeutic agent for Babesiosis to an individual whose anti-Babescia parasite antibody has been detected by the above detection method or an individual who has been diagnosed with Babesiosis (is positive) by the above diagnosis method. The present invention provides a method for treating babesiosis, which comprises:
The dose and administration period of the drug for treating babesiosis to the individual can be set according to the usual usage and dose of the drug for treating babesiosis. Examples of the drug for treating babesiosis include diminazene (trade name Ganazec), clindamycin (trade name dalasin, antilobe), ST mixture (trade name tribrissen, etc.) and the like. In addition, a combination therapy of clindamycin, doxycycline and nitromedazole can be adopted.

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the scope of the present invention is not limited to these examples.
[Example 1]

Production of BgTRAP using a baculovirus-silkworm protein expression system An entry plasmid for transfer which artificially synthesizes a cDNA encoding a region excluding a secretory signal and a transmembrane region in a full-length surface protein of the merozoite of the canine Babesia protozoa Babesia gibsoni Inserted in.
In the present Example, the protein (surface antigen protein of the present invention) in the region excluding the secretory signal and the transmembrane region from the full-length surface protein of the canine Babesia protozoan merozoite is referred to as “BgTRAP”. However, the polynucleotide encoding BgTRAP may be referred to as “BgTRAP”.

At that time, a vector (N-terminal tag vector) in which the H8-STREP-H6-TEV sequence was inserted between the 30K secretion signal and BgTRAP, which was inserted downstream of the DNA sequence encoding the silkworm 30K protein secretion signal, and BgTRAP. Two types of entry vectors, a vector (C-terminal tag vector) into which the H8-STREP-H6-TEV sequence was inserted downstream, were prepared.
Then, the protein coding region containing BgTRAP was inserted into the pDEST8 plasmid for baculovirus transfer by the Gateway LR reaction. As a result of this reaction, a protein coding region containing BgTRAP is inserted into the region sandwiched between the B1 and B2 sequences as shown in FIG. 1 and placed under the control of the polyhedrin promoter derived from baculovirus.

The synthetic gene region containing BgTRAP on this pDEST8 plasmid was inserted into Bacmid containing the baculovirus genome by Tn7 transfer reaction. Specifically, an Escherichia coli strain carrying a plasmid containing baculovirus Bacmid and Tn7 transferase is transformed with pDEST8 plasmid containing a BgTRAP synthetic gene region, and a recombinant Bacmid having a multidrug drug resistance as a marker for a transposition reaction Was selected. Then, recombinant Bacmid was extracted from Escherichia coli by the alkali method, and purified recombinant Bacmid was introduced into the silkworm-derived cultured cells by the lipofection method.

Three days after the transfection, the germinated recombinant baculovirus (P1 virus) was collected in the cell culture supernatant, and the culture cells derived from silkworm were repeatedly infected twice to prepare a stock solution of P3 virus having a high virus concentration. .. 10 microliters of this virus was inoculated into the 5th instar larva of the silkworm, and the serum was collected. The result of confirming the expression level of BgTRAP in this serum by Western blotting is shown in FIG. Next, two-step purification was performed from this recombinant virus-infected larval serum (10 ml or 8 ml) using a His Trap Excel column (His tag purification) and a Strep-Tactin Superflow column (STREP tag purification). 2-6).

The result of dialysis of the purified product and measurement of the protein concentration is shown in FIG. 7. It was possible to recover 4.19 and 0.885 mg of N-terminal tag type BgTRAP and 0.03 mg of C-terminal tag type BgTRAP, respectively. N-terminal tag type BgTRAP was used for the subsequent analysis.
Moreover, the result of having investigated the influence by temperature of BgTRAP is shown in FIG. From FIG. 8, in the range of 0° C. to 42° C., there was no effect of temperature on the purification of BgTRAP.
[Comparative Example 1]
Experiment to confirm expression of E. coli expression system BgTRAP at 25°C and 37°C culture (SDS-PAGE/CBB/WB)

In this comparative example, it was examined whether BgTRAP of Babesia gibsoni could be expressed using Escherichia coli.
Information on the natural full-length antigen protein of Babesia gibsoni is shown in FIG. 9, and a method for constructing an E. coli-expressed BgTRAP construct is shown in FIG.

Culture conditions (1) 37° C. culture In a 250 ml flask, 25 ml of LB-Amp was added at 200 times/min to a preculture solution to OD600=0.02, and each concentration was adjusted to OD600=0.4 (this time 0.438). Add IPTG. The cells were collected 4 hours after the addition.
(2) Culturing at 25° C. A pre-cultured solution at a rate of 200 times/min was added to a 250 ml flask with 25 ml of LB-Amp so that the OD600 was 0.02, and the cells were collected after culturing for 24 hours. IPTG was added from the start of culture.

