CN114805524A - Schistosoma japonicum antigen protein rSjScP92 and application thereof - Google Patents

Abstract

The invention provides a schistosoma japonicum antigen protein rSjScP92 and application thereof. The invention utilizes the schistosoma japonicum whole genome expression profile chip to screen a series of genes highly expressed in schistosoma japonicum schistosomula, wherein the antigen proteins coded by the genes SjScP15, SjScP57 and SjScP92 can be specifically identified by the blood serum of schistosomiasis patients and show stronger positive reaction. ELISA detection shows that the antigen proteins have high sensitivity and specificity in schistosomiasis detection, 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 immunizing mice to attack worms shows that SjScP15, SjScP57 and SjScP92 are expected to be developed into schistosomiasis vaccines.

Description

Schistosoma japonicum antigen protein rSjScP92 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a schistosoma japonicum antigen protein rSjScP92 and application thereof.

Background

Schistosome (Schistosoma), also known as Schistosoma, belongs to the class of the phylum of the flat animal, the family of the Insectia. Schistosomiasis is an endemic parasitic disease caused by the parasitism of schistosoma japonicum in the human body. Of the 6 kinds of blood flukes parasitic on human body, the blood fluke, Schistosoma mansoni and Schistosoma japonicum are distributed most widely and harmfully. Among them, schistosoma japonicum is an epidemic strain of schistosoma japonicum in our country. They are harmful to human health and intensive research on their control is necessary.

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

Compared with the traditional morphological diagnosis method, the enzyme-linked immunosorbent assay (ELISA) diagnosis method has the advantages of simple and rapid operation and higher sensitivity, and can be used for large-scale field monitoring. At present, the most commonly used antigens for schistosomiasis immune diagnosis are Adult insect antigen component (AWA) and egg antigen component (SEA) extracted from schistosome, and because the components of the two crude antigens are complex (consisting of thousands of schistosome proteins) and have serious cross reaction with other parasite infection serum, the specificity of schistosomiasis diagnosis is not high, and the produced reagent is not suitable for standardization. Therefore, there is a need to provide a new detection and diagnosis method to solve the problems of the prior art.

In addition, complete elimination of schistosomiasis, as with other infectious diseases, relies on a strong and effective vaccine, but no schistosomiasis vaccines are currently available. The development of schistosome vaccine requires the development of antigen with protective function, but most of the schistosome proteins are unknown at present. The schistosoma is parasitized in the extracorporeal circulation of the host, and the epidermic protein and secretory protein of the schistosoma and the schistosoma protein in the excrement can induce the humoral immune response of the host. However, when the body of the adult worm develops and matures, the surface membrane of the body of the adult worm is tough and the body of the adult worm has a large volume, so that the antibody generated by the humoral immune response cannot effectively eliminate the adult worm. After the schistosoma japonicum cercaria is drilled into the human body, it can be migrated to lung through skin, and after about 14 days, it can be reached to hepatic portal vein for colonization, and the body of this period is called schistosomulum. The juvenile insect body of 0-14 days is tender and is the best stage of humoral immunity attack, but the antigen released by the juvenile insect body needs 10-14 days to induce humoral immunity reaction, so that the juvenile insect cannot be eliminated at the early stage by the humoral immunity of the host. Therefore, the development of schistosoma japonicum schistosomulum antigens and the development of schistosoma japonicum vaccines aiming at the antigens are expected to help a host to kill the schistosoma japonicum in the schistosomulum stage, and are the key point of schistosoma japonicum vaccine development.

Disclosure of Invention

The invention aims to provide a schistosoma japonicum antigen protein rSjScP92 and application thereof.

The invention has the following conception: screening a series of genes highly expressed in schistosoma japonicum schistosomulum by using a schistosoma japonicum whole genome expression spectrum chip, wherein the schistosomulum is an insect body which contacts with the peripheral circulation of a host earlier and can initiate the humoral immune response of the host earlier to generate a corresponding antibody; and the antigen of the schistosoma japonicum is found to be the best target for developing schistosome vaccines. 3 of the schistosoma japonicum antigen protein genes SjScP15, SjScP57 and SjScP92 are amplified by PCR and recombined and expressed in Escherichia coli, and the obtained recombinant protein has high sensitivity and/or specificity (shown by ELISA test) when used for diagnosing schistosoma japonicum and is a potential diagnostic antigen candidate target. The recombinant protein is used for a schistosoma japonicum infected mouse model test, and the three recombinant proteins are found to have better immune protection and are potential schistosomiasis vaccine candidate antigens.

