CN115043922B - Japanese blood fluke antigen protein rSjScP57 and application thereof - Google Patents

Abstract

The invention provides Schistosoma japonicum antigen protein rSjScP57 and its application. The present invention uses the Schistosoma japonicum whole genome expression profile chip to screen out a series of genes that are highly expressed in Schistosoma japonicum. Among them, the antigen proteins encoded by genes SjScP15, SjScP57 and SjScP92 can be specifically recognized by the serum of schistosomiasis patients and show strong positive reaction. ELISA testing shows that these antigenic proteins have high sensitivity and specificity for detecting schistosomiasis and can be used in the development of diagnostic reagents for schistosomiasis japonicum. The immune protective effects of mice immunized with Schistosoma japonicum antigen proteins SjScP15, SjScP57 and SjScP92 after challenge showed that SjScP15, SjScP57 and SjScP92 are expected to be developed into schistosomiasis vaccines.

Description

Schistosoma japonicum antigenic protein rSjScP57 and its application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to the Schistosoma japonicum antigen protein rSjScP57 and its application.

Background technique

Schistosoma, also known as Schistosoma, belongs to the class Flukes of the phylum Platyhelminthes. Schistosomiasis is an endemic parasitic disease caused by schistosomiasis parasitizing humans. Among the six species of schistosomiasis that parasitize the human body, Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum are the most widely distributed and the most serious. Among them, Schistosoma japonicum is the prevalent strain of schistosomiasis in my country. They are harmful to human health, and it is necessary to conduct in-depth research on their prevention and control.

Diagnosis is a central link in the field of schistosomiasis control. Accurate diagnostic technology not only has important clinical significance for the early detection and early treatment of schistosomiasis patients, but can also provide criteria for judging the level of schistosomiasis endemic areas, and provide essential information and scientific basis for assessing epidemic trends and assessing prevention and control effects. The lack of efficient and accurate diagnostic technology is an important reason why schistosomiasis cannot be completely eliminated. If schistosomiasis patients cannot be diagnosed and treated in time, the eggs contained in their excrement will cause the continued epidemic spread of schistosomiasis. Therefore, it is imperative to develop new schistosomiasis diagnostic methods with high sensitivity and specificity. At present, the diagnosis of schistosomiasis mostly relies on parasite morphological detection methods, such as the modified Kato-Katz method recommended by the World Health Organization (WHO). This method mainly diagnoses the disease by examining the schistosomiasis eggs in the patient's feces or urine. It is not only time-consuming and labor-intensive, but also has low sensitivity for diagnosing mild schistosomiasis infections and is not suitable for large-scale on-site monitoring of schistosomiasis.

Compared with traditional morphological diagnostic methods, the enzyme-linked immunosorbent assay (ELISA) diagnostic method is simple, fast and highly sensitive, and can be used for large-scale on-site monitoring. Currently, the most commonly used antigens used for immunodiagnosis of schistosomiasis are the adult worm antigen (AWA) and the egg antigen (SEA) extracted from the schistosomiasis. The composition of the antigen is complex (composed of thousands of schistosomiasis proteins), and there are serious cross-reactions with other parasite-infected sera. The specificity of schistosomiasis diagnosis is not high, and the produced reagents should not be standardized. Therefore, it is necessary to provide a new detection and diagnosis method to solve the problems of the existing technology.

In addition, like other infectious diseases, the complete elimination of schistosomiasis depends on a strong and effective vaccine, but no schistosomiasis vaccine is currently available. The development of schistosomiasis vaccines requires the development of antigens with protective functions, and currently most of the schistosomiasis proteins are unknown. Schistosoma parasitizes in the host's extracorporeal circulation, and its surface membrane proteins and secretory proteins, as well as schistosome proteins in excreta, can induce the host's humoral immune response. However, when the worms mature, the surface membrane of the worms becomes tougher and the worms become larger, so the antibodies produced by the humoral immune response cannot effectively eliminate the adult worms. Schistosoma cercariae penetrate into the human body and migrate to the lungs through the skin. After about 14 days, they reach the hepatic portal vein and settle. The worms at this stage are called Schistosoma larvae. Child worms aged 0-14 days are relatively delicate and are the best stage for humoral immune attack. However, since the antigens they release require 10-14 days to induce a humoral immune response, the host's humoral immunity cannot clear the child worms at the early stage. Therefore, developing schistosomiasis childhood antigens and developing schistosomiasis vaccines based on these antigens is expected to help the host eliminate the parasites in the childhood stage, which is the key to the development of schistosomiasis vaccines.

Contents of the invention

The object of the present invention is to provide Schistosoma japonicum antigen protein rSjScP57 and its application.