(3) Sample preparation Each sample of 1 ml culture was dissolved in 200 μl of bagbuster and then centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as a soluble fraction. The precipitate was further washed with bagbuster and PBS, dissolved with 200 μl of 1% SDS, and centrifuged at 15,000 rpm for 10 minutes, and the supernatant was used as an insoluble fraction.
(4) SDS-PAGE
The above sample was run at 10 ml/lane for both CBB and WB.
WB was blocked with 5% skim milk at room temperature for 1 hour, primary antibody (anti-GST-tag antibody, 1000-fold dilution) was incubated at 4°C for 2 days, and secondary antibody (anti-rabbit IgG-HRP, 2500-fold dilution) was incubated at room temperature for 1 hour. ..

Results The estimated molecular weight was 100 kda, but no band that appeared to be an expression product was observed in the expected size (Fig. 11).
[Example 2]
Reaction confirmation test using half-strips (strips consisting of a membrane coated with BgTRAP antigen and anti-BgTRAP antibody and an absorbent pad)

In this example, BgTRAP antigen and anti-BgTRAP antibody were applied to the membrane, and the reaction with the colloidal gold-sensitized BgTRAP antigen was confirmed.
(1) Reagent 1) BgTRAP antigen 2) Anti-BgTRAP polyclonal antibody (anti-BgTRAP antibody)
4) Gold colloid (BBI)
5) Membrane (Millipore)
6) Absorption pad (Millipore)
7) Backing sheet (Nippon Engineering)
8) Diluting liquid PIPES-0.1% Tween-0.1% BSA (PTB diluting liquid)

(2) Method 1) Preparation of colloidal gold-sensitized antigen BgTRAP antigen was sensitized at an antigen concentration of 50 μg/mL.
2) Strip preparation BgTRAP antigen and anti-BgTRAP antibody were adjusted to 1 mg/mL, coated on the membrane with a brush, dried at 37°C for 1 hour, and then blocked.
The antibody solid phase membrane was attached to a backing sheet, and an absorption pad was attached to the upper part to prepare a strip.
3) Strip reaction Gold colloid-sensitized antigen was added to the wells of the microplate, and the diluent alone and the anti-BgTRAP antibody adjusted to 10 μg/mL with the diluent were added, and the strip was inserted to develop the reaction solution on the membrane. ..

(3) Results The results of confirming the red line on the membrane generated by the reaction are shown in FIG.
When the reaction between the BgTRAP antigen, the anti-BgTRAP antibody and the colloidal gold-sensitized BgTRAP antigen was confirmed, a clear red test line was confirmed in the sample having an anti-BgTRAP antibody concentration of 10 μg/mL (FIG. 12).
A thin test line was formed with only the PTB diluted solution. This seems to be a non-specific reaction line.
The control line was slightly thin, but it was confirmed as a clear line.

(4) Summary The anti-BgTRAP antibody was successfully detected by half-strip immunochromatography using the BgTRAP antigen prepared in the present invention. ..

[Example 3]
Reaction confirmation test by full strip (a strip composed of a membrane coated with BgTRAP antigen and anti-BgTRAP antibody and an absorption pad, a conjugate pad, and a sample pad) In this example, BgTRAP antigen and anti-BgTRAP antibody were tested. It was applied to a membrane and the reaction with colloidal gold-sensitized BgTRAP antigen was confirmed.

(1) Reagent 1) BgTRAP antigen 2) Anti-BgTRAP polyclonal antibody (anti-BgTRAP antibody)
3) Positive and negative dog serum (5 types each, 10 types in total)
4) Gold colloid (BBI)
5) Membrane (Millipore)
6) Absorption pad (Millipore)
7) Sample Pad (Pall Corporation)
8) Glass fiber pad (Millipore)
9) Backing sheet (Nippon Engineering)
10) Diluent (including PIPES)

(2) Preparation of full strip for immunochromatography 1) Preparation of colloidal gold-sensitized antigen BgTRAP antigen was sensitized at an antigen concentration of 50 μg/mL.
2) Preparation of conjugate pad The colloidal gold-sensitized BgTRAP antigen solution was diluted with a buffer solution, impregnated in a glass fiber pad, and vacuum dried overnight.
3) Preparation of brush coating membrane Coating solution: anti-BgTRAP antibody solution (1.5 mg/mL) for control line BgTRAP antigen solution (1.0 mg/mL) for test line Each coating solution was coated on a membrane (Millipore HF120) with a brush. After drying at 37° C. for 1 hour, blocking was performed.

4) Full strip assembly The coated membrane, the absorbent pad, the conjugate pad, and the sample pad were attached to the backing sheet.
5) Preparation of diluted sample Each dog serum was diluted 100-fold with a diluent.
The following diluted samples were prepared and dispensed into microwells. A full strip (3 mm width) was inserted into this, and the presence or absence of a red line was confirmed.