In order to achieve the object of the present invention, in a first aspect, the present invention provides genes SjScP15, SjScP57 or SjScP92 highly expressed in schistosoma japonicum schistosomulum;

the gene SjScP15 is:

i) 1, SEQ ID NO;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 1 and expresses the same functional protein;

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

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 antigenic protein encoded by the gene SjScP15 (SjScP15) comprises or consists of the amino acid sequence as follows:

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

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

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

The gene SjScP57 is:

i) a nucleotide sequence shown as SEQ ID NO. 2;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 2 and expresses the same functional protein;

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

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 antigenic protein encoded by the gene SjScP57 (rSjScP57) comprises or consists of the amino acid sequence:

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

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

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

The gene SjScP92 is:

i) 3, the nucleotide sequence shown in SEQ ID NO;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 3 and expresses the same functional protein;

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

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 antigenic protein encoded by the gene SjScP92 (rSjScP92) 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 connecting a label at the N end and/or the C end of the i); or

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

Truncated forms of the antigenic proteins encoded by the genes SjScP15, SjScP57, SjScP92, or modified protein derivatives or fusion proteins, and protein variants having the same or similar antigenicity as the antigenic proteins shown in SEQ ID Nos. 4, 5 and 6, are within the scope of the present invention.

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

1) is used for preparing a schistosomiasis japonica detection reagent or a kit;

2) used for preparing schistosomiasis japonica vaccine;

3) used for preparing schistosomiasis japonica medicine;

4) the kit is used for detecting the 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 applied to serodiagnosis as specific schistosome antigens; as immunogen, in preparing schistosome resisting vaccine; as a potential drug action target, in screening chemical and other drug applications; the gene can be used as coding gene of Schistosoma japonicum rSjScP15, rSjScP57 and rSjScP92 protein in gene therapy.

In a third aspect, the invention provides a schistosoma japonicum detection reagent, which contains at least one of the following (i) to (iii):

schistosoma japonicum antigen protein rSjScP15, or a DNA molecule for coding the antigen protein, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule;

schistosoma japonicum antigen protein rSjScP57, or DNA molecule for coding the antigen protein, or recombinant protein produced by recombinant bacteria containing the DNA molecule;

③ schistosoma japonicum antigen protein rSjScP92, or DNA molecule for coding said antigen protein, or recombinant protein produced by recombinant bacteria containing said DNA molecule.

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

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

1) a micropore reaction plate coated with 1-5 mug/mL antigen protein; as coating buffer, a carbonate-bicarbonate buffer (purchased from Sigma under code C3041) containing 0.05% v/v TWEEN20 was used;

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

3) sample diluent: 5-10% BSA solution, using PBS buffer solution as solvent;

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

5) substrate color developing solution: pNPP developing solution;

pNPP was purchased from Sigma under accession number N2640. The preparation method of the pNPP developing solution comprises the following steps: 15mg of pNPP were dissolved in 15mL of 0.1M glycine buffer (0.1M glycine, 1mM MgCl) ₂ ，1mM ZnCl ₂ Dissolving in purified water, and adjusting pH to 10.4).

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

7) positive control: plates were coated with 1. mu.g/mL human immunoglobulin IgG.

Meanwhile, a negative control was set: and (3) coating the plate with corresponding antigen protein, and replacing the enzyme-labeled antibody with sample diluent.

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

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

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

the antigen protein provided by the invention has high detection sensitivity, can still detect the slightly infected schistosome patient (EPG < 100), and has the sensitivity up to 96%.

The detection kit provided by the invention has high specificity, and the diagnosis specificity of schistosomiasis japonica reaches up to 100%.

The kit can be used for early diagnosis of schistosome infection, and the existence of rSjScP15, rSjScP57 and rSjScP92 protein antibodies in serum can be detected after experimental animals are infected for 3 weeks. Simple operation, stable result and high repeatability.

The schistosoma japonica antigenic proteins rSjScP15, rSjScP57 and rSjScP92 are used for immunizing mice, the insect-staining rate and the insect egg load rate can be obviously reduced after the mice are attacked by insects, and the immune protection effect is shown, wherein rSjScP15, rSjScP57 and rSjScP92 are expected to be developed into schistosome vaccines.