The concept of the present invention is as follows: a series of highly expressed genes in Schistosoma japonicum are screened using the Schistosoma japonicum whole-genome expression profile chip. The Schistosoma japonicum is an insect that contacts the host's peripheral circulation earlier and can trigger the host's humoral immune response earlier. Produce corresponding antibodies; and discover the antigen of childworm is the best target for the development of schistosomiasis vaccine. Three of the Schistosoma japonicum antigen protein genes, SjScP15, SjScP57, and SjScP92, were amplified by PCR to amplify the hydrophilic segment of the gene and recombinantly expressed it in Escherichia coli. The resulting recombinant protein was used for the diagnosis of Schistosoma japonicum with high sensitivity and / or specific (as shown by ELISA test), it is a potential diagnostic antigen candidate target. The recombinant proteins were used in a mouse model test of Schistosoma japonicum infection, and it was found that these three recombinant proteins have good immune protection and are potential candidate antigens for schistosomiasis vaccines.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides genes SjScP15, SjScP57 or SjScP92 that are highly expressed in Schistosoma japonicum;

The gene SjScP15 is:

i) The nucleotide sequence shown in SEQ ID NO:1;

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

iii) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO:1 under stringent conditions and expresses the same functional protein in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS solution, hybridize at 65°C, and wash the membrane with this solution; or

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

The antigenic protein (SjScP15) encoded by the gene SjScP15 contains or consists of the following amino acid sequence:

i) The amino acid sequence shown in SEQ ID NO:4;

ii) The amino acid sequence obtained by attaching a tag to the N-terminus and/or C-terminus of i); or

iii) A protein 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) The nucleotide sequence shown in SEQ ID NO:2;

ii) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 2 is substituted, deleted and/or added by one or more nucleotides and expresses the same functional protein;

iii) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO:2 under stringent conditions and expresses the same functional protein in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS solution, hybridize at 65°C, and wash the membrane with this solution; or

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

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

i) The amino acid sequence shown in SEQ ID NO:5;

ii) The amino acid sequence obtained by attaching a tag to the N-terminus and/or C-terminus of i); or

iii) A protein 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 SjScP92 is:

i) The nucleotide sequence shown in SEQ ID NO:3;

ii) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO:3 is substituted, deleted and/or added by one or more nucleotides and expresses the same functional protein;

iii) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO:3 under stringent conditions and expresses the same functional protein in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS solution, hybridize at 65°C, and wash the membrane with this solution; or

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

The antigenic protein (rSjScP92) encoded by the gene SjScP92 contains or consists of the following amino acid sequence:

i) The amino acid sequence shown in SEQ ID NO: 6;

ii) The amino acid sequence obtained by attaching a tag to the N-terminus and/or C-terminus of i); or

iii) A protein 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 or modified protein derivatives or fusion proteins of the antigenic proteins encoded by genes SjScP15, SjScP57, and SjScP92, and have the same or similar antigenicity as the antigenic proteins shown in SEQ ID NO: 4, 5, and 6. All entities belong to the protection scope of the present invention.

In a second aspect, the present invention provides any of the following applications of the antigenic protein:

1) Used to prepare schistosomiasis japonicum detection reagents or kits;

2) Used to prepare schistosomiasis japonicum vaccine;

3) Used to prepare schistosomiasis japonicum drugs;

4) For the detection of Schistosoma japonicum infection;

5) Used to diagnose schistosomiasis japonicum;

6) For the prevention and treatment of schistosomiasis japonicum.

Specifically, the Schistosoma japonicum antigen proteins rSjScP15, rSjScP57, and rSjScP92 of the present invention are used as specific Schistosoma antigens in serological diagnosis; as immunogens, they are used in the preparation of anti-schistosomiasis vaccines; as potential drug targets, Application in screening chemicals and other types of drugs; application in gene therapy as the coding gene for Schistosoma japonicum rSjScP15, rSjScP57, rSjScP92 proteins.

In a third aspect, the present invention provides a Schistosoma japonicum detection reagent, which contains at least one of the following ① to ③:

① Schistosoma japonicum antigen protein rSjScP15, or the DNA molecule encoding the antigen protein, or the recombinant protein produced by the recombinant bacteria containing the DNA molecule;

② Schistosoma japonicum antigen protein rSjScP57, or the DNA molecule encoding the antigen protein, or the recombinant protein produced by the recombinant bacteria containing the DNA molecule;

③ Schistosoma japonicum antigen protein rSjScP92, or the DNA molecule encoding the antigen protein, or the recombinant protein produced by the recombinant bacteria containing the DNA molecule.

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

In a fifth aspect, the present invention provides a schistosomiasis japonicum ELISA immunodiagnostic kit, which kit includes:

1) A microwell reaction plate coated with 1-5 μg/mL antigen protein; use carbonate-bicarbonate buffer (purchased from Sigma, Cat. No. C3041) containing 0.05% v/v TWEEN20 as the coating buffer;

2) Washing buffer: PBST solution, that is, PBS solution containing 0.05% v/v TWEEN20, pH 7.4;

3) Sample diluent: 5-10% BSA solution, prepared with PBS buffer as solvent;

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

5) Substrate chromogenic solution: pNPP chromogenic solution;

pNPP was purchased from Sigma, catalog number N2640. The preparation method of pNPP chromogenic solution is as follows: Dissolve 15mg pNPP in 15mL 0.1M glycine buffer (0.1M glycine, 1mM MgCl ₂ , 1mM ZnCl ₂ , dissolved in purified water, pH 10.4).

