CN114751970B - Japanese blood fluke antigen protein rSjScP15 and application thereof - Google Patents

Abstract

The invention provides a schistosoma japonicum antigen protein rSjScP15 and application thereof. The invention utilizes the whole genome expression profile chip of schistosoma japonicum to screen a series of genes which are highly expressed in schistosoma japonicum, wherein the antigen proteins coded by the genes SjScP15, sjScP57 and SjScP92 can be specifically identified by blood serum of schistosomiasis patient and have stronger positive reaction. ELISA detection shows that the antigen proteins have high sensitivity and specificity for detecting schistosome and can be used for developing schistosomiasis japonica diagnostic reagents. The immune protection effect of the schistosoma japonicum antigen proteins SjScP15, sjScP57 and SjScP92 after the mice are immunized for attack shows that the SjScP15, sjScP57 and SjScP92 are expected to be developed into schistosomiasis vaccines.

Description

Japanese blood fluke antigen protein rSjScP15 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a schistosoma japonicum antigen protein rSjScP15 and application thereof.

Background

Schistosoma (Schistonoma), also known as schistosome, belongs to the class Fasciola of the phylum Platensis. Schistosomiasis is an endemic parasitic disease caused by schistosomiasis mailing to the human body. Of the 6 schistosome species which are parasitic to the human body, the most widely distributed and most serious harmful are the Egyptian schistosome, schistosoma mansoni and Schistosoma japonicum. Wherein, the schistosoma japonica is a schistosome epidemic strain in China. They are a great hazard to human health and require intensive research into their control.

Diagnosis is a central link in the field of schistosomiasis control. The accurate diagnosis technology not only has important clinical significance for early discovery and early treatment of schistosomiasis patients, but also can provide judgment standards for the grades of epidemic areas of schistosomiasis and essential information and scientific basis for evaluating epidemic situations and checking prevention and control effects. The lack of an efficient and accurate diagnostic technique is an important reason why schistosomiasis cannot be completely eliminated. If the schistosomiasis patient cannot be diagnosed and treated in time, the eggs contained in the excrement can cause continuous epidemic spread of the schistosomiasis. Therefore, it is imperative to develop a new diagnosis method of schistosomiasis with high sensitivity and specificity. Currently, the diagnosis of schistosomiasis is mostly dependent on parasite morphology detection methods, such as the modified rattan method (Kato-Katz) recommended by the World Health Organization (WHO). The method is mainly used for diagnosing diseases by checking schistosome eggs in the feces or urine of patients, is time-consuming and labor-consuming, has low sensitivity to mild schistosome infection diagnosis, and is not suitable for large-scale schistosomiasis on-site monitoring.

Compared with the traditional morphological diagnosis method, the enzyme-linked immunosorbent assay (ELISA) diagnosis method has the advantages of simple and quick operation and higher sensitivity, and can be used for large-scale on-site monitoring. At present, the most commonly used antigens for schistosomiasis immune diagnosis are an adult antigen component (Adult worm antigen, AWA) and an egg antigen component (Soluble egg antigen, SEA) of schistosome, and the produced reagent is not suitable for standardization because the two crude antigens have complex components (composed of thousands of schistosome proteins) and have serious cross reaction with other parasite infection serum. Therefore, it is necessary to provide a new detection and diagnosis method to solve the problems of the prior art.

In addition, complete elimination of schistosomiasis, like other infectious diseases, relies on a strong and effective vaccine, but no schistosomiasis vaccine has been developed at present. Development of schistosome vaccine needs to develop antigen with protective function, and most schistosome proteins are unknown at present. The schistosome is in the extracorporeal circulation of host, and its surface film protein, secretion protein and schistosome protein in excrement can induce the humoral immune response of host. However, when the insect body is mature, the surface membrane of the insect body is tough and the volume of the insect body is large, so that antibodies generated by humoral immune reaction cannot effectively clear adult insect bodies. Schistosome cercaria is penetrated into human body and then transferred to lung through skin, and reaches hepatic portal vein to colonise after about 14 days, and the schistosome in this period is called schistosome child. The body of the artemia salina is tender within 0-14 days, which is the best stage of humoral immunity attack, but the host humoral immunity can not remove the artemia salina in the early stage because 10-14 days are needed for the released antigen to induce humoral immunity reaction. Therefore, the development of schistosome child-resistant antigens and schistosome vaccines aiming at the antigens are hopeful to help a host to kill the schistosome in the child-resistant period, and are the key points of the development of the schistosome vaccines.

Disclosure of Invention

The invention aims to provide schistosoma japonicum antigen protein rSjScP15 and application thereof.

The invention is characterized in that: the schistosoma japonicum whole genome expression profile chip is utilized to screen a series of genes which are highly expressed in schistosoma japonicum schistosome, and schistosome is a worm body which is contacted with the peripheral circulation of a host earlier, so that the humoral immune response of the host can be initiated earlier, and corresponding antibodies are generated; and the antigens of the schistosome are found to be the best targets for the development of schistosome vaccines. Among them, 3 Japanese blood fluke antigen protein genes SjScP15, sjScP57 and SjScP92 are amplified by PCR to form hydrophilic segment and expressed in colibacillus, and the obtained recombinant protein has high sensitivity and/or specificity for diagnosing Japanese blood fluke disease (ELISA test shows that it is a potential diagnostic antigen candidate target. The recombinant proteins are used for Japanese schistosome infected mouse model test, and the three recombinant proteins are found to have better immunoprotection and are potential schistosomiasis vaccine candidate antigens.

To achieve the object of the present invention, in a first aspect, the present invention provides a gene SjScP15, sjScP57 or SjScP92 highly expressed in schistosoma japonicum;

the SjScP15 gene is:

i) A nucleotide sequence shown in SEQ ID NO. 1;

ii) the nucleotide sequence shown in SEQ ID NO. 1 is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein;

iii) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 1 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

The antigen protein encoded by the gene SjScP15 (SjScP 15) comprises or consists of the amino acid sequence:

i) An amino acid sequence as shown in SEQ ID NO. 4;

ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)

iii) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).

The gene SjScP57 is:

i) A nucleotide sequence shown in SEQ ID NO. 2;

ii) the nucleotide sequence shown in SEQ ID NO. 2 is substituted, deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein;

iii) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 2 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

The antigen protein encoded by the gene SjScP57 (rSjScP 57) comprises or consists of the amino acid sequence:

i) An amino acid sequence shown as SEQ ID NO. 5;

ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)

iii) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).

The SjScP92 gene is:

i) A nucleotide sequence shown in SEQ ID NO. 3;

ii) the nucleotide sequence shown in SEQ ID NO. 3 is substituted, deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein;

iii) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 3 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

The antigen protein encoded by the gene SjScP92 (rSjScP 92) comprises or consists of the amino acid sequence:

i) An amino acid sequence shown as SEQ ID NO. 6;

ii) an amino acid sequence obtained by ligating a tag at the N-terminal and/or C-terminal of i); or (b)

iii) Proteins with the same function obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence of i) or ii).

Truncated forms of antigen proteins encoded by the genes SjScP15, sjScP57 and SjScP92 or modified protein derivatives or fusion proteins, and protein variants having the same or similar antigenicity as the antigen proteins shown in SEQ ID No. 4, 5 and 6 belong to the protection scope of the present invention.