Drawings

FIG. 1 is a schematic diagram showing the PCR amplification results of the gene sequences of Schistosoma japonicum SjScP15, SjScP57 and SjScP92 in the preferred embodiment of the present invention. Wherein, M: DNA molecular weight standard; 1,2 and 3 are PCR amplification products of SjScP15(832bp), SjScP57(865bp) and SjScP92(728bp) genes respectively.

FIG. 2 is a diagram showing the results of SDS-PAGE analysis of recombinant proteins rSjScP15, rSjScP57 and rSjScP92 in the preferred embodiment of the present invention. Wherein, M: protein molecular weight standard (kDa), 1: rSjScP15, 2: rSjScP57, 3: rSjScP 92.

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 and blood serum of schistosome and blood serum of different schistosome-infected animals, respectively, in a preferred embodiment of the present invention. Wherein, M: protein molecular weight standard (kDa), 1: schistosoma japonicum serum, 2: serum of mice infected with schistosoma japonicum for 42 days, 3: rabbit serum infected with schistosoma japonicum for 42 days, 4: mouse anti-His tag antibody positive control, 5: a normal mouse seronegative control.

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

FIG. 7 is a diagram showing the results of the immunization of mice with recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57, rSjScP92 and rSjSP-13 in the preferred embodiment of the present invention. Wherein A is a comparison graph of the quantity of the insect bodies in the mouse body 42 days after the schistosoma japonicum is infected, and B is a comparison graph of the quantity of the eggs of the liver of the mouse 42 days after the schistosoma japonicum is infected. C is that the schistosome infects the host and then supplies self nutrition by taking the peripheral blood of the host, which causes the lack of host nutrition and the weight loss. In the figure, the PBS group is a PBS-immunized mouse control group, and the BLANK group is a BLANK control group which is not immunized and is not infected with the Japanese schistosoma. P < 0.01, P <0.05, P < 0.0001.

Detailed Description

The general technical process of the invention is as follows:

firstly, the results of chip screening are verified by a qPCR method, and the expression of the SjScP15, SjScP57 and SjScP92 genes in the juvenile worms is confirmed to be obviously higher than the expression level of the worms 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 design tool (https:// www.ncbi.nlm.nih.gov/tools/Primer-blast /). Cloning was then carried out using the Invitrogen-Gateway Technology with clone II kit.

Adding attB site at one end of a primer according to the specification requirement, then carrying out PCR amplification on Schistosoma japonicum SjScP15, SjScP57 and SjScP92 genes, cloning an amplification product to a pDONR221 vector by using BP clone II enzyme provided in the kit after purifying the amplification product, transferring a gene sequence to an expression vector pDEST17 by using LR clone II enzyme provided in the kit after transformation screening and plasmid sequencing identification confirmation, and transferring the gene sequence to a coliform host cell for recombinant protein induced expression after transformation screening and plasmid sequencing identification confirmation; the inclusion body protein is denatured and purified by nickel column affinity chromatography to finally obtain purified recombinant proteins rSjScP15, rSjScP57 and rSjScP92, and the in vitro cloning expression and purification of SjScP15, SjScP57 and SjScP92 genes are completed. Subsequently, Western blot technology is used to verify that the purified rSjScP15, rSjScP57 and rSjScP92 recombinant proteins can be recognized by experimental animal serum infected with schistosoma japonicum and schistosomiasis japonica blood serum. Furthermore, the preparation of schistosomiasis diagnostic reagents and kits was carried out by using purified rSjScP15, rSjScP57 and rSjScP92 recombinant proteins as diagnostic antigens, and the application value thereof in schistosomiasis immunodiagnosis was evaluated by ELISA technical analysis and the change rules of anti-rSjScP 15, rSjScP57 and rSjScP92 antibodies in the serum of infected animals were analyzed.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 cloning of the Schistosoma japonicum SjScP15, SjScP57, SjScP92 genes