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

7) Positive control: plate with 1 μg/mL human immunoglobulin IgG.

At the same time, set up a negative control: coat the plate with the corresponding antigen protein, and replace the enzyme-labeled antibody with the sample diluent.

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

In a seventh aspect, the present invention provides a schistosomiasis vaccine comprising the immunogenic composition. Optionally, adjuvants are included in the vaccine.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(1) The antigen protein detection provided by the present invention has high sensitivity and can still detect mildly infected schistosomiasis patients (EPG <100), with a sensitivity of 96%.

(2) The detection kit provided by the present invention has high specificity, and the diagnostic specificity of schistosomiasis japonicum is as high as 100%.

(3) This kit can be used for the early diagnosis of schistosomiasis infection. The presence of anti-rSjScP15, rSjScP57, and rSjScP92 protein antibodies in the serum of experimental animals can be detected 3 weeks after infection. The operation is simple, the results are stable and the repeatability is high.

(4) Schistosoma japonicum antigenic proteins rSjScP15, rSjScP57, and rSjScP92 immunize mice and can significantly reduce the infection rate and egg load rate after attack. The immune protection effect shows that rSjScP15, rSjScP57, and rSjScP92 are expected to be developed into schistosomiasis vaccines.

Description of the drawings

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

Figure 2 is a schematic diagram of the SDS-PAGE analysis results of rSjScP15, rSjScP57, and rSjScP92 recombinant proteins in the preferred embodiment of the present invention. Among them, M: protein molecular weight standard (kDa), 1: rSjScP15, 2: rSjScP57, 3: rSjScP92.

Figures 3 to 5 respectively show the Western blot analysis results of rSjScP15 (Figure 3), rSjScP57 (Figure 4), rSjScP92 (Figure 5), recombinant protein antigens and sera of schistosomiasis patients and sera of different schistosome-infected animals in the preferred embodiment of the present invention. Schematic diagram. Among them, M: protein molecular weight standard (kDa), 1: Schistosoma japonicum patient serum, 2: mouse serum 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: Normal mouse serum negative control.

Figure 6 is a schematic diagram of the results comparing the rSjSP-13-ELISA kit and the rSjScP15-ELISA, rSjScP57-ELISA and rSjScP92-ELISA kits for the diagnostic evaluation of schistosomiasis japonicum in the preferred embodiment of the present invention. Among them, A is the result of rSjScP57-ELISA kit, B is the result of rSjSP-13-ELISA kit, C is the result of SjScP15-ELISA kit, and D is the result of SjScP92-ELISA kit. Sj is the Schistosoma japonicum patient group, Sj-3M is the Schistosoma japonicum patient group receiving three-month chemotherapy, Cs is the Clonorchis sinensis patient group, and Healthy is the healthy control group. The horizontal line in the figure is the cutoff value of 2.1 times the mean value of the Healthy group. The line above is regarded as positive, and the line below it is regarded as negative.

Figure 7 is a schematic diagram of the immune effect of mice immunized with Schistosoma japonicum rSjScP15, rSjScP57, rSjScP92 and rSjSP-13 recombinant proteins in the preferred embodiment of the present invention. Among them, A is a comparison chart of the number of worms in mice after 42 days of Schistosoma japonicum infection, and B is a comparison chart of the number of eggs in the liver of mice after 42 days of Schistosoma japonicum infection. C means that after schistosomiasis infects a host, it relies on ingesting the host's peripheral blood to supply its own nutrition, causing host nutrition deficiency and weight loss. In the figure, the PBS group is the PBS-immunized mouse control group, and the BLANK group is the blank control group that is neither immunized nor infected with Schistosoma japonicum. * represents P＜0.01, ** represents P＜0.05, *** represents P＜0.0001.

Detailed ways

The overall technical process of the present invention is as follows:

First, the qPCR method was used to verify the results of the chip screening, confirming that the expression levels of SjScP15, SjScP57, and SjScP92 genes in child worms were indeed significantly higher than the expression levels of worms in other stages. Then use SignalP-4.1Server (http://www.cbs.dtu.dk/services/SignalP/) and TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) The sequences corresponding to the signal peptide and hydrophobic region in the corresponding nucleic acid sequence are removed. Use NCBI Primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) for primer design and primer specificity analysis. Then use Invitrogen-GatewayTechnology with clonaseⅡ kit for cloning construction.

