CN110330556B - High-expression gene in schistosoma japonicum schistosomulum and coding protein and application thereof - Google Patents

Abstract

The invention provides a gene highly expressed in schistosoma japonicum schistosomulum, and a coding protein 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 SjScP27, SjScP80, SjScP84 and SjScP88 can be specifically identified by the serum of schistosomiasis patient 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.

Description

High-expression gene in schistosoma japonicum schistosomulum and coding protein and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a highly expressed gene in schistosoma japonicum schistosomulum, and a coding protein and application thereof.

Background

Schistosomiasis is an infectious parasitic disease that seriously threatens human health, and is prevalent in 76 countries and regions of subfira, where 2 hundred million people are infected and nearly 8 hundred million people are threatened. The schistosomiasis japonica is popular in China, and after dozens of years of continuous efforts, the schistosomiasis japonica prevention and treatment work in China has attracted attention, and the schistosomiasis japonica is controlled in popular areas, but the aim of finally eliminating the schistosomiasis japonica is still at all over the world.

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, can also provide judgment standards for the grade of a schistosomiasis epidemic area, and provides necessary information and scientific basis for evaluating epidemic situation and examining prevention and control effects. The lack of efficient and accurate diagnostic techniques is an important reason why schistosomiasis cannot be completely eliminated. With the increase of the prevention and treatment work of schistosomiasis in China, schistosomiasis tends to be a popular trend of low-grade infection in China. If these mildly infected schistosomiasis patients cannot be diagnosed and treated in a timely manner, the eggs contained in their excreta will cause a persistent 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 schistosoma eggs in the excrement or urine of the patients, not only wastes time and labor, but also has lower sensitivity to the 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) which are 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.

Disclosure of Invention

The invention aims to provide a gene highly expressed in schistosomulum japonicum and a coding protein and application thereof.

The invention has the following conception: a series of genes highly expressed in schistosoma japonicum schistosomula are screened by using a schistosoma japonicum whole genome expression profile chip, and the schistosomula 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. The invention amplifies the gene hydrophilic segment of 4 schistosoma japonicum schistosomula protein genes SjScP27, SjScP80, SjScP84 and SjScP88 by PCR, and recombines and expresses the gene hydrophilic segment in colon bacillus, and ELISA test shows that the obtained recombinant protein has high sensitivity and specificity when used for diagnosing schistosomiasis japonica and is a potential diagnostic antigen candidate target.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a gene highly expressed in schistosoma japonicum schistosomula, the gene being selected from at least one of SjScP27, SjScP80, SjScP84, SjScP88, the nucleotide sequences of which are respectively as follows:

gene SjScP 27:

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 which hybridization is performed at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and the membrane is washed 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;

gene SjScP 80:

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 which hybridization is performed at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and the membrane is washed 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;

gene SjScP 84:

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;

gene SjScP 88:

i) 4, the nucleotide sequence shown as 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. 4 and expresses the same functional protein;

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 4 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.

In a second aspect, the present invention provides antigenic proteins encoded by said genes, the amino acid sequences of the antigenic proteins encoded by the genes SjScP27, SjScP80, SjScP84 and SjScP88 are shown in SEQ ID NO. 5-8, respectively.

Truncated forms of the antigenic proteins encoded by the genes SjScP27, SjScP80, SjScP84, SjScP88, 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. 5-8, are within the scope of the invention.

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

1) is used for preparing a schistosoma japonicum detection reagent or a kit;

2) used for preparing the vaccine for resisting the schistosoma japonicum;

3) used for preparing anti-schistosoma japonicum medicine;

4) for the detection of non-diagnostic purpose schistosoma japonicum;

5) can be used for diagnosing schistosomiasis japonica.

Specifically, the schistosoma japonicum SjScP27, SjScP80, SjScP84 and SjScP88 proteins of the invention are used as specific schistosome antigens and applied to serodiagnosis; as immunogen, in preparing schistosome resistant 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 SjScP27, SjScP80, SjScP84 and SjScP88 protein in gene therapy.