According to the gene sequences of SjScP15, SjScP57 and SjScP92 predicted and found by the inventor on the schistosoma japonicum genome, primers are respectively designed and enzyme cutting sites are introduced. As 190-400 bases at the 3' end of the SjScP57 encoding gene are highly conserved, the amino acid sequence of the encoded protein is highly homologous with tyrosine hydroxylase of human and other mammals, and the antibody induced by the immune epitope of the encoded protein is extremely easy to cause nonspecific reaction, on the one hand, the antibody nonspecific reaction can be caused, thereby influencing the specificity of diagnosis; on the other hand, when the protein segment is used for immunizing a host, the induced antibody can nonspecifically influence the tyrosinase activity of the host, and is not beneficial to the research and development of vaccines. Therefore, the non-conserved segment (607-957 bases) of the SjScP57 encoding gene is selected to prepare the recombinant protein for diagnosis and vaccine research in the invention. The SjScP15 and SjScP92 gene encoding proteins have no homologous proteins found in common mammalian hosts, so that primers are designed by using a full-length gene sequence as a template. The primer sequences are 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 section in PF is the attB1 site, designed according to the Invitrogen Gateway Technology with close II kit instructions, for ligation to shuttle vector pDONR 221; the underlined section in PR is attB2 site, designed according to the Invitrogen Gateway Technology with close II kit instructions, for ligation to shuttle vector pDONR 221; specific primers were synthesized by Suzhou Jinzhi Biotechnology, Inc.

Taking 14 days Schistosoma japonicum (Schistosoma japonicum) schistosomula japonica cDNA as a template, carrying out PCR reaction, and amplifying a gene ORF fragment, wherein the reaction system is as follows: 12.5 mul of high fidelity DNA polymerase Mix, 1 mul of cDNA template, 0.5 mul of upstream primer, 0.5 mul of downstream primer, ddH ₂ O10.5. mu.l, total volume 25. mu.l. PCR amplification procedure: 5min at 95 ℃, 30sec at 55 ℃ for annealing, 1min at 72 ℃ for extension, for 35 cycles; finally, extension is carried out for 10min at 72 ℃.

The PCR product was detected by 1.2% agarose gel electrophoresis, and the presence of the desired band was observed, as shown in FIG. 1, M: DNA molecular weight standard, 1: SjScP15, 2: SjScP57, 3: SjScP 92. Agarose gel electrophoresis results show that SjScP15, SjScP57 and SjScP92 respectively have a clear band at 1896bp,354bp and 636bp, and the clear bands are consistent with the sizes of expected target fragments, thereby 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).

The Invitrogen Gateway Technology with clone II kit constructs a shuttle vector in the following reaction system: recovery of DNA product by PCR (dissolved in ddH) ₂ O)150ng, pDONR221 vector (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 is carried out for 1h at 25 ℃. The above-mentioned ligation products were transformed into E.coli DH 5. alpha. competent cells (Beijing Panjin Biotechnology Co., Ltd.), and the transformed competent cells were plated on LB medium plate (containing 50. mu.g/ml kanamycin) and cultured overnight at 37 ℃. Single colonies were picked for PCR identification, and clones identified as positive by PCR were further subjected to the construction of an expression vector using the Invitrogen Gateway Technology with clone II kit. The reaction system is as follows: positive shuttle vector for PCR identification (soluble in ddH) ₂ O)150ng, pDEST17 vector (in ddH) ₂ O)150ng, TE Buffer (pH8.0) was added to a total volume of 8. mu.l, and then 2. mu.l of LRclonase II enzyme was added. After mixing, incubation is carried out for 1h at 25 ℃. Then, 1. mu.l of Protein K (2. mu.g/. mu.l) was added thereto, and the mixture was incubated at 37 ℃ for 10min to remove the Protein. The above-mentioned ligation products were transformed into E.coli B21DE3 competent cells (Beijing Panjin Biotechnology Co., Ltd.), and the transformed competent cells were plated on LB medium plates (containing 50. mu.g/ml ampicillin) and cultured overnight at 37 ℃. Selecting a single colony for PCR identification, and sending the clone identified as positive by PCR to Suzhou Jinzhi Biotechnology limited for DNA sequencing to confirm whether the sequence is correct. The sequencing analysis result shows 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 respectively shown as SEQ ID NO. 1-3, and the amino acid sequences of the antigen proteins coded by the genes are respectively shown as SEQ ID NO. 4-6.

Example 2 expression and purification of recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57, and rSjScP92

Clones identified as positive by the above PCR were inoculated into LB liquid medium (containing 50. mu.g/m)Ampicillin) 15mL, 37 ℃,200 rpm overnight, next day 10mL of the medium was inoculated into 1L of LB medium (containing 50. mu.g/mL ampicillin), and the culture was continued until OD _600nm The value was 0.8, and IPTG was added thereto at a final concentration of 1mM for induction, and the expression was carried out at 18 ℃ and 140rpm for 16 hours, and the cells were collected by centrifugation and frozen at-80 ℃ for further use.