The specific steps are as follows. Add an attB site to one end of the primer according to the instructions, and then perform PCR amplification of the Schistosoma japonicum SjScP15, SjScP57, and SjScP92 genes. After purification, the amplified products are cloned into the pDONR221 vector using the BPClonase II enzyme provided in the kit. After transformation screening and plasmid sequencing identification, the gene sequence is transferred to the expression vector pDEST17 using the LR Clonase II enzyme provided in the kit. After transformation screening and plasmid sequencing identification, it is transformed into E. coli host cells for recombination. Protein induced expression; the inclusion body protein was denatured and purified by nickel column affinity chromatography, and finally the purified recombinant proteins rSjScP15, rSjScP57, and rSjScP92 were obtained, and the in vitro cloning, expression, and purification of the SjScP15, SjScP57, and SjScP92 genes were completed. Subsequently, Western blot technology was used to verify that the purified rSjScP15, rSjScP57, and rSjScP92 recombinant proteins could be recognized by the serum of experimental animals infected with Schistosoma japonicum and the serum of Schistosoma patients. Furthermore, the purified rSjScP15, rSjScP57, and rSjScP92 recombinant proteins were used as diagnostic antigens to prepare schistosomiasis diagnostic reagents and kits, and their application value in schistosomiasis immunodiagnosis was evaluated through ELISA technology analysis and anti-rSjScP15, rSjScP57, The changing pattern of rSjScP92 antibodies in the serum of infected animals.

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

Example 1 Cloning of Schistosoma japonicum SjScP15, SjScP57, and SjScP92 genes

Based on the SjScP15, SjScP57 and SjScP92 gene sequences predicted and discovered by the inventor on the Schistosoma japonicum genome, primers were designed and restriction sites were introduced respectively. Since the 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 to that of human and other mammalian tyrosine hydroxylases, and the immune epitope of the encoded protein can easily induce antibodies. Causes non-specific reactions. On the one hand, it may cause non-specific reactions of antibodies, thereby affecting the specificity of diagnosis; on the other hand, using this protein segment to immunize the host, the antibodies induced may non-specifically affect the host tyrosine protease activity, which is not conducive to vaccines. research and development. Therefore, in the present invention, the non-conservative segment (607-957 bases) of the SjScP57 encoding gene was selected to prepare recombinant proteins for diagnosis and vaccine research. The proteins encoded by the SjScP15 and SjScP92 genes have no homologous proteins found in common mammalian hosts, so the full-length gene sequence was used as a template to design primers. The primer sequences are as follows:

SjScP15:

PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT TGGAAGGTTTCTTACAGCGGA-3'

PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA TGCCGTTATATCACATCCACCA-3'

SjScP57:

PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT TCACGCTACTAGAGCATGCAA-3'

PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA GGAAGTCCTAAAGATGC-3'

SjScP92:

PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT CATGTCACACATTTGGAATGCTAGA-3'

PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA GGTATGAATTCGATAATGAGCC-3'

The underlined part in PF is the attB1 site designed according to the instructions of the Invitrogen Gateway Technology with ClonaseⅡ kit and is used to connect to the shuttle vector pDONR221; the underlined part in PR is the attB2 site designed according to the instructions of the Invitrogen Gateway Technology with ClonaseⅡ kit. Use It was connected to the shuttle vector pDONR221; the specific primer was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

Use Schistosoma japonicum 14-day cDNA as a template to perform a PCR reaction to amplify the gene ORF fragment. The reaction system is as follows: high-fidelity DNA polymerase Mix 12.5μl, cDNA template 1μl, upstream primer 0.5μl, downstream primer 0.5μl , ddH ₂ O 10.5μl, total volume 25μl. PCR amplification program: 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min, a total of 35 cycles; final extension at 72°C for 10 min.

Use 1.2% agarose gel electrophoresis to detect the PCR product and observe whether there is a target band. The results are shown in Figure 1. M: DNA molecular weight standard, 1: SjScP15, 2: SjScP57, 3: SjScP92. The agarose gel electrophoresis results showed that SjScP15, SjScP57, and SjScP92 had a clear band at 1896bp, 354bp, and 636bp respectively, which was consistent with the size of the expected target fragment, indicating that the rSjScP15, rSjScP57, and rSjScP92 genes were successfully amplified from cDNA. ORF fragment.

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

Invitrogen Gateway Technology with ClonaseⅡ kit is used to construct shuttle vectors. The reaction system is as follows: 150ng of PCR recovered DNA product (dissolved in ddH ₂ O), 150ng of pDONR221 vector (dissolved in ddH ₂ O), 2 μl of BPclonase II enzyme, and ddH ₂ O added to the total volume. 10μl. After mixing, incubate at 25°C for 1 hour. The above ligation product was transformed into Escherichia coli DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), the transformed competent cells were spread on LB medium plates (containing 50 μg/ml kanamycin), and cultured at 37°C overnight. Single colonies were picked for PCR identification, and the clones identified as positive by PCR were further used to construct expression vectors using Invitrogen Gateway Technology with ClonaseⅡ kit. The reaction system is as follows: 150 ng of shuttle vector (dissolved in ddH ₂ O) positive by PCR, 150 ng of pDEST17 vector (dissolved in ddH ₂ O), add TE Buffer (pH 8.0) to a total volume of 8 μl, and then add 2 μl of LRclonase II enzyme. After mixing, incubate at 25°C for 1 hour. Then add 1 μl of Protein K (2 μg/μl) and incubate at 37°C for 10 min to remove protein. The above ligation product was transformed into Escherichia coli B21DE3 competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), the transformed competent cells were spread onto LB medium plates (containing 50 μg/ml ampicillin), and cultured at 37°C overnight. Single colonies were picked for PCR identification, and clones identified as positive by PCR were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for DNA sequencing to confirm whether the sequence was correct. Sequencing analysis results showed that the inserted foreign gene fragment sequence was correct, and the recombinant plasmids pDEST17-rSjScP15, pDEST17-rSjScP57, and pDEST17-rSjScP92 were successfully constructed. The nucleotide sequences of genes SjScP15, SjScP57, and SjScP92 are shown in SEQ ID NO: 1-3, respectively, and the amino acid sequences of the antigen proteins they encode are shown in SEQ ID NO: 4-6, respectively.