In a second aspect, the invention provides any one of the following uses of the antigenic protein:

1) For preparing a reagent or kit for detecting schistosomiasis japonica;

2) For preparing a vaccine against schistosomiasis japonica;

3) Is used for preparing medicines for treating schistosomiasis japonica;

4) The method is used for detecting schistosoma japonicum infection;

5) For diagnosing schistosomiasis japonica;

6) Can be used for preventing and treating schistosomiasis japonica.

Specifically, the schistosoma japonicum antigen proteins rSjScP15, rSjScP57 and rSjScP92 are used as specific schistosome antigens and applied to serodiagnosis; as immunogen, in preparing vaccine against schistosome; as potential drug action targets, the method can be applied to screening chemical and other kinds of drugs; as the coding genes of the rSjScP15, rSjScP57 and rSjScP92 proteins of the schistosoma japonica, the gene can be applied to gene therapy.

In a third aspect, the present invention provides a schistosoma japonicum detection reagent comprising at least one of the following (1) to (3):

(1) schistosoma japonica antigen protein rSjScP15, or DNA molecule encoding the antigen protein, or recombinant protein produced by recombinant bacteria containing the DNA molecule;

(2) schistosoma japonica antigen protein rSjScP57, or a DNA molecule encoding the antigen protein, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule;

(3) the schistosoma japonica antigen protein rSjScP92, or a DNA molecule encoding the antigen protein, or a recombinant protein produced by recombinant bacteria containing the DNA molecule.

In a fourth aspect, the present invention provides a kit comprising the schistosoma japonicum detection reagent.

In a fifth aspect, the present invention provides an ELISA immunodiagnostic kit for schistosomiasis japonica, said kit comprising:

1) A microporous reaction plate coated with 1-5 mug/mL antigen protein; carbonate-bicarbonate buffer (available from Sigma, cat. C3041) containing 0.05% v/v TWEEN20 was used as coating buffer;

2) Washing buffer: PBST solution, i.e., PBS solution containing 0.05% v/v TWEEN20, pH7.4;

3) Sample dilution: 5-10% BSA solution, PBS buffer solution is used as solvent for preparation;

4) Enzyme-labeled antibody: an alkaline phosphatase-labeled goat anti-human immunoglobulin antibody;

5) Substrate color development liquid: pNPP color development liquid;

pNPP was purchased from Sigma, cat No. N2640. The preparation method of the pNPP color development liquid comprises the following steps: 15mg of pNPP was dissolved in 15mL of 0.1M glycine buffer (0.1M glycine, 1mM MgCl) ₂ ，1mM ZnCl ₂ Dissolving in purified water, pH 10.4).

6) Reaction termination liquid: 120g/L NaOH aqueous solution;

7) Positive control: human immunoglobulin IgG was coated at 1. Mu.g/mL.

Meanwhile, a negative control was set: and replacing the enzyme-labeled antibody with a sample diluent by using a corresponding antigen protein coated plate.

In a sixth aspect, the invention provides an immunogenic composition comprising the antigenic protein.

In a seventh aspect, the invention provides a schistosome vaccine comprising said immunogenic composition. Optionally, an adjuvant is included in the vaccine.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the antigen protein provided by the invention has high detection sensitivity, can still detect the schistosomiasis patient with mild infection (EPG < 100), and has the sensitivity of 96%.

The detection kit provided by the invention has high specificity, and the diagnosis specificity of the schistosomiasis japonica is as high as 100%.

And thirdly, the kit can be used for early diagnosis of schistosome infection, and the existence of anti-rSjScP 15, rSjScP57 and rSjScP92 protein antibodies in serum can be detected after 3 weeks of infection of experimental animals. Simple operation, stable result and high repeatability.

(IV) the schistosoma japonicum antigen proteins rSjScP15, rSjScP57 and rSjScP92 are used for immunizing mice, the insect-dyeing rate and the egg-loading rate can be obviously reduced after attack, and the immune protection effect shows that the rSjScP15, rSjScP57 and rSjScP92 are hopefully developed into schistosome vaccines.

Drawings

FIG. 1 is a schematic diagram showing the results of PCR amplification of the gene sequences of SjScP15, sjScP57 and SjScP92 of Schistosoma japonicum in the preferred embodiment of the present invention. Wherein M: DNA molecular weight standard; 1,2 and 3 are PCR amplification products of SjScP15 (832 bp), sjScP57 (865 bp) and SjScP92 (728 bp), respectively.

FIG. 2 is a schematic diagram showing the result of SDS-PAGE analysis of rSjScP15, rSjScP57 and rSjScP92 recombinant proteins according to the preferred embodiment of the present invention. Wherein M: protein molecular weight standard (kDa), 1: rSjScP15,2: rSjScP57,3: rSjScP92.

FIGS. 3 to 5 are schematic diagrams showing Western blot analysis results of rSjScP15 (FIG. 3), rSjScP57 (FIG. 4), rSjScP92 (FIG. 5), recombinant protein antigen, schistosomiasis human serum and different schistosome infected animal serum, respectively, in the preferred embodiment of the present invention. Wherein M: protein molecular weight standard (kDa), 1: schistosomiasis japonica human serum, 2: serum of mice infected with schistosoma japonicum for 42 days, 3: rabbit serum from day 42 infected schistosoma japonicum, 4: mouse anti-His tag antibody positive control, 5: normal mouse seronegative control.

FIG. 6 is a schematic diagram showing the results of comparison of rSjSP-13-ELISA kit and rSjScP15-ELISA, rSjScP57-ELISA and rSjScP92-ELISA kit for diagnosis and evaluation of schistosomiasis japonica in a preferred embodiment of the present invention. Wherein A is the result of rSjSP-13-ELISA kit, B is the result of rSjScP15-ELISA kit, C is the result of SjScP57-ELISA kit, and D is the result of SjScP92-ELISA kit. Sj is the group of Japanese schistosomiasis, sj-3M is the group of Japanese schistosomiasis patients with chemotherapy for three months, cs is the group of clonorchis sinensis, and Healthy is the Healthy control group. The horizontal line in the graph is a cutoff value 2.1 times the mean of the health group, and the line is considered positive on the line and negative off the line.

FIG. 7 is a schematic diagram showing the results of immunization effect of Japanese blood fluke rSjScP15, rSjScP57, rSjScP92 and rSjSP-13 recombinant proteins on the immunized mice according to the preferred embodiment of the invention. Wherein A is a comparison graph of the number of insect bodies in the mice after the schistosoma japonicum infection is 42 days, and B is a comparison graph of the number of liver insect eggs in the mice after the schistosoma japonicum infection is 42 days. C is the self nutrition supplied by the peripheral blood of the host after the schistosome infects the host, and causes the lack of the nutrition and the weight reduction of the host. In the figure, PBS group is PBS immunized mouse control group, BLANK group is BLANK control group of non-immunized Japanese blood fluke. * Represents P < 0.01, P <0.05, P < 0.0001.