In a fourth aspect, the present invention provides a schistosoma japonicum detection reagent, which comprises at least one of the following (i) to (iv):

schistosoma japonicum antigen protein SjScP27, 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 SjScP80, or DNA molecule for coding said antigen protein, or recombinant protein produced by recombinant bacterium containing said DNA molecule;

③ schistosoma japonicum antigen protein SjScP84, or DNA molecule for coding said antigen protein, or recombinant protein produced by recombinant bacteria containing said DNA molecule;

schistosoma japonicum antigen protein SjScP88, DNA molecule for coding said antigen protein, or recombinant protein produced by recombinant bacterium containing said DNA molecule.

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

In a sixth 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; using a carbonate-bicarbonate buffer (purchased from Sigma under code number C3041) containing 0-0.05% v/v TWEEN20 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;

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 ZnCl₂Dissolving 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.

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 anti-SjScP 27, SjScP80, SjScP84 and SjScP88 protein antibodies in serum can be detected after experimental animals are infected for 3 weeks.

And (IV) the operation is simple and convenient, the result is stable, and the repeatability is high.

Drawings

FIG. 1 is a diagram showing the PCR amplification results of the gene sequences of Schistosoma japonicum SjScP27, SjScP80, SjScP84 and SjScP88 in example 1 of the present invention. Wherein, M: the DNA molecular weight standards 1,2,3 and 4 are PCR amplification products of SjScP27, SjScP80, SjScP84 and SjScP88 genes respectively.

FIG. 2 is a diagram showing the results of SDS-PAGE analysis of recombinant proteins SjScP27, SjScP80, SjScP84 and SjScP88 in example 2 of the present invention. Wherein, M: protein molecular weight standard, 1: SjScP27, 2: SjScP80, 3: SjScP84, 4: SjScP 88.

FIGS. 3 to 6 are schematic diagrams showing Western blot analysis results of recombinant protein antigens SjScP27 (FIG. 3), SjScP80 (FIG. 4), SjScP84 (FIG. 5) and SjScP88 (FIG. 6) in example 3 of the present invention, and blood serum of schistosome patients and blood serum of different schistosome-infected animals. Wherein, M: protein molecular weight standard, 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. 7 is a diagram showing the results of comparison of the rSP13-ELISA kit with SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA and SjScP88-ELISA kit for the diagnosis and evaluation of schistosomiasis japonica in example 5 of the present invention. Wherein, A is the result of SjScP27-ELISA kit, B is the result of SjScP80-ELISA kit, C is the result of SjScP84-ELISA kit, D is the result of SjScP88-ELISA kit, and E is the result of rSP13-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 of the Healthy group mean, and the line is regarded as positive, and the line is regarded as negative.

FIG. 8 is a diagram showing the results of fluorescent real-time quantitative PCR analysis of the genes SjScP27, SjScP80, SjScP84 and SjScP88 in the eggs (E), cercaria (C), liver infusorium (S), male imago (M) and female imago (F) of Schistosoma japonicum in example 6 of the present invention.

FIG. 9 is a graph showing the results of comparative kinetic analyses of anti-SjScP 27 and SjScP84 protein antibodies in the sera of Babl/c infected mice and New Zealand white rabbits in example 7 of the present invention. Wherein, A and B are kinetic analysis results of anti-SjScP 27 and SjScP84 protein antibodies in the serum of a Babl/C mouse, and C and D are kinetic analysis results of anti-SjScP 27 and SjScP84 protein antibodies in the serum of a white rabbit infected with New Zealand.

FIG. 10 is a graph showing the results of comparative kinetic analyses of anti-SjScP 80 and SjScP88 protein antibodies in the sera of Babl/c infected mice and New Zealand white rabbits in example 7 of the present invention. Wherein, A and B are kinetic analysis results of anti-SjScP 80 and SjScP88 protein antibodies in the serum of a Babl/C mouse, and C and D are kinetic analysis results of anti-SjScP 80 and SjScP88 protein antibodies in the serum of a white rabbit infected with New Zealand, respectively.