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

Inoculate the clone identified as positive by the above PCR into 15 mL of LB liquid medium (containing 50 μg/ml ampicillin) and culture it overnight at 37°C and 200 rpm. The next day, take 10 mL of the medium and transfer it to LB medium (containing 50 μg/ml ampicillin). Penicillin) 1L, continue to culture until the OD _600nm value is 0.8, then add IPTG with a final concentration of 1mM for induction, express at 18°C and 140rpm for 16 hours, collect the cells by centrifugation, and freeze them at -80°C for later use.

Take a small amount of pre- and post-induction bacteria and resuspend them in PBS buffer, add SDS-PAGE loading buffer, mix well and cook in a boiling water bath for 5 minutes to denature the protein.

Add 10 μl of pre-induction and post-induction samples to each loading well, and perform SDS-PAGE analysis (5% stacking gel and 12% separating gel).

The pDEST17-rSjScP15, pDEST17-rSjScP57, and pDEST17-rSjScP92 recombinant plasmids were transformed into expression competent cells respectively. After induction of expression by IPTG, obvious expression bands appeared compared with the cells before induction.

The induced bacteria were resuspended in 40 mL of bacterial lysate, disrupted by sonication, and centrifuged at 12,000 rpm for 30 min at 4°C to collect the inclusion body precipitate and supernatant respectively.

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

Resuspend the inclusion body in PBS solution containing 1% Triton-X100, wash with ultrasonic for 5 minutes, and centrifuge at 12,000 rpm for 15 minutes to collect the inclusion body precipitate;

Suspend the inclusion body in 8M urea, rotate and mix at 4°C overnight to fully dissolve the inclusion body, and centrifuge at 12,000 rpm for 30 minutes to collect the supernatant;

Pass the supernatant through a nickel ion chelating gel column (QIAGEN Company) to bind the rSjScP15, rSjScP57, and rSjScP92 recombinant proteins with 6 histidine tags to the gel column. Wash the impurity proteins with 50mM imidazole, and then wash with 250mM imidazole. Remove the recombinant protein and collect the eluate;

The purified recombinant protein was analyzed by SDS-PAGE to detect the protein purity. The electrophoresis results are shown in Figure 2. The molecular weights of rSjScP15, rSjScP57, and rSjScP92 proteins are approximately 15kDa, 14kDa, and 35kDa respectively. Their molecular weights are consistent with the theoretical relative molecular weight of the target recombinant protein, indicating that the purity has been obtained after purification using a nickel ion chelating gel column. Higher recombinant protein.

Use the BCA protein quantification kit (Thermo Fisher Scientific) to determine the concentration of recombinant protein and operate according to the instructions. The concentration of the purified recombinant protein was determined to be 1.0 mg/mL.

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

SDS-PAGE electrophoresis: Load 100ng of recombinant protein. The electrophoresis conditions are: 100V 20min, 120V 1h.

Membrane transfer: Use the wet transfer method to transfer the proteins in the PAGE gel to the PVDF membrane. The electroporation conditions are: ice bath, 100V 1h.

Blocking: Block the PVDF membrane with 5% skim milk powder at room temperature for 2 hours, and wash it three times with TBST buffer.

Add primary antibody for incubation: add serum from BALB/c mice infected with Schistosoma japonicum for 42 days, serum from New Zealand white rabbits infected with Schistosoma japonicum for 42 days, and serum from patients with Schistosoma japonicum, respectively, and use mouse anti-His-tag antibody (Abimatech Biopharmaceutical Co., Ltd. Company) as the positive control, healthy mouse serum as the negative control (diluted 1:500 with blocking solution), incubate at 4°C overnight, and wash three times with TBST buffer.

Add secondary antibody for incubation: add fluorescently labeled anti-mouse IgG antibody, anti-rabbit IgG antibody, and anti-human IgG antibody (diluted 1:10,000 with blocking solution) respectively, incubate at 37°C for 1 hour in the dark, and wash three times with TBST buffer.

Scanning film: Scan and image using Odyssey infrared laser imaging system.