Detailed Description

The general technical flow of the invention is as follows:

firstly, the result of chip screening is verified by a qPCR method, and the expression level of SjScP15, sjScP57 and SjScP92 genes in the artemia is confirmed to be obviously higher than that of the artemia in other periods. The sequence of the corresponding signal peptide and hydrophobic region in the corresponding nucleic acid sequence was then removed using SignalP-4.1 Server (http:// www.cbs.dtu.dk/services/SignalP /) and TMHMM Server v.2.0 (http:// www.cbs.dtu.dk/services/TMHMM-2.0 /). Primer design and primer specificity analysis were performed using NCBI Primer designing tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast /). Cloning was then performed using Invitrogen-Gateway Technology with clonase II kit.

Adding attB locus on one end of a primer according to the requirements of a specification, carrying out PCR amplification on SjScP15, sjScP57 and SjScP92 genes of schistosoma japonicum, purifying the amplified products, cloning the amplified products onto a pDONR221 vector by using BP Clonase II enzyme provided in a kit, carrying out transformation screening, carrying out plasmid sequencing identification, transferring a gene sequence into an expression vector pDOST 17 by using LR Clonase II enzyme provided in the kit, carrying out transformation screening, carrying out plasmid sequencing identification, and then, carrying out recombinant protein induction expression in a escherichia coli host cell; the inclusion body protein is purified by denaturation and nickel column affinity chromatography, and finally purified recombinant proteins rSjScP15, rSjScP57 and rSjScP92 are obtained, and the in vitro cloning expression and purification of the SjScP15, sjScP57 and SjScP92 genes are completed. Subsequently, western blot technology was used to verify that the purified rSjScP15, rSjScP57, rSjScP92 recombinant proteins could be recognized by the serum of experimental animals infected with Schistosoma japonicum and blood fluke human serum. Furthermore, the purified rSjScP15, rSjScP57 and rSjScP92 recombinant proteins are used as diagnostic antigens for preparing schistosomiasis diagnostic reagents and kits, and the application value of the recombinant proteins in schistosomiasis immunodiagnosis is evaluated by ELISA technology analysis and the change rule of antibodies against rSjScP15, rSjScP57 and rSjScP92 in serum of infected animals is analyzed.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 cloning of the SjScP15, sjScP57 and SjScP92 genes of Schistosoma japonicum

According to the SjScP15, sjScP57 and SjScP92 gene sequences which are found by the inventor in the prediction of the schistosoma japonicum genome, primers are respectively designed and enzyme cutting sites are introduced. Because the 3' -end 190-400 bases of the SjScP57 coding gene is highly conserved, the amino acid sequence of the coding protein is highly homologous with human and other mammal tyrosine hydroxylases, and the antibody induced by the immune epitope of the coded protein is extremely easy to cause nonspecific reaction, on one hand, the nonspecific reaction of the antibody can be possibly caused, so that the diagnosis specificity is influenced; on the other hand, when the protein segment is used for immunizing a host, the induced antibody can nonspecifically influence the activity of host tyrosine proteinase, which is unfavorable for the research and development of vaccines. Therefore, the invention selects the non-conserved segment (607-957 bases) of SjScP57 coding gene to prepare recombinant protein for diagnosis and vaccine research. The SjScP15 and SjScP92 gene encoding proteins have no homologous proteins found in common mammalian hosts, so the primers were designed using the full-length gene sequences as templates. The primer sequences were as follows:

SjScP15：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGGAAGGTTTCTTACAGCGGA-3’

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATGCCGTTATATCACATCCACCA-3’

SjScP57：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCACGCTACTAGAGCATGCAA-3’

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGAAGCTCCTAAAGATGC-3’

SjScP92：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCACACATTTGGAATGCTAGA-3’

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGTATGAATTCGATAATGAGCC-3’

the underlined part of PF is attB1 site designed according to the Invitrogen Gateway Technology with Clonase II kit instructions for ligation to shuttle vector pDONR 221; the underlined portion of PR is the attB2 site designed according to the Invitrogen Gateway Technology with Clonase II kit instructions for ligation to shuttle vector pDONR 221; specific primers were synthesized by su Jin Weizhi biotechnology limited.

PCR reaction is carried out by taking schistosoma japonicum (Schistosoma japonicum) 14 days old schistosome cDNA as a template, and a gene ORF fragment is amplified, wherein the reaction system is as follows: high-fidelity DNA polymerase Mix 12.5 μl, cDNA template 1 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, ddH ₂ O10.5. Mu.l, total volume 25. Mu.l. PCR amplification procedure: 5min at 95 ℃, 30sec of denaturation at 95 ℃, 30sec of annealing at 55 ℃ and 1min of extension at 72 ℃ for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.

The PCR products were detected by electrophoresis on a 1.2% agarose gel to see if the target band was present, and the result is shown in FIG. 1, M: DNA molecular weight standard, 1: sjScP15,2: sjScP57,3: sjScP92. The agarose gel electrophoresis result shows that the SjScP15, sjScP57 and SjScP92 have a clear band at 1896bp,354bp and 636bp respectively, which are consistent with the size of the expected target fragment, thus indicating that the ORF fragments of the rSjScP15, rSjScP57 and rSjScP92 genes are successfully amplified from cDNA.

The PCR product was purified and recovered using AxyPrep DNA gel recovery kit (AXYGEN Co.).

Invitrogen Gateway Technology with Clonase II kit constructs shuttle vector, and the reaction system is as follows: PCR recovery of DNA product (dissolved in ddH ₂ O) 150ng, pDONR221 vector (dissolved in ddH ₂ O) 150ng, BP clonase II enzyme 2. Mu.l, add ddH ₂ O to a total volume of 10. Mu.l. After mixing, incubation was carried out at 25℃for 1h. The above ligation product was transformed into E.coli DH 5. Alpha. Competent cells (Beijing full gold Biotechnology Co., ltd.), and the transformed competent cells were plated on LB medium plates (containing 50. Mu.g/ml kanamycin) and cultured overnight at 37 ℃. And selecting single colony for PCR identification, and constructing an expression vector by using a Invitrogen Gateway Technology with Clonase II kit from the clone which is identified as positive by the PCR identification. The reaction system is as follows: shuttle vector positive for PCR identification (dissolved in ddH ₂ O) 150ng, pDEST17 vector (dissolved in ddH ₂ O) 150ng, TE Buffer (pH 8.0) was added to a total volume of 8. Mu.l, followed by addition of 2. Mu.l of LRclonase II enzyme. After mixing, incubation was carried out at 25℃for 1h. Then 1. Mu.l of Protein K (2. Mu.g/. Mu.l) and 3 were addedThe protein was removed by incubation at 7℃for 10min. The ligation product was transformed into E.coli B21DE3 competent cells (Beijing full gold Biotechnology Co., ltd.), and the transformed competent cells were plated on LB medium plates (50. Mu.g/ml ampicillin) and cultured overnight at 37 ℃. Single colonies were picked for PCR identification, and clones positive for PCR identification were sent to su Jin Weizhi biotechnology limited for DNA sequencing to confirm whether the sequences were correct. Sequencing analysis results show that the sequence of the inserted exogenous gene fragment is correct, and the recombinant plasmids pDEST17-rSjScP15, pDEST17-rSjScP57 and pDEST17-rSjScP92 are successfully constructed. The nucleotide sequences of the genes SjScP15, sjScP57 and SjScP92 are shown in SEQ ID NO. 1-3, and the amino acid sequences of the antigen proteins encoded by the genes are shown in SEQ ID NO. 4-6.