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 SjScP27, SjScP80, SjScP84 and SjScP88 genes in the juvenile worms is confirmed to be obviously higher than the expression level of the worm bodies in other periods. The sequence of the corresponding signal peptide and hydrophobic region in the corresponding nucleic acid sequence was then removed using SignalP-4.1Server (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 SjScP27, SjScP80, SjScP84 and SjScP88 genes, cloning an amplification product to a pDONR221 vector by using BP clone II enzyme provided in the kit after purification, transferring a gene sequence to an expression vector pDOST 17 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 induction 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 SjScP27, SjScP80, SjScP84 and SjScP88, and the in vitro cloning expression and purification of SjScP27, SjScP80, SjScP84 and SjScP88 genes are completed. Subsequently, Western blot technology is utilized to verify that the purified SjScP27, SjScP80, SjScP84 and SjScP88 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 is carried out by using purified SjScP27, SjScP80, SjScP84 and SjScP88 recombinant proteins as diagnostic antigens, the application value of the recombinant proteins in schistosomiasis immunodiagnosis is evaluated by ELISA technical analysis, and the change rule of anti-SjScP 27, SjScP80, SjScP84 and SjScP88 antibodies in the serum of infected animals is analyzed. Finally, the gene expression rules of the SjScP27, SjScP80, SjScP84 and SjScP88 genes at different development stages of schistosoma japonicum are analyzed by using a fluorescent real-time quantitative PCR technology.

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 Schistosoma japonicum SjScP27, SjScP80, SjScP84, and SjScP88 genes

Based on the sequences of SjScP27, SjScP80, SjScP84 and SjScP88 genes (the public transforming gene of S.japonica BU802382, FN315317.1, FN357371.1 and FN357552.1) published in GenBank, primers are respectively designed and introduced into enzyme cutting sites, and the sequences of the primers are as follows:

SjScP27：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAATCTCATAGTATCAGATACAAC-3’，

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATCACTTACCATCAAGATGAAAT-3’

SjScP80：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAAGCCTTTCGTCGGTGATGC-3’，

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTGAAACCCAACTGGACATA-3’

SjScP84：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTCCAGGAAGTTAAATTATGCCGT-3’，

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATCAACTCACCTCACCAACATAA-3’

SjScP88：

PF：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTAATGGAAAACGAAATTTCAAC-3’，

PR：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAACACTCCAAACAGGAACAGG-3’

the underlined section in PF is attB1 site (SEQ ID NO:9 or 10) designed according to the Invitrogen Gateway Technology with clone II kit instructions for ligation to shuttle vector pDONR 221; the underlined section in PR is attB2 site (SEQ ID NO:11) designed according to the Invitrogen Gateway Technology with clone 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 SjScP80 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, ddH₂O10.5. 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: SjScP27, 2: SjScP80, 3: SjScP84, 4: SjScP 88. The agarose gel electrophoresis result shows that SjScP27, SjScP80, SjScP84 and SjScP88 have clear bands at 198bp,811bp,120bp and 720bp respectively, which are consistent with the sizes of expected target fragments, and the ORF fragments of the SjScP27, SjScP80, SjScP84 and SjScP88 genes are successfully amplified from cDNA.

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

The shuttle vector was constructed with the Invitrogen Gateway Technology with clone II kit, as follows: recovery of DNA product by PCR (dissolved in ddH)₂O)150ng, pDONR221 vector (in ddH)₂O)150ng, BP clonase II enzyme 2. mu.l, add ddH₂O to a total volume of 10. mu.l. After mixing, incubation is carried out for 1h at 25 ℃. The above-mentioned ligation product was transformed into E.coli DH 5. alpha. competent cells (Beijing Panjin Biotechnology Co., Ltd.), and the transformed competent cells were plated on LB medium plates (containing 50. mu.g/ml kanamycin) and cultured overnight at 37 ℃. Single colonies were picked for PCR identification, and clones identified as positive by PCR were further subjected to expression vector construction using Invitrogen Gateway Technology with clone II kitAnd (3) a body. The reaction system is as follows: positive shuttle vector for PCR identification (soluble in ddH)₂O)150ng, pDEST17 vector (in ddH)₂O)150ng, TE Buffer (pH8.0) was added to a total volume of 8. mu.l, and then LR clonase II enzyme was added in a volume of 2. mu.l. 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-SjScP27, pDEST17-SjScP80, pDEST17-SjScP84 and pDEST17-SjScP88 are successfully constructed. The nucleotide sequences of the genes SjScP27, SjScP80, SjScP84 and SjScP88 are respectively shown as SEQ ID NO. 1-4, and the amino acid sequences of the antigen proteins coded by the genes are respectively shown as SEQ ID NO. 5-8.