The results are shown in Figures 3 to 5. Figure 3 shows the results of rSjScP15, Figure 4 shows the results of rSjScP57, and Figure 5 shows the results of rSjScP92. Among them, M: protein molecular weight standard (kDa), 1: Schistosoma japonicum patient serum, 2 : Serum of mice infected with Schistosoma japonicum for 42 days, 3: Serum of rabbits infected with Schistosoma japonicum for 42 days, 4: Mouse anti-His tag antibody positive control, 5: Normal mouse serum negative control. Using the mouse anti-His-tag antibody as the positive control, rSjScP15, rSjScP57, and rSjScP92 have obvious recognition bands at 15kDa, 14kDa, and 35kDa respectively. Using healthy mouse serum as the negative control, there is no obvious band. Among them, rSjScP15 and rSjScP57 proteins can be recognized by the serum of BALB/c mice infected with Schistosoma japonicum, the serum of New Zealand white rabbits and the serum of Schistosoma japonicum patients. rSjScP92 can be recognized by human serum, and there is also a recognition band in mouse serum. It shows that the recombinant proteins rSjScP15, rSjScP57 and rSjScP92 have good antigenicity.

Example 4 Preparation of Schistosomiasis japonicum rSjScP15, rSjScP57, rSjScP92 immunodiagnostic kits

1. The main components of the kit include:

Solid-phase carrier coated with antigen: Use carbonate-bicarbonate buffer (purchased from Sigma, Cat. No. C3041) containing 0-0.05% v/v TWEEN20 as the coating buffer, dilute the target protein to 1 μg/mL, Coated in polystyrene reaction wells, 100μL/well.

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

Sample diluent: 10% BSA solution, prepared with PBS buffer as solvent.

Enzyme-labeled antibodies: alkaline phosphatase-labeled sheep anti-human immunoglobulin antibodies (α, γ and μ-chainspecific) antibodies (Sigma).

Substrate chromogenic solution: pNPP chromogenic solution.

pNPP was purchased from Sigma, catalog number N2640. The preparation method of pNPP chromogenic solution is as follows: Dissolve 15mg pNPP in 15mL 0.1M glycine buffer (0.1M glycine, 1mM MgCl ₂ , 1mM ZnCl ₂ , dissolved in purified water, pH 10.4).

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

Positive control: coat the plate with human immunoglobulin IgG (1μg/mL).

Negative control: Wrap the plate with the corresponding antigen protein, and replace the enzyme-labeled antibody with the sample diluent.

2. Operating procedures and detection methods of the kit:

Blocking: Spin the liquid in the wells dry, add 200 μL/well of diluent, seal at 37°C for 2 hours, wash three times with PBST, 200 μL/well, 2 min/time, and pat dry the last time.

Add the sample to be tested: dilute the serum and diluent of the sample to be tested at a ratio of 1:100, and set up positive, negative and blank controls (only add sample diluent). Add each sample at 100 μL/well, and test 3 samples in parallel. Repeat wells and incubate at 37°C for 1 hour.

Washing: Spin the liquid in the wells dry, wash four times with PBST buffer, 200 μL/well, 2 min/time, and pat dry the last time.

Add enzyme-labeled antibodies: Add alkaline phosphatase-labeled goat anti-human immunoglobulin (α, γ and μ-chainspecific) antibodies, 100 μL/well, 37°C, incubate for 1 hour, wash as above, and pat dry.

Color development: Add enzyme substrate chromogenic solution, 100 μL/well, and incubate at 37°C in the dark for 30 minutes. Add stop solution, 25 μL/well.

Data reading and processing: Use a microplate reader to read the OD _405nm value, and use 2.1 times the average OD value of the negative control sample as the critical value (cut-off) for judging negative and positive.

Example 5 Evaluation of rSjScP15, rSjScP57, rSjScP92-ELISA kits for immunodiagnosis of schistosomiasis japonicum

1. Sensitivity of the kit

The modified Kato-Katz method was used to collect 35 sera from patients with Schistosoma japonicum positive for fecal eggs in Hunan Province. The sensitivity of each kit was evaluated and compared with the rSjSP-13-ELISA kit (rSjSP-13-ELISA kit). The preparation method is the same as in Example 4. The amino acid sequence of rSjSP-13 antigen protein is shown in SEQ ID NO: 7) for comparison. The ELISA experimental operation is the same as in Example 3.

The results are shown in Table 1 and Figure 6. Sj is the Schistosoma japonicum patient group, and Healthy is the healthy control group (35 normal human serum samples from Heilongjiang Province). The horizontal straight line running through the entire coordinate system in the figure is the cutoff value of 2.1 times the mean value of the Healthy group. This line is regarded as positive, and the line below is regarded as negative. Among 20 sera from Schistosoma japonicum patients, 18 were diagnosed positive by the rSjSP-13-ELISA kit (Figure 6A). The sensitivity of the rSjSP-13-ELISA kit in diagnosing Schistosomiasis japonicum was 90.0% (95% confidence interval, 66.9 %-98.3%), this result is close to the sensitivity of rSjSP-13-ELISA kit reported in the literature, 90.4% (95% confidence interval, 75.0%-95.0%); 20 samples were diagnosed as rSjScP15-ELISA and rSjScP57-ELISA kits Positive (Figure 6B and Figure 6C, 95% confidence interval, 80.0%-100%). 19 were diagnosed as positive by the rSjScP92-ELISA kit (Fig. 6D, 95% confidence interval, 73.1%-99.7%). The sensitivities of rSjScP15, rSjScP57 and rSjScP92-ELISA kits were significantly higher than those of rSjSP-13-ELISA kit. The results show that the sensitivity of the rSjScP57-ELISA kit is the same as that of the rSjSP-13-ELISA kit.