EXAMPLE 2 expression and purification of recombinant protein of Schistosoma japonicum rSjScP15, rSjScP57, rSjScP92

Clones identified as positive by the PCR were inoculated into 15mL of LB liquid medium (containing 50. Mu.g/mL ampicillin) and cultured overnight at 37℃and 200rpm, and 10mL of the medium was transferred into 1L of LB medium (containing 50. Mu.g/mL ampicillin) the next day, followed by further culturing until OD _600nm The value is 0.8, IPTG with the final concentration of 1mM is added for induction, the expression is carried out for 16 hours at 140rpm at 18 ℃, the thalli are collected by centrifugation, and the thalli are frozen at-80 ℃ for standby.

And respectively taking a small amount of pre-induction and post-induction bacteria, suspending in PBS buffer solution, adding SDS-PAGE loading buffer solution, uniformly mixing, and boiling in a boiling water bath for 5min to denature protein.

10 μl of each of the pre-and post-induction samples was added to each well for SDS-PAGE analysis (5% gel concentrate and 12% gel separator).

The pDEST17-rSjScP15, pDEST17-rSjScP57 and pDEST17-rSjScP92 recombinant plasmids are respectively transformed into expression competent cells, and obvious expression bands appear on thalli after IPTG induction expression compared with thalli before induction.

The induced bacteria were resuspended in 40mL of bacterial lysate, sonicated, centrifuged at 12,000rpm for 30min at 4℃and the inclusion body pellet and supernatant were collected, respectively.

SDS-PAGE analysis of inclusion body pellet and supernatant was performed to identify the solubility of recombinant protein. The results show that recombinant proteins are mainly present in inclusion body pellet.

The inclusion body was resuspended in PBS containing 1% Triton-X100, washed ultrasonically for 5min, and centrifuged at 12,000rpm for 15min to collect inclusion body pellet;

suspending inclusion body in 8M urea, rotating at 4deg.C, mixing overnight, dissolving inclusion body completely, centrifuging at 12,000rpm for 30min, and collecting supernatant;

passing the supernatant through a nickel ion chelating gel column (QIAGEN company) to bind rSjScP15, rSjScP57 and rSjScP92 recombinant proteins with 6 histidine tags on the gel column, washing the hybrid protein with 50mM imidazole, eluting the recombinant protein with 250mM imidazole, and collecting the eluate;

the purity of the recombinant protein obtained by purification is detected by SDS-PAGE analysis. As shown in FIG. 2, the molecular weights of rSjScP15, rSjScP57 and rSjScP92 proteins are about 15kDa,14kDa and 35kDa respectively, and the molecular weights of the rSjScP15, rSjScP57 and rSjScP92 proteins are consistent with the theoretical relative molecular weights of the target recombinant proteins, which indicates that the recombinant proteins with higher purity are obtained after purification by a nickel ion chelating gel column.

The concentration of the recombinant protein was measured using BCA protein assay kit (Thermo Fisher Scientific company) and the procedure was performed according to the instructions. The concentration of the recombinant protein obtained by measurement and purification was 1.0mg/mL.

EXAMPLE 3 detection of antigenicity of recombinant proteins of schistosoma japonicum rSjScP15, rSjScP57 and rSjScP92

SDS-PAGE electrophoresis: taking 100ng of recombinant protein for loading, and carrying out electrophoresis under the following conditions: 100V 20min,120V 1h.

Transferring: transferring proteins in the PAGE gel to a PVDF membrane by adopting a wet transfer method, wherein the electrotransfer conditions are as follows: ice bath, 100v 1h.

Closing: PVDF membranes were blocked with 5% nonfat dry milk for 2h at room temperature and washed 3 times with tbst buffer.

Adding an antibody for incubation: BALB/c mouse serum, new Zealand white rabbit serum and schistosomiasis japonica human serum, which are infected with schistosoma japonica 42 days, were added, respectively, and incubated overnight at 4℃with a positive control of a mouse anti-His-tag antibody (Aibi biological medicine Co., ltd.) and a negative control of a healthy mouse serum (diluted with blocking solution 1:500), and washed 3 times with TBST buffer.

Adding secondary antibody for incubation: fluorescent-labeled anti-mouse IgG antibody, anti-rabbit IgG antibody and anti-human IgG antibody (diluted 1:10,000 with blocking solution) were added separately, incubated at 37℃for 1h in the dark, and washed 3 times with TBST buffer.

Film sweeping: scanning imaging was performed using an Odyssey infrared laser imaging system.

As shown in fig. 3 to 5, fig. 3 shows the result of rSjScP15, fig. 4 shows the result of rSjScP57, and fig. 5 shows the result of rSjScP92, where M: protein molecular weight standard (kDa), 1: schistosomiasis japonica human serum, 2: serum of mice infected with schistosoma japonicum for 42 days, 3: rabbit serum from day 42 infected schistosoma japonicum, 4: mouse anti-His tag antibody positive control, 5: normal mouse seronegative control. With mouse anti-His-tag antibodies as positive controls, rSjScP15, rSjScP57, rSjScP92, there was a distinct recognition band at 15kDa,14kDa,35kDa, respectively, and healthy mouse serum as negative control, without distinct bands. Wherein rSjScP15 and rSjScP57 proteins can be recognized by BALB/c mouse serum, new Zealand white rabbit serum and Japanese schistosome human serum infected with Japanese schistosome, rSjScP92 can be recognized by human serum, and the mouse serum also has recognition bands. The recombinant proteins rSjScP15, rSjScP57 and rSjScP92 are proved to have good antigenicity.

EXAMPLE 4 preparation of Japanese schistosomiasis rSjScP15, rSjScP57, rSjScP92 immunodiagnosis kit

1. The main components of the kit comprise:

solid phase carrier for coating antigen: the target protein was diluted to 1. Mu.g/mL with a carbonate-bicarbonate buffer (purchased from Sigma, cat. C3041) containing 0-0.05% v/v TWEEN20 as coating buffer, and coated in polystyrene reaction wells at 100. Mu.L/well.

Washing buffer: PBST solution, i.e., PBS solution containing 0.05% v/v TWEEN20, pH7.4.

Sample dilution: 10% BSA solution in PBS buffer as solvent.

Enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-human immunoglobulin antibody (α, γ and μ -chain specific) antibody (Sigma).

Substrate color development liquid: pNPP color development liquid.

pNPP was purchased from Sigma, cat No. N2640. The preparation method of the pNPP color development liquid comprises the following steps: 15mg of pNPP was dissolved in 15mL of 0.1M glycine buffer (0.1M glycine, 1mM MgCl) ₂ ，1mM ZnCl ₂ Dissolving in purified water, pH 10.4).

Reaction termination liquid: 120g/L NaOH aqueous solution.

Positive control: plates were coated with human immunoglobulin IgG (1. Mu.g/mL).

Negative control: and replacing the enzyme-labeled antibody with a sample diluent by using a corresponding antigen protein coated plate.

2. The operation procedure and detection method of the kit are as follows:

closing: the liquid in the well was dried, 200. Mu.L/well of diluent was added, the well was blocked for 2 hours at 37℃and washed three times with PBST, 200. Mu.L/well, 2 min/time and finally the well was dried by a beat.

