Clonorchis sinensis antigen and application thereof

Abstract

The invention relates to a clonorchis sinensis specific antigen and application thereof. The invention firstly discloses a clonorchis sinensis antigen and a separated gene for coding the antigen, the nucleotide sequence of the gene and the amino acid sequence of the antigen are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, and a core component polypeptide, namely Cs3P polypeptide, of the specific antigen is further synthesized by using a polypeptide synthesis technology; the polypeptide contains a repetitive tandem polypeptide consisting of 10 specific amino acids in the amino acid sequence of the specific antigen, and has high sensitivity and specificity, and the invention also discloses application of the synthesized polypeptide in preparation of products for detecting, diagnosing or preventing clonorchiasis sinensis. The antigen and the synthetic polypeptide are good in specificity and high in sensitivity, and the constructed colloidal gold immunochromatography test strip can simply, rapidly and accurately detect clonorchiasis sinensis infection and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biological genetic engineering, and in particular to a Clonorchis sinensis antigen and application thereof. Background Art

[0002] Clonorchiasis is a zoonotic parasitic disease caused by Clonorchis sinensis (Cs) parasitizing humans and livestock. Clonorchiasis is mainly distributed in Asia, such as China, Japan, South Korea, North Korea, Vietnam, Southeast Asia and other countries. Due to the flow of immigrants and the increasingly frequent global tourism and economic activities, there are more and more cases of the disease reported in some non-endemic areas and developed countries (including North America and Western Europe).

[0003] Adult Clonorchis sinensis parasites in the hepatobiliary ducts of humans or other definitive hosts. The secretions / metabolites of the worms and the mechanical stimulation of the worms themselves can cause inflammatory reactions in the bile ducts, especially in the secondary bile ducts. Moderately infected bile ducts may show localized dilatation, bile duct epithelial hyperplasia, duct wall thickening, and ductal stenosis. If combined with bacterial infection, it can cause cholangitis and cholangiohepatitis, and surrounding fibrous tissue hyperplasia. In the late stage, cirrhosis and hepatic ascites may occur, and cholecystitis, cholangitis, and bile duct obstruction may also occur. Evidence shows that infection with Clonorchis sinensis can increase the risk of bile duct cancer and liver cancer in patients. Hepatobiliary tumors associated with Clonorchis sinensis infection are an important public health problem in areas where Clonorchis sinensis is endemic.

[0004] Since many patients with clonorchiasis have no obvious clinical symptoms and are difficult to differentiate from other diseases such as hepatitis, misdiagnosis and mistreatment are prone to occur, resulting in delayed treatment, causing great pain and economic losses to patients and their families. For the diagnosis of patients with clonorchiasis, traditional stool examination is still the main method for diagnosing clonorchiasis, but the eggs of clonorchiasis are small and easy to miss, and require well-trained professionals to detect, with a low detection rate, and often easy to miss or misdiagnose. Therefore, etiological testing cannot meet the needs of clinical diagnosis.

[0005] Data show that the excretory secretory antigens (ESA) of some parasites can be used for serological diagnosis of parasitic diseases. Min-Ho CHOI compared and evaluated the diagnostic value of the ELISA method for detecting antibodies using ESA and the ELISA method for detecting antibodies using crude adult antigens (CA) of Clonorchis sinensis. The specificity of the ELISA method for detecting antibodies using ESA is significantly higher than that of the ELISA method for detecting antibodies using CA (the former has a specificity of 93.1% and the latter has a specificity of 87.8%). However, the preparation of traditional worm-derived antigens is complex, the quality controllability is low, and the preparation volume is limited, making it difficult to meet the needs of large-scale disease detection. Therefore, the use of biological technology or chemical synthesis technology to construct specific recombinant antigens or artificially synthesized polypeptide antigens is the only way to find suitable diagnostic antigens. Summary of the invention

[0006] The present invention aims at the deficiencies of the prior art and actual needs, and provides a Clonorchis sinensis antigen, a polypeptide (Cs3P polypeptide) and its application. The antigen described in the present invention has good specificity, high sensitivity, good repeatability, and can be mass-produced. The constructed kit and colloidal gold immunochromatographic test strip can quickly and accurately detect Clonorchis sinensis infection, solving the problem that it is difficult to quickly and accurately diagnose Clonorchis sinensis in the existing diagnostic technology.

[0007] The present invention provides an isolated gene, the sequence of which is shown as SEQ ID NO.3.

[0008] The present invention also provides a Clonorchis sinensis antigen, which is encoded by the above-mentioned isolated gene, and its amino acid sequence is shown in SEQ ID NO.4.

[0009] The present invention also provides an antibody which specifically binds to the above-mentioned Clonorchis sinensis antigen.

[0010] The present invention also provides a detection reagent or a kit, which contains the above-mentioned isolated gene, the above-mentioned antigen, or the above-mentioned antibody.

[0011] The present invention also provides a polypeptide, whose amino acid sequence is shown in SEQ ID NO.9.

[0012] The present invention also provides a synthetic polypeptide, whose amino acid sequence is shown in SEQ ID NO.5.