Example 2 expression and purification of recombinant proteins of S.japonicum SjScP27, SjScP80, SjScP84, SjScP88

The clone identified as positive by the PCR was inoculated into 15mL of LB liquid medium (containing 50. mu.g/mL ampicillin), cultured overnight at 37 ℃ and 200rpm, 10mL of the medium was taken the next day and transferred into 1L of LB medium (containing 50. mu.g/mL ampicillin), and the culture was continued until OD_600nmThe value was 0.8, and IPTG was added to the cells to a final concentration of 1mM for induction, and the cells were expressed at 18 ℃ and 140rpm for 16 hours, centrifuged to collect the cells, and frozen at-80 ℃ for further use.

And (3) taking a small amount of thalli before and after induction to be resuspended in PBS buffer solution, adding SDS-PAGE loading buffer solution, mixing uniformly, 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 gel concentrate and 12% for gel isolate).

The recombinant plasmids pDEST17-SjScP27, pDEST17-SjScP80, pDEST17-SjScP84 and pDEST17-SjScP88 are respectively transformed into expression competent cells, and after IPTG induction expression, a remarkable expression band appears in the cells compared with 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 the recombinant SjScP80 protein with 6 histidine tags to the gel column, washing the hybrid protein with 50mM imidazole, eluting the recombinant protein with 250mM imidazole, and collecting the eluate;

the purity of the purified recombinant protein is detected by SDS-PAGE analysis. The electrophoresis results are shown in FIG. 2, the molecular weights of the SjScP27, SjScP80, SjScP84 and SjScP88 proteins are about 10.2kDa, 33.0kDa, 7.1kDa and 29.5kDa respectively, 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 antigenic detection of recombinant proteins of Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88

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, 100V1 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.

As shown in FIGS. 3 to 6, the positive control of mouse anti-His-tag antibody showed significant recognition bands at 10.2kDa, 33.0kDa, 7.1kDa and 29.5kDa for SjScP27, SjScP80, SjScP84 and SjScP88, respectively, and the negative control of serum from healthy mice showed no significant band. Wherein the SjScP27, SjScP80 and SjScP88 proteins can be recognized by BALB/c mouse serum infected with schistosoma japonicum, New Zealand white rabbit serum and schistosoma japonicum serum, the SjScP84 can be recognized by human serum, and weak recognition bands are also found in the rabbit serum. The recombinant proteins SjScP27, SjScP80, SjScP84 and SjScP88 are proved to have good antigenicity.

Example 4 preparation of SjScP27, SjScP80, SjScP84, SjScP88-ELISA Immunodiagnostic kit

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 ZnCl₂Dissolving 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_405nmThe 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 evaluation of immunodiagnostic value of SjScP27, SjScP80, SjScP84, SjScP88 ELISA kit for Japanese schistosomiasis

1. Sensitivity of the kit

35 parts of human serum of the positive schistosoma japonicum virus of the dung beetle eggs in Hunan province are collected by using a modified rattan adding method (Kato-Katz), the sensitivity of the kit is evaluated, and compared with an rSP13-ELISA kit (the preparation method of the rSP13-ELISA kit is the same as that of the embodiment 4, and the amino acid sequence of the rSP13 antigen protein is shown in SEQ ID NO:12), and the ELISA experimental operation is the same as that of the embodiment 3.

The results are shown in Table 1 and FIG. 7, 32 of the 35 blood serum of schistosoma japonicum patients are positive by the rSP13-ELISA kit, the sensitivity of the rSP13-ELISA kit for diagnosing schistosoma japonicum is 91.4% (95% confidence interval, 75.8% -97.7%), and the results are close to 90.4% (95% confidence interval, 75.0% -95.0%) of the sensitivity of the rSP13-ELISA kit reported in the literature; 34 parts of the protein were positively diagnosed by the SjScP27-ELISA kit, and the sensitivity of the SjScP27-ELISA kit was 97.1% (95% confidence interval, 83.4% -99.9%); 35 were diagnosed as positive by the SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kits, all three kits having a sensitivity of 100% (95% confidence interval, 87.7% -100%).