Adding a sample to be tested: the serum and the diluent of the sample to be detected are diluted according to the proportion of 1:100, positive, negative and blank controls (only sample diluent is added) are simultaneously arranged, each sample is added according to 100 mu L/hole, 3 compound holes are detected in parallel for each sample, and the sample is incubated for 1h at 37 ℃.

Washing: the liquid in the wells was drained, washed four times with PBST buffer, 200. Mu.L/well, 2 min/time, and finally patted dry.

Adding enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-human immunoglobulin (α, γ and μ -chain specific) antibody was added, 100 μl/well, incubated at 37 ℃ for 1h, washed as above, and patted dry.

Color development: enzyme substrate chromogenic solution, 100. Mu.L/well, was added and incubated at 37℃for 30min in the dark, and stop solution, 25. Mu.L/well was added.

Data reading and processing: reading OD with an ELISA reader _405nm The value was 2.1 times the OD mean value of the negative control sample as a critical value (cut-off) for negative and positive judgment.

EXAMPLE 5 immunodiagnostic evaluation of rSjScP15, rSjScP57, rSjScP92-ELISA kits for Japanese schistosomiasis

1. Sensitivity of the kit

35 parts of human serum of the schistosomiasis japonica positive to the egg of the fecal test of Hunan province was collected by using a modified rattan adding method (Kato-Katz), the sensitivity of each kit was evaluated, and compared with an rSjSP-13-ELISA kit (the preparation method of the rSjSP-13-ELISA kit is the same as that of example 4, the amino acid sequence of the rSjSP-13 antigen protein is shown as SEQ ID NO: 7), and ELISA experimental operation is the same as that of example 3.

The results are shown in Table 1 and FIG. 6, sj is the Japanese schistosomiasis group, and Healthy is the Healthy control group (35 serum samples from normal people in Heilongjiang province). The horizontal line through the whole coordinate system in the figure is the cut off value of 2.1 times the mean of the health group, and the line is considered positive on the line and negative off the line. Of 20 human serum of schistosomiasis japonica, 18 were diagnosed positive by rSjSP-13-ELISA kit (fig. 6A), the sensitivity of rSjSP-13-ELISA kit to schistosomiasis japonica was 90.0% (95% confidence interval, 66.9% -98.3%), the result was close to the literature reported sensitivity of rSjSP-13-ELISA kit 90.4% (95% confidence interval, 75.0% -95.0%); 20 parts were diagnosed as positive by rSjScP15-ELISA and rSjScP57-ELISA kits (FIGS. 6B and 6C,95% confidence interval, 80.0% -100%). 19 parts were diagnosed as positive by rSjScP92-ELISA kit (FIG. 6D,95% confidence interval, 73.1% -99.7%). The sensitivity of the rSjScP15, rSjScP57 and rSjScP92-ELISA kit is significantly higher than that of the rSjSP-13-ELISA kit. The results showed that the sensitivity of the rSjScP57-ELISA kit was the same as that of the rSjSP-13-ELISA kit.

TABLE 1 comparative analysis of sensitivity of rSjSP-13-ELISA to rSjScP15, rSjScP57, rSjScP92-ELISA kit

* Sensitivity was significantly higher than for the rSP13-ELISA kit, p <0.05.

2. Kit specificity

The false positive rate of each kit in normal population and the cross reaction condition of the serum of other parasite patients were evaluated by using 20 parts of collected serum samples of normal persons in Healthy group Heilongjiang province and 18 parts of serum of clonorchiasis sinensis patients in Guangdong province, and compared with rSjSP-13-ELISA kit, and ELISA experimental operation was the same as in example 3.

The results are shown in Table 2 and FIG. 6, where Cs is the group of clonorchis sinensis patients. 20 normal human serum was diagnosed as negative by rSjSP-13-ELISA, rSjScP57-ELISA, rSjScP15-ELISA and rSjScP92-ELISA kits, and the specificity of each of the four kits was 100% (95% confidence interval, 80.0% -100%). 15 clonorchis sinensis patients serum is diagnosed as negative by four kits, and has no cross reaction.

TABLE 2 comparative analysis of rSjSP-13-ELISA with SjScP15, sjScP57, sjScP92-ELISA kit specificity

3. Value of the kit in assessing the progress of schistosomiasis japonica

The above 20 cases of schistosomiasis japonica patients were collected again 3 months after chemotherapy in a conventional treatment manner, and the value of the kit in evaluating the progress of schistosomiasis japonica was evaluated and compared with the rSjSP-13-ELISA kit, and the ELISA experimental procedure was the same as in example 3. As a result, as shown in FIG. 6, sj-3M was a group of three months of chemotherapy for Japanese schistosomiasis patients. The OD value of the patients after the SjScP15-ELISA and the rSjSP-13-ELISA kit are obviously reduced compared with the OD value of the patients after the SjScP15-ELISA and the SjScP92-ELISA kit are not obviously changed compared with the OD value of the patients after the SjScP15-ELISA and the SjScP92-ELISA kit are subjected to the chemotherapy.

EXAMPLE 6 evaluation of protective Effect of recombinant proteins of Japanese blood fluke rSjScP15, rSjScP57 and rSjScP92 as antigen immunized mice

1. Preparation of antigen immunized mouse model

a) Immunization

Immunization of Balb/c mice; the orbit was negative serum prior to immunization.

Taking 60ug of immunogen (initial 60ug for boosting 30 ug), diluting to 200ul with normal saline, and adding equal volume of Freund's adjuvant (Freund's complete adjuvant for initial immunization and Freund's incomplete adjuvant for boosting); mixing the solution with adjuvant to form water-in-oil.

The well mixed immunogen is injected subcutaneously into the back and the abdomen for immunization, and the number of the immunization points is 8-10.

b) The orbit takes negative blood

The mouse is held by one hand and the eyeball of the mouse is exposed; the other hand holds a capillary tube to insert into the back angle of the eye of the mouse, and the user slowly rotates the back angle of the eye of the mouse by tilting 45 degrees. The force and posture of the hand were slowly adjusted to allow blood to flow into the capillary, and after almost filling, the capillary was immediately placed in a 1.5ml centrifuge tube, and negative blood was collected at 20ul.

Standing whole blood at room temperature for 30min-120min, centrifuging at 5000rpm for 10min, and collecting serum.

c) ELISA assay titers

And (3) preparation of a reagent:

coating liquid: sodium carbonate-sodium bicarbonate buffer, ph9.6.

PBS buffer, pH7.4.

Sealing liquid: 2% BSA or PBS containing skimmed milk powder.

Washing liquid: PBST (i.e., PBS solution containing 0.05% v/v TWEEN 20).

Color development liquid: 1%A solution +10% solution B (solution A: DMSO containing 1% TMB; solution B: 0.1% H) ₂ O ₂ Citric acid buffer of (c).

Stop solution: 2M sulfuric acid.

And (2) secondary antibody: goat anti-mouse IgG/HRP.

The experimental steps are as follows:

1) Diluting the antigen with the coating solution to a final concentration of 2ug/ml,100 ul/well, 4 ℃ overnight; after which the washing liquid was washed 2 times.

2) Sealing the sealing liquid, 200 ul/hole, incubating the box at 37 ℃ for 2 hours; after which the washing liquid was washed 1 time.