(3) Test method Negative serum (1 individual), other infection negative serum (4 individuals) and dog Babesia positive serum (5 individuals) were diluted 100-fold with a diluent, and 60 μL of each was dispensed into microwells. A full strip (3 mm width) was inserted into the microwell and 5 minutes later, the presence or absence of a red line was confirmed.
The "other infection negative serum" is serum of a dog that is not infected with Babesia parasite but is infected with other infectious diseases.

(4) Results The results of confirming the red line on the membrane generated by the reaction are shown in FIG.
When the reaction between BgTRAP antigen, anti-BgTRAP antibody and colloidal gold-sensitized BgTRAP antigen was confirmed, a clear red test line was confirmed in all serum samples of Babesia protozoa-infected (Babeciasis)-positive dogs (lanes 6 to 10). ..
A thin test line was formed with only the PTB diluted solution. (Non-specific reaction line)
The control line was confirmed as a clear line in all samples (lanes 1 to 5). In addition, in FIG. 13, N-5 (lane 5) is the result of the serum of the dog infected with the filarial parasite.

(5) Summary It was confirmed by full strip immunochromatography that the dog serum sample reacted well.

Claims (21)

1. A protein in which the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminus have been removed from the amino acid sequence of the full-length surface antigen protein of Babesia parasite.
2. The following protein (a), (b) or (c).
   (A) a protein consisting of any of the amino acid sequences represented by SEQ ID NO: 2n (n represents an integer of 1 to 4); and (b) represented by SEQ ID NO: 2n (n represents an integer of 1 to 4). A protein (c) SEQ ID NO: 2n (n is an integer of 1 to 4) which consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any amino acid sequence, and which reacts with an antibody against Babesia parasite A protein consisting of an amino acid sequence having a homology of 80% or more with any of the amino acid sequences shown in (3) and reacting with an antibody against Babesia parasite
3. A polynucleotide encoding the protein according to claim 1 or 2.
4. The polynucleotide of (d), (e) or (f) below.
   (D) Polynucleotide consisting of any of the base sequences represented by SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) (e) SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) Polynucleotide (f), which hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to any of the nucleotide sequences shown in (4) under stringent conditions, and which encodes a protein which reacts with an antibody against Babesia parasite (f) SEQ ID NO: 2n-1 (n represents an integer of 1 to 4) and a protein comprising a nucleotide sequence having a homology of 80% or more with a polynucleotide comprising any of the nucleotide sequences represented by 2n-1 and reacting with an antibody against a Babesia parasite. Encoding polynucleotide
5. A vector containing the polynucleotide according to claim 3 or 4.
6. The vector according to claim 5, which is a viral vector.
7. A silkworm host containing the polynucleotide according to claim 3 or 4, or the vector according to claim 5 or 6.
8. A method for producing the silkworm host according to claim 7, which comprises a step of culturing or breeding the silkworm host and recovering the protein according to claim 1 from the silkworm host after the culture or breeding.
9. A protein produced by the method according to claim 8.
10. A reagent for detecting an antibody against Babesia protozoa, which comprises the protein according to claim 1, 2 or 9.
11. A reagent for diagnosing babesiosis, which comprises the protein according to claim 1, 2 or 9.
12. The reagent according to claim 10 or 11 for use in immunochromatography.
13. A method for detecting an antibody against Babesia protozoa, which comprises reacting a sample collected from a test animal with the protein according to claim 1, 2 or 9 or the reagent according to claim 10 or 11.
14. A method for diagnosing babesiosis using the detection result detected by the method according to claim 13 as an index.
15. A method for treating babesiosis, which comprises administering a therapeutic drug for babesiosis to a test animal in which babesiosis is diagnosed by the method according to claim 14.
16. A vaccine for treating or preventing babesiosis, which comprises the protein according to claim 1, 2 or 9.
17. A kit for detecting an antibody against Babesia parasite, which comprises the protein according to claim 1, 2 or 9.
18. A diagnostic kit for babesiosis, which comprises the protein according to claim 1, 2 or 9.
19. The kit according to claim 17 or 18 for use in immunochromatography.
20. A protein obtained by removing the amino acid sequence of the N-terminal signal peptide and the amino acid sequence of the region from the transmembrane region to the C-terminal of the amino acid sequence of the full-length surface antigen protein of Babesia protozoa for the detection or diagnosis of Babesiosis.
21. The following protein (a), (b) or (c) for detecting or diagnosing babesiosis.
    (A) a protein consisting of any of the amino acid sequences represented by SEQ ID NO: 2n (n represents an integer of 1 to 4); and (b) represented by SEQ ID NO: 2n (n represents an integer of 1 to 4). A protein (c) SEQ ID NO: 2n (n is an integer of 1 to 4) which consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in any amino acid sequence, and which reacts with an antibody against Babesia parasite A protein having an amino acid sequence showing 80% or more homology with any of the amino acid sequences represented by

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