The results show that the sensitivity of the SjScP27, SjScP80, SjScP84 and SjScP88-ELISA kit is obviously higher than that of the rSP13-ELISA kit, and p is less than 0.05.

TABLE 1 comparative analysis of rSP13-ELISA with SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kit sensitivity

*: the sensitivity of the SjSAPLLP5-ELISA kit is obviously higher than that of the rSP13-ELISA kit, and p is less than 0.05.

2. Specificity of the kit

35 parts of normal human serum samples of Heilongjiang province and 18 parts of serum of clonorchiasis patients of Guangdong province are collected, the false positive rate of the kit in normal people and the cross reaction condition with serum of other parasite patients are evaluated, and compared with the rSP13-ELISA kit, the ELISA experimental operation is the same as that of example 3.

As shown in Table 2 and FIG. 7, 35 normal human sera were negative by rSP13-ELISA and SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kits, and the specificity of each of the five kits was 100% (95% confidence interval, 87.7% -100%); 18 parts of clonorchis sinensis patient serum is diagnosed as negative by the five kits, and no cross reaction occurs.

TABLE 2 comparative analysis of rSP13-ELISA with the specificity of SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kits

3. Value of kit in evaluating schistosomiasis japonica progression status

The above 35 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 rSP13-ELISA kit, the ELISA procedure was the same as that in example 3. As shown in Table 3 and FIG. 7, the OD values of patients treated with the SjScP80-ELISA, SjScP88-ELISA, rSP13-ELISA kit were significantly reduced from those at the infection stage. The SjScP27-ELISA and SjScP84-ELISA kits did not differ significantly between the current infection and post-chemotherapy stages.

TABLE 3 comparative analysis of the value of rSP13-ELISA with SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kits for assessing the progress of schistosomiasis japonica

Example 6 analysis of expression rules of Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 genes

Total RNA of Schistosoma japonicum eggs, cercaria, liver stage trichuris and 42-day adult males and females is extracted by using an RNeasy kit (QIAGEN company), DNA is removed by DNase digestion, cDNA is synthesized by using a SuperScriptTM III reverse transcription kit (Invitrogen company), and the transcription levels of SjScP27, SjScP80, SjScP84 and SjScP88 genes in different development stages of Schistosoma japonicum are detected by using 7300Real-Time PCR (Applied Biosystems).

The SjScP27, SjScP80, SjScP84 and SjScP88 gene primers are respectively as follows:

SjScP27 upstream primer: 5'-AATCTCATAGTATCAGATACAAC-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TCACTTACCATCAAGATGAAAT-3', respectively;

SjScP80 upstream primer: 5'-CGTCCTACAGTGGGCATTCT-3' the flow of the air in the air conditioner,

a downstream primer: 5'-AAGAACGCCGAACCAGAACC-3', respectively;

SjScP84 upstream primer: 5'-TTCCAGGAAGTTAAATTATGCCGT-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TCAACTCACCTCACCAACATAA-3', respectively;

SjScP88 upstream primer: 5'-GGTAATGGAAAACGAAATTTCAACA-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TTCCAACCAGCTGTTGTCCA-3', respectively;

the proteasome regulatory subunit gene PSMD4 is used as an internal reference, the upstream primer is 5'-CCTCACCAACAATTTCCACATCT-3', and the downstream primer is 5'-GATCACTTATAGCCTTGCGAACAT-3'.

And (3) PCR reaction system: SYBR Green Mix (Agilent Co.) 12.5. mu.l, cDNA template 1. mu.l, forward primer 0.5. mu.l, reverse primer 0.5. mu.l, ddH₂O10.5. mu.l, total volume 25. mu.l.

PCR amplification procedure: at 95 ℃ for 10 min; 95 ℃, 30sec, 60 ℃, 1min (40 cycles).

Data analysis was performed using SDS 1.4 software (Applied Biosystems).

As shown in FIG. 8, the genes SjScP27, SjScP80, SjScP84 and SjScP88 of Schistosoma japonicum are mainly highly expressed in the schistosoma japonicum stage, are relatively low in the other stages (egg, cercaria and adult stages), are key genes in the early development stage of Schistosoma japonicum, and are potential anti-schistosome drug targets and vaccine candidate antigens.