[0013] The present invention also provides a colloidal gold immunochromatographic test strip, which contains a polypeptide with an amino acid sequence as shown in SEQ ID NO.9 or a synthetic polypeptide with an amino acid sequence as shown in SEQ ID NO.5 as an antigen.

[0014] The present invention also provides an antibody which specifically binds to the above-mentioned polypeptide or synthetic polypeptide.

[0015] The present invention also provides a detection reagent or a test kit, which contains the above-mentioned polypeptide or synthetic polypeptide, or the above-mentioned antibody that specifically binds to the above-mentioned polypeptide or synthetic polypeptide.

[0016] The present invention also provides a use of the above antigen or the above polypeptide or synthetic polypeptide or the above colloidal gold immunochromatographic test strip or the above detection reagent or kit in the preparation of a product for detecting, diagnosing or preventing Clonorchiasis sinensis.

[0017] The present invention also provides an application of an antibody specifically binding to the above-mentioned Clonorchis sinensis antigen or the above-mentioned polypeptide or synthetic polypeptide in the preparation of a product for detecting, diagnosing or treating Clonorchiasis sinensis.

[0018] The present invention performs bioinformatics analysis on the reported diagnostic antigens for clonorchiasis sinensis, and finds that some antigens have complete or incomplete tandem repeat polypeptides, and are secretory proteins, that is, they contain signal peptide sequences at the same time. The present invention searches for transcriptome data of the adult stage of Clonorchiasis sinensis with the dual characteristics of tandem repeat polypeptides and signal peptides, and further identifies through immunological methods, and finds a novel Clonorchiasis sinensis-specific antigen-Cs3P antigen, which provides a new method for the diagnosis of clonorchiasis sinensis, and further uses polypeptide synthesis technology to synthesize the core component of Cs3P antigen. The antigen and synthetic polypeptide (Cs3P polypeptide) of the present invention have good antigenic reactivity. When the synthetic polypeptide is prepared into a colloidal gold immunochromatographic test strip, it has good specificity, high sensitivity, and can be mass-produced, and can quickly and accurately detect clonorchiasis sinensis infection, solving the problem that it is difficult to quickly and accurately diagnose clonorchiasis sinensis in existing diagnostic technologies, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Signal peptide prediction results.

[0020] Figure 2 : Blasting sequence comparison analysis of the amino acid sequence deduced from contig1018.4380.

[0021] Figure 3 : RT-PCR result of Cs3P antigen gene, M: DNA Marker, 1-2: amplified cDNA fragment.

[0022] Figure 4 : Mass spectrometry identification of Cs3P polypeptide.

[0023] Figure 5 : HPLC identification diagram of Cs3P polypeptide.

[0024] Figure 6:Results of positive infection serum and normal human serum tested by Cs3P polypeptide-colloidal gold immunochromatographic test strip. The upper row in the figure shows the results of positive infection serum, and the lower row in the figure shows the results of healthy serum. C is the quality control line, and T is the detection line. During the test, only the C line is colored and it is judged as negative, the C and T lines are both colored and it is judged as positive, and the C line is not judged as invalid. DETAILED DESCRIPTION

[0025] The specific implementation and technical scheme of the present invention are further described in detail below in conjunction with the accompanying drawings and specific examples, and the specific examples provide preferred implementations. In the present invention, unless otherwise specified, the raw materials can be purchased from conventional commercial products, and the equipment and testing methods are conventional equipment and methods in the art.

[0026] Example 1: Collection of Clonorchis sinensis adults

[0027] The metacercariae of Clonorchis sinensis were isolated from the whitebait fish caught in Hengxian County, Guangxi Zhuang Autonomous Region, by pepsin digestion. The experimental dogs were orally infected and the feces were examined for worm eggs on the 42nd day. The dogs were killed on the 60th day, and the livers were removed. Adult Clonorchis sinensis were isolated from the bile ducts, rinsed repeatedly with 0.2 mol / L pH 7.4 phosphate buffer (PBS), and stored in liquid nitrogen.

[0028] Example 2: Isolation and purification of mRNA

[0029] The total RNA of Clonorchis sinensis was extracted using TRIzol reagent from Invitrogen Company, and mRNA was separated and purified from the total RNA according to the instructions of Oligitex mRNA Spin-Column Kit.

[0030] Example 3: Transcriptome sequencing and assembly of Clonorchis sinensis adults

[0031] The sequencing and analysis of the transcriptome was completed by Shanghai Ouyi Biomedical Technology Co., Ltd. Large-scale transcriptome sequencing was completed using a Roche 454 sequencer. The raw data of the obtained transcriptome was preprocessed to remove the adapter sequence and primer sequence, and to remove low-quality and short-length sequences. A total of 97,278 sequences were obtained after processing. The final assembly was 9,534 contigs and 13,027 singlets.