3) The polyclonal serum was diluted 2-fold from 200-fold (diluted in PBS), the blank (blank) was PBS, and the negative (negative) was diluted 200-fold (diluted in PBS) with negative serum; all are 100 ul/hole, incubator at 37 ℃ for 1h; after which the washing liquid was washed 3 times.

4) Adding PBS to dilute 20000 times of secondary antibody, 100 ul/hole, incubator at 37 ℃ for 1h; after removal, the mixture was washed 3 times with a washing solution.

5) And (3) developing, wherein the developing time is 5-15min, and the developing liquid is 100 ul/hole.

6) The wells were terminated by adding 50ul of stop solution per well.

7) The absorbance is measured at two wavelengths (450, 630), the stored data is recorded, and a mapping analysis is performed.

The titer is a dilution multiple corresponding to a maximum OD value of 1/2.

2. Immune protective action evaluation

a) Attack insects

The positive oncomelania is raised for one week at room temperature and then collected in a conical flask, and then irradiated by warm light for 2-4 hours to release cercaria in vivo.

The abdominal hair of the mice was plucked, and the abdominal skin was exposed.

40 cercarias/mice were stained on coverslips with a fungus-removing ring under a microscope.

The coverslip was applied to the skin of the abdominal wall of the mice for 5min to promote penetration of cercaria into the skin.

Infected mice were routinely kept for 42 days in the BSL2 laboratory.

b) Evaluation of antigen immunoprotection

Each mouse was weighed after 42 days of feeding (5 per group of experiments, 3 total replicates).

After anesthetizing the mice, the portal vein is cut, PBS is injected through cardiac puncture, the insect bodies are flushed out of the body, and the insect bodies are counted.

The livers are picked up, sheared and ground by a 200-mesh copper net, 20ml of 5% KOH solution is added into each 1g of the livers, the livers are evenly mixed, the mixture is incubated at 37 ℃ for overnight digestion, and the number of eggs of the livers is counted by microscopic examination.

The rSjSP-13 recombinant protein immune group and the BALNK protein-free empty control immune group are used as comparison. The results are shown in tables 3 to 4 and FIG. 7A.

Specifically, as shown in table 3 and fig. 7A, the mice in the rSjScP15, rSjScP57, rSjScP92 immunized group had significantly higher rate of reduction than the PBS immunized control group, reaching 38.1%, 34.8% and 33.4% respectively, and the rate of reduction was calculated as: (1-total number of mature worms/total number of mature worms in the blank group) ×100%. In this example, the number of infected cercarias per mice was 40, and the total number of adult worms was based on PBS control group, since the number of surviving worms in each mouse after infection was generally less than 40 due to the influence of cercarias activity and sex ratio and host immune system. The insect reduction rate of the rSjSP-13 recombinant protein immune group is only 3.8 percent respectively. Therefore, the rSjScP15, rSjScP57 and rSjScP92 recombinant proteins have better insect-reducing effect than rSjSP-13 group after immunization.

The biggest hazard of schistosomiasis is that eggs lay after the worm body is mature, and the eggs deposit in the liver to lead to liver fibrosis so as to cause a series of pathological changes. As shown in table 4 and fig. 7B, the number of eggs in liver of rSjScP15, rSjScP57, rSjScP92 immunized mice was significantly reduced compared with PBS immunized control group, and the egg reduction rates reached 46.0%, 48.9% and 62.3%, respectively, and the egg reduction rate calculation formulas were: (1-total number of eggs/total number of eggs in PBS group) ×100%. The egg drop rate of the rSjSP-13 recombinant protein immune group is only 4.9 percent. Therefore, the effect of reducing the eggs after the rSjScP15, rSjScP57 and rSjScP92 recombinant proteins are immunized is superior to that of the rSjSP-13 group.

The schistosome is fed with self nutrition by taking peripheral blood of the host after being infected with the schistosome, so that the host is deficient in nutrition and weight is reduced. As shown in fig. 7C, rSjScP15, rSjScP57, rSjScP92 immunized mice had significantly higher body weight than PBS immunized control. The BLANK group is a normal BLANK control mice that were not immunized nor infected with schistosome. The weight of the rSjSP-13 recombinant protein immunized group mice is not significantly different from that of the PBS immunized control group. From this, the rSjScP15, rSjScP57 and rSjScP92 proteins of the present invention had better weight-increasing effect than rSjSP-13.

The number of mature worms in the mice is shown in tables 3 to 4, and the data in table 3 is a summary of experimental data obtained in three independent experiments, 5 mice per experiment, and 15 mice in total in 3 experiments.

TABLE 3 statistics of the number of adults (only) in Japanese blood fluke infected mice

TABLE 4 statistics of liver egg numbers (individuals) of schistosoma japonicum infected mice

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of pathogenic biology of national academy of medical science