Example 7 kinetic analysis of anti-SjScP 27, SjScP80, SjScP84, SjScP88 protein antibodies in sera of infected animals

Serum was collected from 6 Babl/c mice and 5 New Zealand white rabbits at 1-8 weeks before and after infection with Schistosoma japonicum, and ELISA was performed as in example 3.

As shown in FIGS. 9-10, 6 Babl/c mice and 5 New Zealand white rabbits were able to detect the corresponding protein antibodies in serum by SjScP27, SjScP80, SjScP84, SjScP88-ELISA kit 2 weeks after infection with Schistosoma japonicum, and the test results show that the kit has potential value for diagnosing early infection with schistosomiasis. Wherein the serum corresponding protein antibody begins to be reduced at 8 weeks of the SjScP80 and SjScP88-ELISA kit, and the serum antibody level is obviously reduced at 7 weeks of the SjScP27-ELISA kit. The three kits are suggested to have potential value in distinguishing acute infection from chronic infection.

Example 8 application of the combination assays of SjScP27, SjScP80, SjScP84 and SjScP88 ELISA

The antigen proteins adopted in the application are all high-expression gene coding antigens in the phase of the juvenile insect, the expression level of the antigens in the phase of the juvenile insect is higher, the expression is reduced after the insect body develops into an adult, and the corresponding antigen level is reduced. Animal serum antibody kinetic analysis in example 7 showed that the four protein antibodies were detected in serum by corresponding ELISA kits at two weeks of animal infection. Therefore, the four kits have potential diagnostic value in the early infection of the schistosoma japonicum.

In the existing schistosome diagnosis, the OD value of SjScP80 and SjScP88 is obviously reduced when the ELISA is detected in a patient group with three months of chemotherapy, but SjScP27 and SjScP84 are not reduced.

The decrease in OD values of SjScP80 and SjScP88 following chemotherapy indicates a decrease in somatic death following praziquantel treatment, and thus a decrease in the corresponding antigen, and thus a gradual decrease in the patient's antibody levels. Is helpful for the evaluation of therapeutic effect. The decrease in antibody titer after chemotherapy, while helpful in evaluating efficacy, will not be detectable when the antibody titer is below a certain level.

The OD value of SjScP27 and SjScP84 after chemotherapy is not reduced, which is helpful for the evaluation of the previous infection. Therefore, the combination of antigen proteins such as SjScP80 and SjScP88 with SjScP27 and SjScP84 can help to distinguish between a present infection and a previous infection.

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> high expression gene in schistosoma japonicum schistosomulum, coding protein and application thereof