Respectively taking a small amount of thalli before and after induction, re-suspending the thalli in PBS buffer solution, adding SDS-PAGE sample buffer solution, uniformly mixing, and boiling in a boiling water bath for 5min to denature proteins.

Mu.l of each pre-and post-induction sample was added to each well and analyzed by SDS-PAGE (5% for concentrated gel and 12% for gel separation).

The recombinant plasmids pDEST17-rSjScP15, pDEST17-rSjScP57 and pDEST17-rSjScP92 were transformed to express competent cells, and after IPTG induction expression, a clear expression band appeared in the cells before induction.

The induced thallus is resuspended in 40mL of bacterial lysate, and is subjected to ultrasonic disruption, centrifugation at 4 ℃ and 12,000rpm for 30min, and the inclusion body precipitate and the supernatant are collected respectively.

The inclusion body pellet and supernatant were subjected to SDS-PAGE analysis to identify the solubility of the recombinant protein. The results show that the recombinant protein is mainly present in the inclusion body pellet.

Resuspending the inclusion bodies in PBS solution containing 1% Triton-X100, ultrasonically washing for 5min, centrifuging at 12,000rpm for 15min, and collecting the inclusion body precipitate;

resuspending the inclusion bodies in 8M urea, rotating and mixing the inclusion bodies evenly at 4 ℃ overnight, fully dissolving the inclusion bodies, centrifuging the mixture at 12,000rpm for 30min, and collecting supernatant;

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

the purity of the purified recombinant protein is detected by SDS-PAGE analysis. The electrophoresis result is shown in figure 2, the molecular weights of the rSjScP15, rSjScP57 and rSjScP92 proteins are respectively about 15kDa, 14kDa and 35kDa, and the molecular weights are consistent with the theoretical relative molecular weight of the target recombinant protein, which indicates that the recombinant protein with high purity is obtained after being purified by a nickel ion chelating gel column.

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

Example 3 detection of antigenicity of recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57 and rSjScP92

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

Film transfer: transferring the protein in the PAGE gel to a PVDF membrane by adopting a wet transfer method, wherein the electric transfer conditions are as follows: ice-bath, 100V 1 h.

And (3) sealing: PVDF membrane with 5% skimmed milk powder room temperature closed for 2h, TBST buffer washing 3 times.

Adding primary antibody for incubation: adding 42 days BALB/c mouse serum infected with schistosoma japonicum, 42 days New Zealand white rabbit serum infected with schistosoma japonicum and schistosoma japonicum serum, respectively, using mouse anti-His-tag antibody (Abiramate biomedicine Co., Ltd.) as positive control, using healthy mouse serum as negative control (diluted with confining liquid 1: 500), incubating overnight at 4 deg.C, and washing with TBST buffer solution 3 times.

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

Sweeping the membrane: scanning and imaging by using an Odyssey infrared laser imaging system.

The results are shown in fig. 3 to 5, fig. 3 shows the results of rSjScP15, fig. 4 shows the results of rSjScP57, and fig. 5 shows the results of rSjScP92, where M: protein molecular weight standard (kDa), 1: schistosoma japonicum serum, 2: serum of mice infected with schistosoma japonicum for 42 days, 3: rabbit serum infected with schistosoma japonicum for 42 days, 4: mouse anti-His tag antibody positive control, 5: a normal mouse seronegative control. Mouse anti-His-tag antibody is used as a positive control, and rSjScP15, rSjScP57 and rSjScP92 respectively have obvious recognition bands at 15kDa, 14kDa and 35kDa, and healthy mouse serum is used as a negative control, and no obvious band exists. Wherein the rSjScP15 and rSjScP57 proteins can be recognized by BALB/c mouse serum infected with schistosoma japonicum, New Zealand rabbit serum and schistosoma japonicum serum, and the rSjScP92 can be recognized by human serum, and also has recognition bands in mouse serum. The recombinant proteins rSjScP15, rSjScP57 and rSjScP92 are proved to have good antigenicity.

Example 4 preparation of immunodiagnosis kits for Japanese schistosomiasis rSjScP15, rSjScP57, and rSjScP92

1. The main components of the kit comprise:

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

Washing buffer solution: PBST solution, i.e., PBS solution containing 0.05% v/v TWEEN20, pH 7.4.

Sample diluent: 10% BSA solution was prepared using PBS buffer as a solvent.