Table 1 Comparative analysis of sensitivity of rSjSP-13-ELISA and rSjScP15, rSjScP57, rSjScP92-ELISA kits

*The sensitivity is significantly higher than rSP13-ELISA kit, p<0.05.

2. Kit specificity

The above-mentioned Healthy group collected 20 normal human serum samples from Heilongjiang Province and 18 clonorchiasis patient sera from Guangdong Province to evaluate the false positive rate of each kit in the normal population and its crossover with sera from patients with other parasites. The reaction situation was compared with the rSjSP-13-ELISA kit. The ELISA experimental operation was the same as in Example 3.

The results are shown in Table 2 and Figure 6. Cs is the Clonorchis sinensis patient group. All 20 normal human sera were diagnosed as negative by rSjSP-13-ELISA, rSjScP57-ELISA, rSjScP15-ELISA and rSjScP92-ELISA kits. The specificity of the four kits was 100% (95% confidence interval, 80.0% - 100%). All 15 sera of patients with Clonorchis sinensis were diagnosed as negative by the four kits, and there was no cross-reaction.

Table 2 Comparative analysis of specificity of rSjSP-13-ELISA and SjScP15, SjScP57, SjScP92-ELISA kits

3. The value of the test kit in assessing the progression of schistosomiasis japonicum

The above 20 patients with Schistosoma japonicum were treated with conventional treatment and serum was collected again after 3 months of chemotherapy to evaluate the value of this kit in assessing the progression of Schistosomiasis japonicum and compare it with the rSjSP-13-ELISA kit. ELISA experiment The operation is the same as in Example 3. The results are shown in Figure 6. Sj-3M is the three-month chemotherapy group for Schistosoma japonicum patients. The OD values of patients after chemotherapy with SjScP15-ELISA and rSjSP-13-ELISA kits were significantly lower than those at the current infection stage. There was no significant change in the OD values of patients after chemotherapy with SjScP15-ELISA and SjScP92-ELISA kits compared with the current infection stage.

Example 6 Evaluation of the protective effect of Schistosoma japonicum rSjScP15, rSjScP57, rSjScP92 recombinant proteins as antigens in immunized mice

1. Preparation of antigen immunized mouse model

a)Immune

Balb/c mice were used for immunization; negative serum was collected from the orbit before immunization.

Take 60ug of immunogen (60ug for primary immunization and 30ug for booster), dilute it to 200ul with normal saline, and then add an equal volume of Freund's adjuvant (Freund's complete adjuvant for primary immunization, Freund's incomplete adjuvant for boosting immunization); mix with Use a homogeneous instrument to mix the solution and adjuvant to form water-in-oil.

Inject the mixed immunogen subcutaneously into the back and abdomen at 8-10 points.

b) Take negative blood from the orbit

Hold the mouse firmly with one hand and make its eyeball pop out; insert a capillary tube into the back corner of the mouse's eye with the other hand, tilt it at 45° and slowly screw it in from the back corner of the mouse's eye. Slowly adjust the strength and posture of your hands to allow the blood to flow into the capillary. After it is almost full, immediately place the capillary into a 1.5ml centrifuge tube and take 20ul of negative blood.

After the whole blood is allowed to stand at room temperature for 30-120 minutes, it is centrifuged at 5000 rpm for 10 minutes to collect the serum.

c) ELISA determination of potency

Reagent preparation:

Coating solution: sodium carbonate-sodium bicarbonate buffer, pH9.6.

PBS buffer, pH7.4.

Blocking solution: 2% BSA or PBS containing skim milk powder.

Washing solution: PBST (i.e. PBS solution containing 0.05% v/v TWEEN20).

Chromogenic solution: 1% A solution + 10% B solution (A solution: DMSO containing 1% TMB; B solution: citric acid buffer containing 0.1% H ₂ O ₂ ).

Stop solution: 2M sulfuric acid.

Secondary antibody: goat anti-mouse IgG/HRP.

Experimental steps:

1) Dilute the antigen with coating solution to a final concentration of 2ug/ml, 100ul/well, and incubate at 4°C overnight; then wash twice with washing solution.

2) Block with blocking solution, 200ul/well, incubate at 37°C for 2 hours; then wash once with washing solution.

3) The polyantiserum is diluted 2-fold starting from 200-fold (diluted in PBS), the blank control is PBS, and the negative control is the negative serum diluted 200-fold (diluted in PBS); both are 100ul/well , 37℃ incubator, 1h; then wash with washing liquid 3 times.