<130> KHP191112980.2

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 201

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 1

ttcaatctca tagtatcaga tacaacaaca aaatcatatg aatataactt aataaatgat 60

aatgatgatg atgatgtaat aaaagatgtg acaactgaat taagagattt aaattggcca 120

aatttattac caaaatcatc taatcaatca gaaaataaaa cttatcatat tcaaacagta 180

tttcatcttg atggtaagtg a 201

<210> 2

<211> 834

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 2

ttaaagcctt tcgtcggtga tgctgtatat ggatatggga ttactaactg tggtattggt 60

ttatgcgaaa agaagcaatg caaaaataat gctgcttgtt tgcaaacatc cggctcgact 120

gtcatctgtc agtgtccttt gggaacttat ggacaattat gtgaaaaaag aggtgagcta 180

attataccgt cctacagtgg gcattcttac acagagtata ttggattaag tgggacatca 240

tcatcattta ctaccttgga actccaatac agaccaataa aacccgatgg gctgatttta 300

tatgaaggtt atagtcatga cagtcgtggt gattttttgg ctattatttt ggttaatgga 360

tacgttgtag tggcattcga cttaggttct ggttcagcgt tcttaaagag tgaggatcaa 420

atgaaattaa atgagtggca tttaatacgt ttctggcgaa ttggtcgtgt gggttatcta 480

caagttgact taaatgagca tattatgaca aactactcat ttggtttaca agttcaacta 540

acattaactt actttttatt tcttggcgga catccaaatt taaatattgt gtcaacacat 600

ataaaagaat atattggatt cccaaataat ctaagtattg gattccaagg atgcataagt 660

aaaattgaaa caaacggaat actgattgat cctatcaaga atgcgatagg aggtgcaaat 720

gtttataatt gcgcaggaca agaatgtggt ttttcaaatc ctgtatgttt aaacaacgga 780

aaatgtgtac aatactcaaa tggctataag tgtatatgtc cagttgggtt tcac 834

<210> 3

<211> 123

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 3

ttcttccagg aagttaaatt atgccgtaag acatcaaatg aaaaattcgg tttaacactt 60

tgttatcgac aaggtgatac atgcgataca agttgtaatg tttatgttgg tgaggtgagt 120

tga 123

<210> 4

<211> 720

<212> DNA

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 4

ttggtaatgg aaaacgaaat ttcaacaaag aaaagttcat acaaaactaa tataccagtg 60

aattatgatg gtgcagtagt aggtttagct agacgatcaa cattatttcc atttacaaca 120

cctttaccag gtatttcaac agctgcttta caattggaac atttaatagg taaaaataaa 180

aaatggacaa cagctggttg gaaattaagt tgtgaatcag gattagtatt tcgtactcaa 240

ttacaagata aagaatatgt ttacttgaat gcagctgaat ttggtgaatt agaagttgtt 300

cgagatttat tagatgatac aaaacttgat gtgaattgtg tagattatat gggacgtaat 360

gctttactac tggcaatgaa aaatgaaaat attgatttag tagaactatt agtgaataga 420

ttagattttt atgctgttga agatgcctta ttaaatgcaa ttagtcaaca aaaaaatcat 480

ttagttaaat taattgttga tcatccacaa tatattagaa tggaaaaaca atgcaaaagt 540

aaagtgtctg gaccatcaag tggaacaaat aatcgtggaa aacgatcaca attttcttca 600

gacatttcac ctttaatgct tgcagcacat attaacaatc atgaaattat tcagttatta 660

ttagatagag ggttaaaact agaaatgcca catgatcgtg cctgttcctg tttggagtgt 720

<210> 5

<211> 66

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 5

Phe Asn Leu Ile Val Ser Asp Thr Thr Thr Lys Ser Tyr Glu Tyr Asn

1 5 10 15

Leu Ile Asn Asp Asn Asp Asp Asp Asp Val Ile Lys Asp Val Thr Thr

20 25 30

Glu Leu Arg Asp Leu Asn Trp Pro Asn Leu Leu Pro Lys Ser Ser Asn

35 40 45

Gln Ser Glu Asn Lys Thr Tyr His Ile Gln Thr Val Phe His Leu Asp

50 55 60

Gly Lys

65

<210> 6

<211> 278

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 6

Leu Lys Pro Phe Val Gly Asp Ala Val Tyr Gly Tyr Gly Ile Thr Asn

1 5 10 15

Cys Gly Ile Gly Leu Cys Glu Lys Lys Gln Cys Lys Asn Asn Ala Ala

20 25 30

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

35 40 45

Thr Tyr Gly Gln Leu Cys Glu Lys Arg Gly Glu Leu Ile Ile Pro Ser

50 55 60

Tyr Ser Gly His Ser Tyr Thr Glu Tyr Ile Gly Leu Ser Gly Thr Ser

65 70 75 80

Ser Ser Phe Thr Thr Leu Glu Leu Gln Tyr Arg Pro Ile Lys Pro Asp

85 90 95

Gly Leu Ile Leu Tyr Glu Gly Tyr Ser His Asp Ser Arg Gly Asp Phe

100 105 110

Leu Ala Ile Ile Leu Val Asn Gly Tyr Val Val Val Ala Phe Asp Leu