Enzyme-labeled antibody: alkaline phosphatase-labeled goat anti-human immunoglobulin antibody (. alpha.,. gamma. and. mu. -chain specific) antibody (Sigma Co.).

Substrate color developing solution: pNPP developing solution.

pNPP was purchased from Sigma under accession number N2640. The preparation method of the pNPP developing solution comprises the following steps: 15mg of pNPP were dissolved in 15mL of 0.1M glycine buffer (0.1M glycine, 1mM MgCl) ₂ ，1mM ZnCl ₂ Dissolving in purified water, and adjusting pH to 10.4).

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

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

Negative control: and (3) coating the plate with corresponding antigen protein, and replacing the enzyme-labeled antibody with sample diluent.

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

and (3) sealing: spin-drying the liquid in the wells, adding 200. mu.L/well of diluent, sealing at 37 ℃ for 2h, washing with PBST three times, 200. mu.L/well for 2 min/time, and finally patting dry.

Adding a sample to be tested: diluting the serum and the diluent of a sample to be detected according to a ratio of 1:100, simultaneously setting positive, negative and blank controls (only adding the diluent), adding each sample according to 100 mu L/hole, detecting 3 multiple holes of each sample in parallel, and incubating for 1h at 37 ℃.

Washing: spin-dry the well, wash with PBST buffer four times, 200. mu.L/well, 2 min/time, and dry with patting.

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

Color development: adding enzyme substrate developing solution, incubating at 37 deg.C in dark for 30min, adding stop solution, and incubating at 25 μ L/well.

Data reading and processing: OD reading with microplate reader _405nm The value of the negative control sample is 2.1 times the OD mean value, and is used as a cut-off value for determining the negative and positive.

Example 5 rSjScP15, rSjScP57, rSjScP 92-evaluation of immunodiagnosis of schistosomiasis japonica by ELISA kit

1. Sensitivity of the kit

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

As shown in Table 1 and FIG. 6, Sj is a Japanese blood fluke patient group, and health is a Healthy control group (35 parts of human serum sample from Heilongjiang province). The horizontal straight line running through the whole coordinate system in the figure is cut off value 2.1 times of the Healthy group mean value, the line is regarded as positive, and the line is regarded as negative. 20 parts of blood serum of schistosoma japonicum patients, 18 parts of blood serum is diagnosed to be positive by the rSjSP-13-ELISA kit (figure 6A), the sensitivity of the rSjSP-13-ELISA kit for diagnosing schistosoma japonicum is 90.0% (95% confidence interval, 66.9% -98.3%), and the result is close to 90.4% (95% confidence interval, 75.0% -95.0%) of the sensitivity of the rSjSP-13-ELISA kit reported by the literature; 20 of the cells were diagnosed as positive by the rSjScP15-ELISA and rSjScP57-ELISA kits (FIG. 6B and FIG. 6C, 95% confidence intervals, 80.0% -100%). 19 were diagnosed as positive by the rSjScP92-ELISA kit (FIG. 6D, 95% confidence interval, 73.1% -99.7%). The sensitivity of the rSjScP15, the rSjScP57 and the rSjScP92-ELISA kit is obviously 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 rSjSP-13-ELISA with rSjScP15, rSjScP57, rSjScP92-ELISA kit sensitivity

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

2. Specificity of the kit

The collected 20 parts of human serum samples of Healthy's Heilongjiang province and 18 parts of human serum of clonorchis sinensis disease patients in Guangdong province were used to evaluate the false positive rate of each kit in normal population and the cross reaction with other parasite patients' serum, and compared with rSjSP-13-ELISA kit, the ELISA experiment was performed as in example 3.

As shown in Table 2 and FIG. 6, Cs is a group of Clonorchis sinensis patients. 20 normal human sera were all diagnosed as negative by the rSjSP-13-ELISA, rSjScP57-ELISA, rSjScP15-ELISA and rSjScP92-ELISA kits, all four kits having a specificity of 100% (95% confidence interval, 80.0% -100%). 15 parts of clonorchis sinensis human serum is diagnosed as negative by the four kits, and no cross reaction occurs.

