Schistosoma japonicum katsurada recombinant antigens rSjCB1 and rSjSAP4 and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to schistosoma japonicum recombinant antigens rSjCB1 and rSjSAP4 and application thereof. The schistosoma japonicum recombinant antigen rSjCB1 is used as a detection antigen for schistosomiasis diagnosis, has sensitivity of 97.87% and specificity of 81% on sheep schistosoma japonicum, and has no cross reaction with sheep serum infected by pichia pastoris and haemonchus contortus. The combined detection result of the rSjCB1 and the rSjSAP4 shows that the sensitivity is 93.62%, and the specificity is 92%, so that the recombinant antigen rSjCB1, the rSjCB1 and the rSjSAP4 prepared by the invention are used for diagnosing the sheep schistosomiasis, are high in sensitivity and strong in specificity, and have important significance in preventing and treating the sheep schistosomiasis.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the recombinant antigens rSjCB1 and rSjSAP4 of Schistosoma japonicum and their application in the detection of specific antibodies against schistosomiasis in sheep. Background Technology

[0002] Schistosomiasis is a zoonotic parasitic disease caused by schistosomes, and it is a neglected tropical disease. Many countries worldwide consider the elimination of schistosomiasis a significant public health issue. In China, the main pathogen of schistosomiasis is *Schistosoma japonicum*, which can infect multiple definitive hosts, including humans, various livestock (such as goats and cattle), and wild animals. Currently, the prevalence of schistosomiasis in China is effectively controlled, but in some lake and swamp areas, challenges remain in controlling infectious sources such as wild animals and snails, and the risk of recurring outbreaks persists. Sheep are among the livestock species raised in China in recent years that have seen rapid growth in schistosomiasis-endemic areas, and due to their farming methods and environment, they are more likely to become infected with schistosomes. Therefore, a high-throughput and sensitive-specific monitoring and detection method, such as ELISA or colloidal gold test strips, is needed.

[0003] In the diagnosis of schistosomiasis in sheep, traditional fecal egg detection methods are time-consuming and labor-intensive, making it difficult to meet the needs of large-scale screening. In recent years, serological diagnostic techniques have become a research hotspot due to their high sensitivity and convenience. Serological methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic strips (ICT) have been widely used in the serological diagnosis and epidemiological surveillance of schistosomiasis. Currently, the main serological diagnostic method for animal schistosomiasis japonicus is based on SEA (soluble egg antigens, SEA). SEA is a commonly used diagnostic antigen in schistosomiasis diagnosis, but SEA still has problems such as cumbersome preparation, difficulty in quantification, and complex composition. In contrast, recombinant proteins have advantages such as simple preparation, convenient quantification, and clear composition. Therefore, establishing a recombinant protein-based method for animal schistosomiasis surveillance and serological diagnosis is of great significance.

[0004] In recent years, several promising targets for schistosomiasis immunodiagnostic purposes have been identified. Sj23, a member of the tetraspanome protein family, is expressed in the body of *Schistosoma japonicum* and has a molecular weight of 23 kDa. It is considered an important candidate target for the development of schistosomiasis vaccines and serological diagnostic methods. SjSAP4 has recently been identified as an important biomarker for immunodiagnosis, exhibiting high sensitivity and specificity, and excellent diagnostic performance in *Schistosoma japonicum*. Sj32 and SjCB1 are digestive enzymes of *Schistosoma japonicum*, secreted in the host's intestinal tract. They possess good immunogenicity and antigenicity, and previous studies have shown them to have high diagnostic target potential. Due to significant species differences between humans and other animals such as cattle, sheep, and rodents, immune responses to different antigens vary. Therefore, comparative studies on the clinical diagnostic performance of promising immunodiagnostic targets are needed to screen for candidate antigens for the monitoring and diagnosis of ovine *Schistosoma japonicum*. Summary of the Invention

[0005] This invention aims to solve the technical problem of the lack of sensitive and specific antigens for the diagnosis of schistosomiasis in sheep, and provides two recombinant antigens of Schistosoma japonicum, rSjCB1 and rSjSAP4. These recombinant antigens are used to detect specific antibodies against schistosomiasis in sheep and have high sensitivity and strong specificity.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0007] In one aspect of the invention, two recombinant antigens of Schistosoma japonicum are provided, which are prepared by expression of a recombinant vector containing a CB1 gene fragment of Schistosoma japonicum and a recombinant vector containing a SAP4 gene fragment, wherein the sequence of the CB1 gene fragment is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, and the sequence of the SAP4 gene fragment is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.4.