4) Add secondary antibody diluted 20,000 times in PBS, 100ul/well, incubate at 37°C for 1 hour; remove and wash 3 times with washing solution.

5) Color development, 100ul of color development solution/well, color development time is 5-15 minutes.

6) Add 50ul stop solution to each well to stop.

7) Measure the absorbance value at dual wavelengths (450, 630), record and save the data, and do graph analysis.

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

2. Evaluation of immunoprotective effects

a) Attack insects

The positive snails were raised at room temperature for a week, then collected in an Erlenmeyer flask and irradiated with warm light for 2-4 hours to release the cercariae in their bodies.

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

Under the microscope, use a bacterial loop to pick up 40 cercariae/mouse on the coverslip.

The coverslip was attached to the mouse abdominal skin for 5 minutes to promote cercariae penetration into the skin.

Infected mice were routinely raised in a BSL2 laboratory for 42 days.

b) Evaluate the protective effect of antigen immunity

After 42 days of feeding, the weight of each mouse was measured (5 mice per group, repeated 3 times in total).

After anesthetizing the mice, the hepatic portal vein was cut, and the heart was punctured and perfused with PBS to flush the worms out of the body and count the worms.

Remove the liver, cut it into pieces and then grind it with a 200-mesh copper mesh. Add 20ml of 5% KOH solution to every 1g of liver tissue, mix well, incubate at 37°C overnight for digestion, and count the number of eggs in the liver tissue under a microscope.

The rSjSP-13 recombinant protein immunization group and the BALNK protein-free control immunization group were used as comparisons. The results are shown in Table 3-Table 4 and Figure 7A.

Specifically, as shown in Table 3 and Figure 7A, the worm reduction rates of mice in the rSjScP15, rSjScP57, and rSjScP92 immunized groups were significantly higher than those in the PBS immunized control group, reaching 38.1%, 34.8%, and 33.4% respectively. The calculation formula for the worm reduction rate is: (1-Total number of mature worms/Total number of mature worms in the blank control group)×100%. In this example, the number of infected cercariae was 40 cercariae per mouse. Due to the influence of cercariae activity and sex ratio and the host immune system, the number of viable cercariae in each mouse after infection is generally less than 40, so the total number of mature cercariae is expressed in PBS. The control group is the standard. However, the worm reduction rate in the rSjSP-13 recombinant protein immunization group was only 3.8%. It can be seen from this that the rSjScP15, rSjScP57 and rSjScP92 recombinant proteins of the present invention have better worm reduction effects than the rSjSP-13 group after immunization.

The greatest harm of schistosomiasis is that the parasites lay eggs after they mature. The eggs are deposited in the liver, leading to liver fibrosis and a series of pathological changes. As shown in Table 4 and Figure 7B, the number of liver eggs in mice immunized with rSjScP15, rSjScP57, and rSjScP92 was significantly lower than that in the PBS immunized control group. The egg reduction rates reached 46.0%, 48.9%, and 62.3% respectively. The calculation formula for the egg reduction rate is: (1-total number of eggs/total number of eggs in PBS group)×100%. However, the egg reduction rate in the rSjSP-13 recombinant protein immunization group was only 4.9%. It can be seen from this that the rSjScP15, rSjScP57 and rSjScP92 recombinant proteins of the present invention are better than the rSjSP-13 group in reducing insect eggs after immunization.

After schistosomiasis infects a host, it relies on ingesting the host's peripheral blood to supply its own nutrition, causing host nutrition deficiency and weight loss. As shown in Figure 7C, the body weight of mice immunized with rSjScP15, rSjScP57, and rSjScP92 was significantly higher than that of the PBS immunized control group. The BLANK group is normal blank control mice that are neither immunized nor infected with schistosomiasis. There was no significant statistical difference in the body weight of mice in the rSjSP-13 recombinant protein immunization group compared with the PBS immunization control group. It can be seen from this that the effect of increasing body weight after immunization with rSjScP15, rSjScP57 and rSjScP92 proteins of the present invention is better than that of the rSjSP-13 group.

The number of mature worms in mice is shown in Tables 3 and 4. The data in Table 3 is a summary of the experimental data obtained from three independent experiments. Each experiment was conducted with 5 mice, for a total of 15 mice in the three experiments.

Table 3 Statistics of the number of adult worms (individuals) in mice infected with Schistosoma japonicum

Table 4 Statistical table of the number (number) of liver eggs in mice infected with Schistosoma japonicum

Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made based on the present invention, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

sequence list

<110> Institute of Pathogen Biology, Chinese Academy of Medical Sciences

<120> Schistosoma japonicum antigenic protein rSjScP57 and its application

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

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

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

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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

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

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

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

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

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Ala Asn Tyr Ile Trp Glu Ile

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

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

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

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Ile

Claims (8)

1. Schistosoma japonicum antigen protein rSjScP57, characterized in that it consists of the amino acid sequence of i) or ii):

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

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

2. The nucleotide sequence of the coding gene SjScP57 of the schistosoma japonicum antigen protein rSjScP57 is shown as SEQ ID NO. 2.

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.