TABLE 2 comparative analysis of the specificity of rSjSP-13-ELISA with SjScP15, SjScP57, SjScP92-ELISA kits

3. Value of kit in evaluating schistosomiasis japonica progression status

The 20 cases of schistosoma japonicum patients were treated with chemotherapy for 3 months, and then serum was collected again to evaluate the value of the kit in evaluating the progress of schistosoma japonicum, and compared with the rSjSP-13-ELISA kit, the ELISA procedure was the same as in example 3. The results are shown in FIG. 6, where Sj-3M was a three-month group of chemotherapy for schistosoma japonicum patients. The OD value of the patients treated by the SjScP15-ELISA kit and the rSjSP-13-ELISA kit is obviously reduced compared with the current infection stage, and the OD value of the patients treated by the SjScP15-ELISA kit and the SjScP92-ELISA kit is not obviously changed compared with the current infection stage.

Example 6 evaluation of the protective Effect of recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57, and rSjScP92 as antigens in mice immunized with

1. Preparation of antigen-immunized mouse model

a) Immunization

Immunizing Balb/c mice; negative serum was taken from the orbit prior to immunization.

60ug of immunogen (60 ug of prime boost 30ug) was taken, diluted to 200ul with physiological saline, and added with equal volume of Freund's adjuvant (Freund's complete adjuvant for prime boost, Freund's incomplete adjuvant for boost); the solution and adjuvant are mixed by a mixing instrument to form water-in-oil.

And (3) performing subcutaneous injection immunization on the back and the abdomen of the uniformly mixed immunogen, and beating 8-10 points.

b) Negative blood from orbit

One hand holds the mouse firmly and makes the eyeball pop out; the other hand holds a capillary tube and inserts the capillary tube into the back angle of the mouse eye, and inclines for 45 degrees and slowly rotates in from the back angle of the mouse eye. The force and posture of the hand are slowly adjusted to make the blood flow into the capillary, and after the capillary is almost filled, the capillary is immediately placed into a 1.5ml centrifuge tube, and 20ul of negative blood is taken.

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

c) ELISA assay potency

Preparing a reagent:

coating liquid: sodium carbonate-sodium bicarbonate buffer, ph 9.6.

PBS buffer, pH 7.4.

Sealing liquid: 2% BSA or PBS with skim milk powder.

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

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

Stopping liquid: 2M sulfuric acid.

Secondary antibody: goat anti-mouse IgG/HRP.

The experimental steps are as follows:

1) diluting antigen with coating solution to final concentration of 2ug/ml, 100 ul/well, 4 deg.C overnight; then washed 2 times with wash solution.

2) Sealing with sealing liquid, 200 ul/hole, incubating at 37 ℃ for 2 h; then washed 1 time with wash solution.

3) The multiple antisera were diluted in a 2-fold gradient starting from 200 fold (in PBS), with a blank control (blank) in PBS and a negative control (negative) in 200-fold dilution of negative serum (in PBS); 100 ul/hole, 37 ℃ incubator for 1 h; then washed 3 times with wash solution.

4) Adding secondary antibody which is 20000 times diluted by PBS (phosphate buffer solution), 100 ul/hole, incubating at 37 ℃ for 1 h; after removal, the plate was washed 3 times with a washing solution.

5) Developing with 100 ul/hole developing solution for 5-15 min.

6) Stop with 50ul of stop solution per well.

7) Absorbance values were measured at both wavelengths (450, 630), and the stored data was recorded and plotted for analysis.

The titer is 1/2 dilution factor corresponding to the maximum OD value.

2. Evaluation of immunoprotection

a) Attack worm

The positive oncomelania are raised for one week at room temperature and then collected in a conical flask, and the cercariae in the body of the oncomelania are released after 2-4 hours of warm light irradiation.

The abdominal hair of the mice was removed and the abdominal skin was exposed.

The ring was used to pick up 40 cercaria/mouse on a coverslip under the microscope.

The cover glass was applied to the abdominal wall skin of mice for 5min to facilitate the penetration of cercaria into the skin.

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

b) Evaluation of antigen immunoprotection

After 42 days of feeding, each mouse was weighed (5 experiments per group, 3 replicates).

After anesthetizing the mice, the hepatic portal vein was cut, PBS was perfused by cardiac puncture, and the worms were flushed out of the body and counted.

Picking liver, cutting, grinding with 200 mesh copper net, adding 20ml of 5% KOH solution into 1g liver tissue, mixing, incubating at 37 deg.C overnight to digest tissue, and counting the number of eggs in liver tissue by microscopic examination.

rSjSP-13 recombinant protein immunization groups and BALNK protein-free empty control immunization groups were used as controls. The results are shown in tables 3 to 4 and FIG. 7A.