[0008] Therefore, the present invention provides a recombinant antigen of Schistosoma japonicum, which is a recombinant antigen with the amino acid sequence shown in SEQ ID NO.2 or SEQ ID No.4.

[0009] Also provided are polynucleotides encoding the recombinant antigen, specifically, whose nucleotide sequences are shown in SEQ ID NO.1 or SEQ ID No.3.

[0010] The present invention further provides a recombinant vector containing the aforementioned polynucleotide.

[0011] The present invention further provides a host cell comprising the recombinant vector described above.

[0012] The present invention also provides the application of a recombinant antigen of Schistosoma japonicum in the preparation of a product for detecting specific antibodies against schistosomiasis japonicum, wherein the recombinant antigen is the recombinant antigen protein of Schistosoma japonicum CB1 with the amino acid sequence shown in SEQ ID NO.1, and / or the recombinant antigen protein of Schistosoma japonicum SAP4 with the amino acid sequence shown in SEQ ID NO.3.

[0013] The present invention particularly provides a product for detecting specific antibodies against schistosomiasis japonicus, comprising the recombinant antigen protein of Schistosoma japonicus CB1 with the amino acid sequence shown in SEQ ID NO.1, and / or the recombinant antigen protein of Schistosoma japonicus SAP4 with the amino acid sequence shown in SEQ ID NO.3.

[0014] Specifically, the product is an ELISA detection kit or a detection chip. Specifically, the detection chip is a protein microarray or a liquid phase chip.

[0015] Preferably, it is used for the detection of a protein mixture consisting of Schistosoma japonicum CB1 recombinant antigen protein and Schistosoma japonicum SAP4 recombinant antigen protein.

[0016] Specifically, when using the combined detection of recombinant antigen protein CB1 and recombinant antigen protein of Schistosoma japonicum, the following are included: ELISA plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, and colorimetric solution.

[0017] The recombinant antigen rSjCB1 from Schistosoma japonicum of this invention, used as a diagnostic antigen for schistosomiasis, exhibits a sensitivity of 97.87% and a specificity of 81% for sheep schistosomiasis, while showing no cross-reactivity with serum from sheep infected with *Schistosome simonii* or *Haemaphysema contortus*. Combined detection of rSjCB1 and rSjSAP4 showed a sensitivity of 93.62% and a specificity of 92%, indicating that the recombinant antigen rSjCB1 and the specific antibody detected by combining rSjCB1 and rSjSAP4 of this invention are highly sensitive and specific for the diagnosis of sheep schistosomiasis, and are of great significance for the prevention and control of sheep schistosomiasis. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a graph showing the results of SDS-PAGE analysis of the phase expression levels of recombinant proteins in Example 1 of this invention;

[0020] Figure 2 This is a graph showing the results of Western blotting detection of recombinant protein antigenicity in Example 1 of this invention;

[0021] Figure 3This is a graph showing the results of sheep schistosomiasis detection for the four recombinant proteins and SEA in Example 2 of this invention;

[0022] Figure 4 This is a ROC curve analysis diagram of four recombinant proteins and SEA in sheep schistosomiasis according to Example 2 of the present invention;

[0023] Figure 5 This is a graph showing the results of the combined detection of sheep schistosomiasis by rSjCB1 and rSjSAP4 in Example 4 of the present invention.

[0024] Figure 6 This is the ROC curve analysis diagram of the combined detection of sheep schistosomiasis by rSjCB1 and rSjSAP4 in Embodiment 4 of the present invention. Detailed Implementation

[0025] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0026] Example 1: Discovery of antigens CB1 and SAP4

[0027] Based on previous research results in our laboratory and the expression distribution, biological function, and other characteristics of various candidate proteins in Schistosoma japonicum, the inventors selected four Schistosoma japonicum antigens, SjCB1, SjSAP4, Sj32, and Sj23, as well as SEA, a commonly used diagnostic antigen for schistosomiasis japonicum, to compare their diagnostic capabilities for schistosomiasis japonicum in sheep.