<120> Japanese blood fluke antigen protein rSjScP15 and use thereof

<130> KHP221112772.5

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1896

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 1

atgagtaaca acggaatata tgatgatttc aatgattgcc ttaagatagg aatcctacat 60

attccatctg gaaggtttct tacagcggaa gtattcaaca atgaaattaa tatctcggga 120

acagcattac gtcgacgtca agtgtggaca ttgattactg atcgaaagca tccggatact 180

ttgtttttac aaaattcttt gggtcgattt ttatcagtta ataaagatgg aaaaattagt 240

gcatctttga aatcagattc tgaagaacgt tttcgtttag aatttgcgcc agatggtaca 300

ggtgaatggg cttttaaatc tgatacctac ggtttttatc tcagtggttc agacactcaa 360

gtttcgtgtt tttccaaatc tcctgtttgg tggtcaattc gcttagctac tcaccctcaa 420

gttcatattc gtcaccagtt ccgttcacgt tacttacgtc ttcttaatga tggtcttgaa 480

cttcgagccg atcagtcata tccatggggc ccagaatctg ttatttggat cgaacaacct 540

ggaatggttg tgagtcaaag ttttggtcga agtccaaata ctccacttgt tggcaaagct 600

gctgtttcac gtcttggtcg tgttgctttc agaatgccaa acggaaaata tttaaaacct 660

actggagaaa tgtgtgatga aatggatgac tcaacattat tttcttttga atatcgtcct 720

ggtaatcctg gtatattcgc ttttcgtgat caaaaaggtt actatttaac aacaatagga 780

cctggaacaa ctaaagttaa aacaaataca aatattggca aagaagaact atttctcatt 840

gaacatgctg ccttgcaaat tggtatattg gcgcataatg gcaaatttgc atctgttaaa 900

caaggtattg aaatctctgc aaaccagcat gaattagacg aaacagctat cttccaatta 960

gaatatatcg gtgggacggg tttcaatgca ctggaggctg tagcatcttc cactccaaca 1020

tcagtccaac tgtctggtca agatactggt gctgactcat ctacttccaa tttgtgtttg 1080

ataaatagta accattcgat tactaacggc gtccattcaa cagatatcta ctttctaact 1140

acgggatttt ggcgtctacg ttcacgtagt ggaaagttat ggaaacttac tccgtcatct 1200

ggcgtaaaga atactgcttc tgatggagat caagaaagtc tattcgaaat gttaactgtg 1260

aataagatca gtgataaacg aattcgtcaa atcgtattcc gttcaaatag tggtccatcc 1320

tctagtggac agtctttaac agctcgtaaa cttggcgccg tatcaactag tggtcgtcta 1380

attgatgaga gtcaatcacc atcttctgat gagcttttca atattgtttt aactaacaga 1440

acatctattg tctttctttc ttcattgact ggtggcttct tagttcgtgg gaaacagaat 1500

gtacttgatt caaatggtgt agcttatgaa ccattttata ttcatgtaac taaacgacaa 1560

acttataaat tttttgcacg caatcaaaat tcatcctatt ctctatgggc agctggtatg 1620

gatggctccc tacaattgga acctactcac ctaacacctc ttgatgatgg tgacataatc 1680

aatgaaggtg atgataatat caacgatttg aaatatgaat ttcaacttca ttttcttggt 1740

catggtcgtg ttctaattca atcgttaaat agtcaacgtg attcaatctc cttagtcaaa 1800

tctgaaatta aaggcgatgt gaagttggat gtaccaggag gaggaggtag tagtggtgga 1860

tgtgatataa cggcaaatta tatttgggaa atttaa 1896

<210> 2

<211> 369

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 2

atgaaggaca ttcacgctac tagagcatgc aaagaataca ttgatggatt tcagctatta 60

gaaaaatatt gcaactatac ttctgaaagc ataccacaac ttcaatcagt ctctgaattt 120

atgcatcgta catcaggatt tcgtattcga cctgttgctg gtttagttac accaagagat 180

tttttagcga gtctagcatt tagagtattc caaagtactc aatatatacg tcatcattca 240

cgtcctatgc atacaccaga accagattgc atccatgaac tcattggtca tgtgccgatg 300

ttagtaaaca gagaattcgc tgatttttct caagaactgg gtttagcatc tttaggagct 360

tccgaagaa 369

<210> 3

<211> 654

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 3

atgtcacaca tttggaatgc tagatttcca tattctaata atttaggaat ctgttcaaat 60

caaacgataa cgaattcaca tgattgtatc aagactgaac catgttttcc tgaagaaatg 120

gaaaggattc ctatgaaaga aatcaataat atagagtcac acatccgaac tgattattct 180

gtacaaagtt atgagtacaa tgtcccttca acaaacatca cttcatattc agataatgct 240

ttacactatc ccgtgaaaag tggttattcc acttgtcagt tgtcaggttg cacatgttgt 300

tatcagagta gcatgacacc aaactattct tacattccac ctaatactta tttttttgga 360

cacagctatt catctgaata tttaattggc aatatccaac atccttccaa ctatcgtcaa 420

acatatgacc atgattcacc gtatttactg accacaaatc aaagatactc cccagatgaa 480

gctgagatga cagacattga gaataaaaag ggaaaccaga caataaaaga tgaaccattg 540

aataaaaagt atagatgtac ttttccaggc tgtgaaaaaa gatatctgaa gtcaagtcat 600

ttaaaggctc attatcgaat tcataccgat tacaatgaag aaatatcttt ctaa 654

<210> 4

<211> 631

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 4

Met Ser Asn Asn Gly Ile Tyr Asp Asp Phe Asn Asp Cys Leu Lys Ile

1 5 10 15

Gly Ile Leu His Ile Pro Ser Gly Arg Phe Leu Thr Ala Glu Val Phe

20 25 30

Asn Asn Glu Ile Asn Ile Ser Gly Thr Ala Leu Arg Arg Arg Gln Val

35 40 45

Trp Thr Leu Ile Thr Asp Arg Lys His Pro Asp Thr Leu Phe Leu Gln

50 55 60

Asn Ser Leu Gly Arg Phe Leu Ser Val Asn Lys Asp Gly Lys Ile Ser

65 70 75 80

Ala Ser Leu Lys Ser Asp Ser Glu Glu Arg Phe Arg Leu Glu Phe Ala

85 90 95

Pro Asp Gly Thr Gly Glu Trp Ala Phe Lys Ser Asp Thr Tyr Gly Phe

100 105 110

Tyr Leu Ser Gly Ser Asp Thr Gln Val Ser Cys Phe Ser Lys Ser Pro

115 120 125

Val Trp Trp Ser Ile Arg Leu Ala Thr His Pro Gln Val His Ile Arg

130 135 140

His Gln Phe Arg Ser Arg Tyr Leu Arg Leu Leu Asn Asp Gly Leu Glu

145 150 155 160

Leu Arg Ala Asp Gln Ser Tyr Pro Trp Gly Pro Glu Ser Val Ile Trp

165 170 175

Ile Glu Gln Pro Gly Met Val Val Ser Gln Ser Phe Gly Arg Ser Pro

180 185 190

Asn Thr Pro Leu Val Gly Lys Ala Ala Val Ser Arg Leu Gly Arg Val

195 200 205

Ala Phe Arg Met Pro Asn Gly Lys Tyr Leu Lys Pro Thr Gly Glu Met

210 215 220

Cys Asp Glu Met Asp Asp Ser Thr Leu Phe Ser Phe Glu Tyr Arg Pro

225 230 235 240

Gly Asn Pro Gly Ile Phe Ala Phe Arg Asp Gln Lys Gly Tyr Tyr Leu

245 250 255

Thr Thr Ile Gly Pro Gly Thr Thr Lys Val Lys Thr Asn Thr Asn Ile

260 265 270

Gly Lys Glu Glu Leu Phe Leu Ile Glu His Ala Ala Leu Gln Ile Gly

275 280 285

Ile Leu Ala His Asn Gly Lys Phe Ala Ser Val Lys Gln Gly Ile Glu

290 295 300

Ile Ser Ala Asn Gln His Glu Leu Asp Glu Thr Ala Ile Phe Gln Leu

305 310 315 320

Glu Tyr Ile Gly Gly Thr Gly Phe Asn Ala Leu Glu Ala Val Ala Ser

325 330 335

Ser Thr Pro Thr Ser Val Gln Leu Ser Gly Gln Asp Thr Gly Ala Asp

340 345 350

Ser Ser Thr Ser Asn Leu Cys Leu Ile Asn Ser Asn His Ser Ile Thr

355 360 365

Asn Gly Val His Ser Thr Asp Ile Tyr Phe Leu Thr Thr Gly Phe Trp

370 375 380

Arg Leu Arg Ser Arg Ser Gly Lys Leu Trp Lys Leu Thr Pro Ser Ser

385 390 395 400