Specifically, as shown in table 3 and fig. 7A, the pest reduction rate of the mice in the rSjScP15, rSjScP57 and rSjScP92 immune group is significantly increased to 38.1%, 34.8% and 33.4% respectively compared with that in the PBS immune control group, and the pest reduction rate is calculated according to the following formula: (1-total mature polypide/total mature polypide of blank control) x 100%. In this example, the number of infected cercaria was 40 cercaria/mouse, and the total number of mature larvae was based on the PBS control group, because the active and sex ratio of cercaria and the influence of host immune system were generally less than 40. And the reduction rate of the rSjSP-13 recombinant protein immune group is only 3.8 percent respectively. Therefore, the recombinant proteins rSjScP15, rSjScP57 and rSjScP92 of the invention have better insect-reducing effect after immunization than the rSjSP-13 group.

The biggest harm of schistosomiasis is that the worm body lays eggs after being mature, and the deposition of worm eggs in the liver causes liver fibrosis so as to trigger a series of pathological changes. As shown in table 4 and fig. 7B, the number of eggs of liver worms of mice immunized with rSjScP15, rSjScP57, and rSjScP92 was significantly reduced compared to the PBS-immunized control group, and the egg reduction rates reached 46.0%, 48.9%, and 62.3%, respectively, and the calculation formula of the egg reduction rate was: (1-total number of eggs/total number of eggs in PBS group) × 100%. And the egg reduction rate of the rSjSP-13 recombinant protein immune group is only 4.9 percent. Therefore, the effect of reducing the insect eggs after the rSjScP15, the rSjScP57 and the rSjScP92 recombinant proteins are immunized is better than that of the rSjSP-13 group.

After the schistosome infects the host, the schistosome can supply self nutrition by taking the peripheral blood of the host, thus causing the lack of the host nutrition and the reduction of the body weight. As shown in fig. 7C, the weight of the mice immunized with rSjScP15, rSjScP57, and rSjScP92 was significantly increased compared to the PBS-immunized control group. The BLANK group was normal control mice that were not immunized nor infected with schistosoma. The body weight of mice in the rSjSP-13 recombinant protein immune group has no significant statistical difference compared with that of a PBS immune control group. Therefore, the rSjScP15, rSjScP57 and rSjScP92 proteins of the invention have better body weight-improving effect than the rSjSP-13 group after being immunized.

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

TABLE 3 statistical table of the number of adults (only) in mice infected with schistosoma japonicum

TABLE 4 statistical table of the number (number) of eggs of mouse liver infected with schistosoma japonicum

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of pathogenic biology of Chinese academy of medical sciences

<120> schistosoma japonicum antigen protein rSjScP92 and application thereof

<130> KHP221112776.9

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<170> SIPOSequenceListing 1.0

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acagcattac gtcgacgtca agtgtggaca ttgattactg atcgaaagca tccggatact 180

ttgtttttac aaaattcttt gggtcgattt ttatcagtta ataaagatgg aaaaattagt 240

gcatctttga aatcagattc tgaagaacgt tttcgtttag aatttgcgcc agatggtaca 300

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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

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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

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<210> 4

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<213> Schistosoma japonicum (Schistosoma japonica)

<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

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

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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

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

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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 japonica)

<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 japonica)

<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. Schistosoma japonicum antigen protein rSjScP92, comprising or consisting of the amino acid sequence:

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

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

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

2. The gene SjScP92 highly expressed in schistosoma japonicum is characterized in that:

i) 3, the nucleotide sequence shown in SEQ ID NO;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 3 and expresses the same functional protein;

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

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.

3. The antigenic protein of claim 1 for use as a medicament for any one of:

1) is used for preparing a schistosomiasis japonica detection reagent or a kit;

2) used for preparing schistosomiasis japonica vaccine;

3) is used for preparing the schistosomiasis japonica medicine.

4. The schistosoma japonicum detection reagent is characterized by comprising the following components in parts by weight: 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. The schistosomiasis japonica ELISA immunodiagnosis kit is characterized by comprising:

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

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

3) sample diluent: 5-10% BSA solution, using PBS buffer solution as solvent;

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

5) substrate color developing solution: pNPP developing solution;

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

7) positive control: coating with 1. mu.g/mL human immunoglobulin IgG;

8) negative control: and (3) coating the plate with corresponding antigen protein, and replacing the enzyme-labeled antibody with sample diluent.

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

8. Schistosomiasis vaccine, characterized in that it comprises an immunogenic composition according to claim 7.

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