[0028] Based on the known sequences of the obtained genes in the NCBI (or Genedb) database, gene fragments containing the screened cds sequence were selected to construct recombinant expression plasmids pCold-I and pET-21a. IPTG was used to induce expression in BL21, and the expressed recombinant proteins rSjCB1 and rSjSAP4 were purified. The molecular weights were approximately 33 kDa and 23 kDa, respectively. Western blotting showed that the recombinant proteins rSjCB1 and rSjSAP4 had good antigenicity. The ELISA method was used to evaluate the detection efficacy of recombinant protein rSjCB1 against sheep schistosomiasis. The results showed that the sensitivity of recombinant protein rSjCB1 against sheep schistosomiasis was 97.87% (92 / 94) and the specificity was 81% (81 / 100). The combined detection results of rSjCB1 and rSjSAP4 showed a sensitivity of 93.62% (88 / 94) and a specificity of 92% (92 / 100), indicating that the recombinant protein rSjCB1 of this invention has high diagnostic application value in the detection of sheep schistosomiasis.

[0029] Example 2: Expression and purification of candidate antigens for schistosomiasis japonicus in livestock

[0030] 1. Materials

[0031] The glycerol-expressing bacteria pCold-I-SjCB1-BL21, pET-21a-SjSAP4, pET-28a-Sj32, and pGEX-GST-Sj23 were preserved in our laboratory.

[0032] 2. Methods

[0033] 2.1 Prokaryotic expression phase analysis of recombinant proteins rSjCB1, rSjSAP4, rSj32 and rGST-Sj23

[0034] Transfer 5 μl of correctly identified positive bacterial culture to 5 ml of LB liquid medium (containing 1% kanamycin and ampicillin), and incubate at 200 rpm for 2-3 hours in a 37°C constant temperature shaker. When the colonies reach the logarithmic growth phase (OD50), the culture is ready for further incubation. 600 When the concentration of the protein is 0.6-0.8, take 1 ml of bacterial culture (0h) and add IPTG to the bacterial culture to a final concentration of 1 mM to induce expression. Take 1 ml of bacterial culture at 2h, 4h and 6h after induction. Centrifuge the 0h, 2h, 4h and 6h bacterial cultures at 4℃ and 12000 g for 5 min, remove the supernatant, add 40 μl of deionized water to resuspend the precipitate, add 10 μl of 5× protein loading buffer and mix well. Boil in boiling water for 5 min. Analyze the optimal induction time of recombinant protein by SDS-PAGE electrophoresis.

[0035] 2.2 Extensive prokaryotic expression of recombinant proteins rSjCB1, rSjSAP4, rSj32, and rGST-Sj23

[0036] Transfer 5 ml of correctly identified positive bacterial suspensions (pCold-I-SjCB1-BL21, pET-21a-SjSAP4, pET-28a-Sj32, pGEX-GST-Sj23 / Bl21) to 400 ml of LB liquid medium (containing 1% kanamycin and ampicillin), and incubate at 200 rpm for 2-3 hours in a 37°C shaker. When the monoclonal positive colonies in the medium reach the logarithmic growth phase (OD50), the culture is considered complete. 600When the concentration of the protein was 0.6-0.8, IPTG was added to the bacterial culture to a final concentration of 1 mM, and expression was induced for 4 h. After induction, the bacterial culture was transferred to 400 ml centrifuge tubes and centrifuged at 6000 rpm for 30 min at 4 °C. The supernatant was discarded. The precipitate of each tube was resuspended in 20 ml of 1×PBS, and the mixture was subjected to 5 freeze-thaw cycles and sonicated on ice for 30 min (5 s sonication, 9 s pause). The mixture was then centrifuged at 12000 g for 20 min at 4 °C, and the supernatant and precipitate were collected. The precipitate was resuspended in 6M urea. After resuspending the precipitate in 6M urea, the mixture was centrifuged at 12000 g for 15 min at 4 °C, and the supernatant was collected. 40 μl of the supernatant was sonicated, and the precipitate was dissolved in 6M urea. 10 μl of 5× protein loading buffer was added and mixed well. The mixture was boiled in water for 5 min and then analyzed by SDS-PAGE electrophoresis.