115 120 125

Gly Ser Gly Ser Ala Phe Leu Lys Ser Glu Asp Gln Met Lys Leu Asn

130 135 140

Glu Trp His Leu Ile Arg Phe Trp Arg Ile Gly Arg Val Gly Tyr Leu

145 150 155 160

Gln Val Asp Leu Asn Glu His Ile Met Thr Asn Tyr Ser Phe Gly Leu

165 170 175

Gln Val Gln Leu Thr Leu Thr Tyr Phe Leu Phe Leu Gly Gly His Pro

180 185 190

Asn Leu Asn Ile Val Ser Thr His Ile Lys Glu Tyr Ile Gly Phe Pro

195 200 205

Asn Asn Leu Ser Ile Gly Phe Gln Gly Cys Ile Ser Lys Ile Glu Thr

210 215 220

Asn Gly Ile Leu Ile Asp Pro Ile Lys Asn Ala Ile Gly Gly Ala Asn

225 230 235 240

Val Tyr Asn Cys Ala Gly Gln Glu Cys Gly Phe Ser Asn Pro Val Cys

245 250 255

Leu Asn Asn Gly Lys Cys Val Gln Tyr Ser Asn Gly Tyr Lys Cys Ile

260 265 270

Cys Pro Val Gly Phe His

275

<210> 7

<211> 40

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 7

Phe Phe Gln Glu Val Lys Leu Cys Arg Lys Thr Ser Asn Glu Lys Phe

1 5 10 15

Gly Leu Thr Leu Cys Tyr Arg Gln Gly Asp Thr Cys Asp Thr Ser Cys

20 25 30

Asn Val Tyr Val Gly Glu Val Ser

35 40

<210> 8

<211> 240

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 8

Leu Val Met Glu Asn Glu Ile Ser Thr Lys Lys Ser Ser Tyr Lys Thr

1 5 10 15

Asn Ile Pro Val Asn Tyr Asp Gly Ala Val Val Gly Leu Ala Arg Arg

20 25 30

Ser Thr Leu Phe Pro Phe Thr Thr Pro Leu Pro Gly Ile Ser Thr Ala

35 40 45

Ala Leu Gln Leu Glu His Leu Ile Gly Lys Asn Lys Lys Trp Thr Thr

50 55 60

Ala Gly Trp Lys Leu Ser Cys Glu Ser Gly Leu Val Phe Arg Thr Gln

65 70 75 80

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

85 90 95

Leu Glu Val Val Arg Asp Leu Leu Asp Asp Thr Lys Leu Asp Val Asn

100 105 110

Cys Val Asp Tyr Met Gly Arg Asn Ala Leu Leu Leu Ala Met Lys Asn

115 120 125

Glu Asn Ile Asp Leu Val Glu Leu Leu Val Asn Arg Leu Asp Phe Tyr

130 135 140

Ala Val Glu Asp Ala Leu Leu Asn Ala Ile Ser Gln Gln Lys Asn His

145 150 155 160

Leu Val Lys Leu Ile Val Asp His Pro Gln Tyr Ile Arg Met Glu Lys

165 170 175

Gln Cys Lys Ser Lys Val Ser Gly Pro Ser Ser Gly Thr Asn Asn Arg

180 185 190

Gly Lys Arg Ser Gln Phe Ser Ser Asp Ile Ser Pro Leu Met Leu Ala

195 200 205

Ala His Ile Asn Asn His Glu Ile Ile Gln Leu Leu Leu Asp Arg Gly

210 215 220

Leu Lys Leu Glu Met Pro His Asp Arg Ala Cys Ser Cys Leu Glu Cys

225 230 235 240

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggggacaagt ttgtacaaaa aagcaggctt c 31

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggggacaagt ttgtacaaaa aagcaggctt 30

<210> 11

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggggaccact ttgtacaaga aagctgggtc cta 33

<210> 12

<211> 177

<212> PRT

<213> Schistosoma japonicum (Schistosoma japonica)

<400> 12

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 (6)

1. The nucleotide sequence of the gene SjScP27 highly expressed in schistosoma japonicum schistosomula is shown in SEQ ID NO. 1.

2. The antigenic protein encoded by the gene of claim 1, wherein the amino acid sequence is as shown in SEQ ID NO. 5.

3. The use of the antigenic protein of claim 2 in the preparation of a schistosoma japonicum detection reagent or kit.

4. The schistosoma japonicum detection reagent is characterized by comprising the following components in parts by weight: schistosoma japonicum antigen protein SjScP27, or a DNA molecule for coding the antigen protein, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule;

wherein the amino acid sequence of the antigen protein SjScP27 is shown in SEQ ID NO. 5.

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 2; using a carbonate-bicarbonate buffer containing 0-0.05% v/v TWEEN20 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.

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