Gly Val Lys Asn Thr Ala Ser Asp Gly Asp Gln Glu Ser Leu Phe Glu

405 410 415

Met Leu Thr Val Asn Lys Ile Ser Asp Lys Arg Ile Arg Gln Ile Val

420 425 430

Phe Arg Ser Asn Ser Gly Pro Ser Ser Ser Gly Gln Ser Leu Thr Ala

435 440 445

Arg Lys Leu Gly Ala Val Ser Thr Ser Gly Arg Leu Ile Asp Glu Ser

450 455 460

Gln Ser Pro Ser Ser Asp Glu Leu Phe Asn Ile Val Leu Thr Asn Arg

465 470 475 480

Thr Ser Ile Val Phe Leu Ser Ser Leu Thr Gly Gly Phe Leu Val Arg

485 490 495

Gly Lys Gln Asn Val Leu Asp Ser Asn Gly Val Ala Tyr Glu Pro Phe

500 505 510

Tyr Ile His Val Thr Lys Arg Gln Thr Tyr Lys Phe Phe Ala Arg Asn

515 520 525

Gln Asn Ser Ser Tyr Ser Leu Trp Ala Ala Gly Met Asp Gly Ser Leu

530 535 540

Gln Leu Glu Pro Thr His Leu Thr Pro Leu Asp Asp Gly Asp Ile Ile

545 550 555 560

Asn Glu Gly Asp Asp Asn Ile Asn Asp Leu Lys Tyr Glu Phe Gln Leu

565 570 575

His Phe Leu Gly His Gly Arg Val Leu Ile Gln Ser Leu Asn Ser Gln

580 585 590

Arg Asp Ser Ile Ser Leu Val Lys Ser Glu Ile Lys Gly Asp Val Lys

595 600 605

Leu Asp Val Pro Gly Gly Gly Gly Ser Ser Gly Gly Cys Asp Ile Thr

610 615 620

Ala Asn Tyr Ile Trp Glu Ile

625 630

<210> 5

<211> 123

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 5

Met Lys Asp Ile His Ala Thr Arg Ala Cys Lys Glu Tyr Ile Asp Gly

1 5 10 15

Phe Gln Leu Leu Glu Lys Tyr Cys Asn Tyr Thr Ser Glu Ser Ile Pro

20 25 30

Gln Leu Gln Ser Val Ser Glu Phe Met His Arg Thr Ser Gly Phe Arg

35 40 45

Ile Arg Pro Val Ala Gly Leu Val Thr Pro Arg Asp Phe Leu Ala Ser

50 55 60

Leu Ala Phe Arg Val Phe Gln Ser Thr Gln Tyr Ile Arg His His Ser

65 70 75 80

Arg Pro Met His Thr Pro Glu Pro Asp Cys Ile His Glu Leu Ile Gly

85 90 95

His Val Pro Met Leu Val Asn Arg Glu Phe Ala Asp Phe Ser Gln Glu

100 105 110

Leu Gly Leu Ala Ser Leu Gly Ala Ser Glu Glu

115 120

<210> 6

<211> 217

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonicum)

<400> 6

Met Ser His Ile Trp Asn Ala Arg Phe Pro Tyr Ser Asn Asn Leu Gly

1 5 10 15

Ile Cys Ser Asn Gln Thr Ile Thr Asn Ser His Asp Cys Ile Lys Thr

20 25 30

Glu Pro Cys Phe Pro Glu Glu Met Glu Arg Ile Pro Met Lys Glu Ile

35 40 45

Asn Asn Ile Glu Ser His Ile Arg Thr Asp Tyr Ser Val Gln Ser Tyr

50 55 60

Glu Tyr Asn Val Pro Ser Thr Asn Ile Thr Ser Tyr Ser Asp Asn Ala

65 70 75 80

Leu His Tyr Pro Val Lys Ser Gly Tyr Ser Thr Cys Gln Leu Ser Gly

85 90 95

Cys Thr Cys Cys Tyr Gln Ser Ser Met Thr Pro Asn Tyr Ser Tyr Ile

100 105 110

Pro Pro Asn Thr Tyr Phe Phe Gly His Ser Tyr Ser Ser Glu Tyr Leu

115 120 125

Ile Gly Asn Ile Gln His Pro Ser Asn Tyr Arg Gln Thr Tyr Asp His

130 135 140

Asp Ser Pro Tyr Leu Leu Thr Thr Asn Gln Arg Tyr Ser Pro Asp Glu

145 150 155 160

Ala Glu Met Thr Asp Ile Glu Asn Lys Lys Gly Asn Gln Thr Ile Lys

165 170 175

Asp Glu Pro Leu Asn Lys Lys Tyr Arg Cys Thr Phe Pro Gly Cys Glu

180 185 190

Lys Arg Tyr Leu Lys Ser Ser His Leu Lys Ala His Tyr Arg Ile His

195 200 205

Thr Asp Tyr Asn Glu Glu Ile Ser Phe

210 215

<210> 7

<211> 177

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Met Leu Lys Arg Leu Phe Ile Leu Ile Val Ile Leu Gly Val Asn Glu

1 5 10 15

Val Thr Leu Gly Leu Glu Asn Ser Val Ser Pro Leu Lys Gln Pro Asn

20 25 30

Cys Arg Leu Leu Cys Gly Thr Cys Leu Phe Met Gly Arg Met Thr Lys

35 40 45

Val Phe Leu Glu Ser Glu Pro Phe Ile Pro Ile Met Ala Arg Ile Ile

50 55 60

Ser Pro Leu Cys His Leu Ile Pro Asn Glu Glu Cys Lys His Asn Cys

65 70 75 80

Leu Asn Val Thr His Glu Leu Pro Arg Glu Ile Lys Thr Trp Ala Lys

85 90 95

His Met Asn Val Ser His Asp Cys Ser Lys Leu Gly Leu Cys His Lys

100 105 110

Asn His Ser Met Val Ser Ser Phe Glu Phe Thr Ser Phe Leu Lys Glu

115 120 125

His Met Asn Tyr Trp Leu Ser Leu Asp Gln Asn Gly Lys Tyr Lys Asn

130 135 140

Thr Phe Ile Lys Asn Leu Cys Lys His His Ala Ala Asp Thr Asp Lys

145 150 155 160

Cys Ile Glu Thr Leu Glu Thr Ile Val Lys Phe Leu Val Gln Phe Thr

165 170 175

Ile

Claims (8)

1. The Japanese blood fluke antigen protein rSjScP15 is characterized in that the amino acid sequence is shown as SEQ ID NO. 4.

2. The coding gene of the schistosoma japonicum antigen protein rSjScP15 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

3. The antigen protein of claim 1 for any one of the following applications:

1) For preparing a reagent or kit for detecting schistosomiasis japonica;

2) For preparing a vaccine against schistosomiasis japonica;

3) Is used for preparing medicines for treating schistosomiasis japonica.

4. A schistosoma japonica detection reagent, characterized in that the detection reagent comprises: the antigenic protein of claim 1, or a DNA molecule encoding said antigenic protein, or a recombinant protein produced by a recombinant bacterium comprising said DNA molecule.

5. A kit comprising the detection reagent according to claim 4.

6. An ELISA immunodiagnosis kit for schistosomiasis japonica, characterized in that the kit comprises:

1) A microwell reaction plate coated with 1-5 μg/mL of the antigen protein of claim 1; using a carbonate-bicarbonate buffer containing 0.05% v/vTWEEN20 as the coating buffer;

2) Washing buffer: PBST solution, i.e., PBS solution containing 0.05% v/v TWEEN20, pH7.4;

3) Sample dilution: 5% -10% BSA solution, and PBS buffer solution is used as solvent for preparation;

4) Enzyme-labeled antibody: an alkaline phosphatase-labeled goat anti-human immunoglobulin antibody;

5) Substrate color development liquid: pNPP color development liquid;

6) Reaction termination liquid: 120g/L NaOH aqueous solution;

7) Positive control: human immunoglobulin IgG coated plate at 1 μg/mL;

8) Negative control: and replacing the enzyme-labeled antibody with a sample diluent by using a corresponding antigen protein coated plate.

7. An immunogenic composition comprising the antigenic protein of claim 1.

8. Schistosomiasis vaccine, characterized in that it comprises the immunogenic composition of claim 7.

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