[0037] 2.3 Purification of recombinant proteins rSjCB1, rSjSAP4, rSj32 and rGST-Sj23

[0038] Based on the results of SDS-PAGE electrophoresis, the form in which the target protein exists was determined. The recombinant protein was then analyzed and purified using a Ni-NTAhis Bind Resin column (GST Bind Resin was used on the Sj23). 2 ml of the suspended resin was loaded into the column, and the resin was allowed to settle naturally under gravity. When the level of the resin preservative solution dropped to the resin surface, the Ni-NTAhis Bind Resin was operated in the following sequence:

[0039] (a) 3 times the volume of sterilized deionized water

[0040] (b) 5 times the volume of 1× ionization buffer (Ni 2+ )

[0041] (c) 3 times the volume of 1× binding buffer (recombinant protein is added to the precipitate with a final concentration of 6M urea).

[0042] Perform column chromatography on the prepared recombinant protein solution in the following order:

[0043] (a) Once the binding buffer has descended to the surface of the chromatography medium, carefully add the prepared extract (repeat the addition 3 times).

[0044] (b) 10 times the volume of 1× binding buffer (recombinant protein is added to the precipitate with a final concentration of 6M urea).

[0045] (c) 6 times the volume of 1× wash buffer (recombinant protein is added to the precipitate with a final concentration of 6M urea).

[0046] (d) Elute the target protein with 6 times the volume of 1× elution buffer (add 6M urea to the precipitate for recombinant protein), and transfer 1 ml of the elution buffer to 1.5 ml EP tubes.

[0047] (e) Wash the column with 6 volumes of 1× stripping buffer (recombinant protein was added to the precipitate with a final concentration of 6M urea).

[0048] (f) 3 times the volume of sterilized deionized water

[0049] GST Bind Resin is performed in the following order:

[0050] (a) 10 ml PBS (pH=7.3), equilibrate 3 times.

[0051] (b) Add recombinant protein solution to the column (repeat 3 times).

[0052] (c) Wash 3 times with 10 ml PBS (pH=7.3)

[0053] (d) Add 1 ml of GSH-containing Tris buffer (pH=8.0) 7 times, sequentially transferring 1 ml to each 1.5 ml EP tube.

[0054] (e) Wash 3 times with 10 ml PBS (pH=7.3).

[0055] (f) Wash twice with 10 ml of deionized water.

[0056] Both resins were stored in 20% ethanol, ensuring the liquid level was above the column bed medium; an appropriate amount of the target protein was eluted, and SDS-PAGE electrophoresis was performed to identify the purification effect.

[0057] 2.4 Western Blot detection of the immunogenicity of recombinant proteins rSjCB1, rSjSAP4, rSj32 and rGST-Sj23

[0058] Recombinant proteins rSjCB1, rSjSAP4, rSj32, and rGST-Sj23 were electrophoresed by SDS-PAGE and then electrotransferred onto a PVDF membrane at 260 mA for 75 min. After transfer, the NC membrane was blocked at room temperature for 1 h with 1% skim milk powder-TBST solution and washed three times with TBST for 5 min each time. Primary antibodies were used as normal mouse serum and positive serum from mice infected with Schistosoma japonicum for 42 days, and incubated overnight at 4°C. After incubation, the membrane was washed three times with TBST for 5 min each time. Secondary antibodies were used as hRP-labeled goat anti-mouse IgG, and the membrane was incubated at room temperature for 1 h. After incubation, the membrane was washed four times with TBST for 5 min each time. Color development was performed using chromogenic buffer, and the results were photographed and saved.

[0059] 3. Results

[0060] 3.1 Resuscitation and Identification of Recombinant Protein Expression Bacteria

[0061] The recombinant protein expression bacteria containing rSjCB1, rSjSAP4, rSj32 and rGST-Sj23 were revived and cultured, and the bacterial culture was sequenced by the biotechnology company. The sequencing results were verified to be correct by NCBI.

[0062] 3.2 Expression and temporal analysis of recombinant plasmids pET-21a-SjSAP4, pET-28a-Sj32, pCold-I-SjCB1, and pGEX-GST-Sj23 / Bl21 in Escherichia coli

[0063] SDS-PAGE electrophoresis results showed that, after IPTG induction, recombinant protein rSjCB1 showed a specific band at 33 kDa, recombinant protein rSjSAP4 showed a specific band at 23 kDa, recombinant protein rSj32 showed a specific band at 40 kDa, and recombinant protein rGST-Sj23 showed a specific band at 35.6 kDa. Furthermore, all four proteins showed optimal expression 4 hours after induction. Figure 1 ).

[0064] 3.3 Antigenicity analysis of recombinant proteins rSjCB1, rSjSAP4, rSj32 and rGST-Sj23

[0065] Western blot analysis of rSjCB1, rSjSAP4, rSj32, and rGST-Sj23 showed that all four recombinant proteins could be recognized by mouse Schistosoma japonicum positive serum, but not by mouse negative serum. After NC membrane staining, recombinant protein rSjCB1 showed a specific band at 33 kDa, recombinant protein rSjSAP4 showed a specific band at 23 kDa, recombinant protein rSj32 showed a specific band at 40 kDa, and recombinant protein rGST-Sj23 showed a specific band at 35.6 kDa. These results indicate that recombinant proteins rSjCB1, rSjSAP4, rSj32, and rGST-Sj23 possess good antigenicity. Figure 2 ).

[0066] Example 3: Diagnostic efficacy evaluation of recombinant antigens rSjCB1, rSjSAP4, rSj32, and rGST-Sj23

[0067] 1. Establishment of ELISA detection method

[0068] 1.1 ELISA Detection Methods and Procedures

[0069] After diluting the antigen with coating buffer, coat each well with 100 μl of antigen and incubate overnight at 4°C. Wash the plate three times with PBST, shaking for 5 min each time. Shake the plate to remove excess water, add 1% gelatin blocking buffer to each well, and incubate at 37°C for 1 h. Wash the plate three times with PBST, shaking for 5 min each time. Add diluted primary antibody serum to each well and incubate at 37°C for 1 h. Wash the plate three times with PBST, shaking for 5 min each time. Add diluted HRP-labeled secondary antibody to each well and incubate at 37°C for 1 h. Wash the plate three times with PBST, shaking for 5 min each time. Add 100 μl of TMB chromogenic buffer to each well and incubate in the dark until color development begins in the negative wells (10-15 min). Stop the reaction by adding 150 μl of stop solution to each well. Measure the OD using a microplate reader. 450 value.

[0070] 1.2 Optimization conditions for the square array experiment

[0071] Each antigen was diluted with ELISA coating buffer to coat each well with 50 ng, 100 ng, and 200 ng of antigen, respectively. Standard positive and negative sera were diluted 1:50, 1:100, and 1:200. HRP-labeled protein G was diluted 1:2500 and 1:5000. Sheep samples were measured for both standard positive and standard negative results, and the average values ​​and the ratio of the average OD values ​​(P / N) of the positive and negative sera were calculated.

[0072] 1.3 Determining the ELISA Operating Procedures and Result Interpretation

[0073] Optimized reaction conditions were used as the ELISA procedure.

[0074] Result determination: Using serum from healthy lambs as a control, according to... Calculate the OD cutoff value. Sheep serum with an OD value higher than the OD cutoff value is defined as positive, and sheep serum with an OD value lower than the OD cutoff value is defined as negative.

[0075] 1.4 ELISA detection of specific antibodies against Schistosoma japonicum from sheep using candidate antigens

[0076] Candidate antigens were evaluated using ELISA under optimized conditions. The sensitivity and specificity of recombinant antigens rSjCB1, rSjSAP4, rSj32, and rGST-Sj23 for detecting ovine schistosomiasis japonicus, as well as their cross-reactivity for detecting ovine Haemophilus contortus and ovine Epistylis fascioliasis, were assessed.

[0077] 2. Results

[0078] Recombinant protein rSjCB1 is used for the detection of sheep schistosomiasis. It has high sensitivity, strong specificity and good effect, which is better than the traditional SEA. The following are the specific performance data of its use in detecting sheep schistosomiasis. Its optimal coating amount is 100ng, the optimal dilution for detecting sheep serum is 1:200, and the optimal dilution concentration of HRP-labeled protein G is 1:5000.

[0079] 2.1 ELISA detection of ovine schistosomiasis using candidate antigens

[0080] Ninety-four positive serum samples and one hundred negative serum samples from sheep were collected and detected by ELISA using recombinant proteins rSjCB1, rSjSAP4, rSj32, rGST-Sj23, and SEA, respectively. Figure 3 ). Among the five antigens used in detecting sheep serum, SjSAP4 showed a sensitivity of 39.36% (95% CI 29.44-49.98) and a specificity of 98% (95% CI 92.96-99.76); Sj32 showed a sensitivity of 95.75% (95% CI 89.46-98.83) and a specificity of 42% (95% CI 32.20-52.29); SjCB1 showed a sensitivity of 97.87% (95% CI 92.53-99.74) and a specificity of 81% (95% CI 71.93-88.16); Sj23 showed a sensitivity of 48.94% (95% CI 38.48-59.46) and a specificity of 92% (95% CI 84.84-96.48); and SEA showed a sensitivity of 78.72% (95% CI 95%). The specificity was 69% (95% CI 58.97-77.87). SjCB1 had the highest sensitivity, while SjSAP4 had the highest specificity. Receiver operating characteristic (ROC) analysis was used to preliminarily evaluate the diagnostic potential of rSjCB1, rSjSAP4, rSj32, rGST-Sj23, and the SEA antigen. The results showed that SjCB1 had the largest area under the curve (AUC) and the highest diagnostic potential, while the other antigens had lower diagnostic potential. Figure 4 )

[0081] Table 1. Recombinant proteins rSjCB1, rSjSAP4, rSj32, rGST-Sj23, and SEA as diagnostic antigens for detecting sheep schistosomiasis.

[0082]

[0083] Note: Sensitivity = positive rate in the infected group; Specificity = negative rate in the non-infected group.

[0084] 2.2 Candidate Antigen ELISA

[0085] When testing standard positive sheep serum for Haemaphysema contortus and Epistylis infection, all four recombinant protein antigens were negative.

[0086] Example 4: Combined detection of Schistosoma japonicum-specific antibodies using rSjCB1 and rSjSAP4

[0087] Based on the high sensitivity of rSjCB1 and the high specificity of rSjSAP4, as shown in Example 3, the combined diagnostic efficacy of rSjCB1 and rSjSAP4 was analyzed by detecting sheep serum samples coated with equal amounts of rSjCB1 and rSjSAP4 (100ng + 100ng, 100μl per well). The implementation method is described in Example 3.

[0088] The results of combined detection of sheep schistosomiasis using rSjCB1 and rSjSAP4 are shown in the figure below. Figure 5 As shown, the joint detection results of rSjCB1 and rSjSAP4 are as follows: Figure 6 As shown in the results, the sensitivity was 93.62% (95% CI 86.62-97.62), the specificity was 92% (95% CI 84.84-96.48), and the AUC value was 0.9472.

[0089] Table 2. Recombinant protein rSjCB1+SAP4 as a diagnostic antigen for detecting sheep schistosomiasis.

[0090]

[0091] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A recombinant antigen of Schistosoma japonicum, characterized in that, The recombinant antigen is a Schistosoma japonicum CB1 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO. 1, and / or a Schistosoma japonicum SAP4 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO.

3.

2. A polynucleotide encoding the recombinant antigen of claim 1, in particular with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO.

3.

3. A recombinant vector containing the polynucleotide of claim 2.

4. A host cell comprising the recombinant vector of claim 3.

5. Use of a recombinant antigen of Schistosoma japonicum in the preparation of a product for detecting specific antibodies of sheep Schistosoma japonicum disease, characterized in that, The recombinant antigen is a Schistosoma japonicum CB1 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO. 1, and / or a Schistosoma japonicum SAP4 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO.

3.

6. A product for detecting specific antibodies to Japanese blood fluke disease of sheep, characterized by, The recombinant antigen is a Schistosoma japonicum CB1 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO. 1, and / or a Schistosoma japonicum SAP4 recombinant antigen protein with the amino acid sequence shown in SEQ ID NO.

3.

7. The product of claim 6, wherein, The product is an ELISA detection kit or a detection chip.

8. The product of claim 7, wherein, It is used for combined detection of Schistosoma japonicum CB1 recombinant antigen protein and Schistosoma japonicum SAP4 recombinant antigen protein.

9. The product of claim 8, wherein, When the combined detection of Schistosoma japonicum CB1 recombinant antigen protein and Schistosoma japonicum SAP4 recombinant antigen protein is used, it comprises an ELISA enzyme-labeled plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, and color developing solution.

10. The product according to any one of claims 7 to 9, characterized in that, The detection chip is a protein microarray or a liquid chip.

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