[0032] Example 4: Searching for candidate antigen genes

[0033] For the 9534 contigs and 13027 singlets of the transcriptome, we first used Tandem RepeatsFinder software (version 3.3.0.0) to search for tandem repeat sequences, with parameter settings of "Alignment Parameters [match, mismatch, indel]" to 2, 7, 7; "Minimum Alignment Score To Report Repeat" to 50; and "Maximum Period Size" to 2000. Then, cDNA sequences with a Minimum Alignment Score To Report Repeat value greater than 50 were searched for open reading frame sequences using NCBI's Open Reading Frame Finder (http: / / www.ncbi.nlm.nih.gov / projects / gorf / ); finally, the amino acid sequence was predicted using SignalP 5.0 software (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ) to determine whether it contained a signal peptide sequence.

[0034] Using the above strategy, multiple assembled sequences containing tandem repeat sequences and signal peptide sequence structures were found in the transcriptome. Among them, the contig1018.4380 sequence was a novel gene sequence after blast comparison. The sequence is 641 bp in length, 11-382 bp is an open reading frame, and the nucleotide sequence of contig1018.4380 is shown in SEQ ID NO: 1. The deduced amino acid sequence of contig1018.4380 is shown in SEQ ID NO: 2.

[0035] SEQ ID NO: 1

[0036] >contig1018.4380

[0037] AACAATAGCAATGAAGTTCCTGAAACTTACTATCATTGCAGCCCTCTTCCTCAATGTTCTGTGTTCAGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGCTCAACGACCGTTTAGTCACTGGATCGCTGGTTGGATCTTGGTTCTCCTGGCAGTCAAGGCCTCCGATCACTTCTGATTCCCATGTGAAAACGGACGCGCATCGCCCCAAAATACTATGTGTGTTGAGCTTTGATATCATGCAACTAACGTAAGCTTTTTGCCATTTTGTTGAATTTCACTTAATTTCGCAAACGATTGTTTTTTGCGCTACTCCAAACACTGTCTGCATAATCCACTGTTTTTCACGTTTTCGGATAATGAAATACATTTTGTAACCCATAAGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0038] SEQ ID NO:2

[0039] Met-Lys-Phe-Leu-Lys-Leu-Thr-Ile-Ile-Ala-Ala-Leu-Phe-Leu-Asn-Val-Leu-Cys-Ser-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly- Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys- Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala- Pro-Pro-Pro-Glu-Ser-Ala-Ala-Gln-Arg-Pro-Phe-Ser-His-Trp-Ile-Ala-Gly-Trp-Ile-Leu-Val-Leu-Leu-Ala-Val-Lys-Ala-Ser-Asp-His-Phe. The abbreviation is: SEQ ID NO: 2

[0040] MKFLKLTIIAALFLNVLCSGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAP PPESAGGKAPPPESAGGKAPPPESAAQRPFSHWIAGWILVLLAVKASDHF.

[0041] The contig 1018.4380 sequence has a group of tandem repeats located at 68-307 bp, with a repeat unit of 30 bp (i.e., 10 aa, the corresponding amino acid sequence is GGKAPPPESA) (SEQ ID NO.9), and a total of 8 repeats. The amino acid sequence deduced from the open reading frame sequence contained in contig 1018.4380 was analyzed online using SignalP 5.0 software. The probability of a signal peptide was 98.59%, and the cleavage point was located between the 20th and 21st amino acids: i.e., CSG-GK, with a probability of 0.5793, indicating that 1-20 aa in the amino acid sequence is a signal peptide structure (see Figure 1 ).

[0042] Example 5: blastp sequence alignment analysis of the amino acid sequence deduced from contig1018.4380

[0043] The amino acid sequence deduced from contig1018.4380 was analyzed by online blastp sequence alignment at NCBI ( Figure 2 ). The amino acid sequence deduced from contig1018.4380 was not highly homologous to other homologous protein sequences by blastp sequence comparison analysis. The blastp sequence comparison results indicated that the contig1018.4380 sequence represented a novel gene.

[0044] Example 6: Isolation of Cs3P antigen gene

[0045] According to the nucleotide sequence of contig1018.4380, a reverse transcription primer (Cs3PGSP: 5'-ATGCGCGTCCGTTT-3') (SEQ ID NO.6) was designed and the reverse transcription primer was amplified at 42°C using Invitrogen's SuperScript TM II RT reverse transcriptase was used to reverse transcribe the total RNA extracted from Clonorchis sinensis, and the resulting cDNA was used as a template and a forward primer (Cs3PAF: 5'-CCGACATG TCAGGCGGCAAGGCACCA -3') (SEQ ID NO.7) and reverse primer (Cs3PAR: 5'-CCGCTCGAG GAAGTGATCGGAGGCCTTG -3') (SEQ ID NO.8) was further amplified by PCR. Reaction conditions: 95℃5min; 94℃1min; 56℃1min; 72℃3min, 35 cycles in total; 72℃7min. RT-PCR results are shown in Figure 3 The obtained PCR fragment was connected with the pGEM T-easy vector to construct the T_Cs3P plasmid, which was transformed into E. coli DH5α, and the monoclonal colonies were selected for sequencing (sequencing by BGI), and the gene sequence fragment of the mature peptide of the target protein was obtained. According to the arrangement characteristics of its amino acids, it was named Cs3P antigen. The obtained TA clone was sequenced to obtain the nucleotide sequence of the mature peptide of the protein, as shown in SEQ ID NO: 3. The deduced amino acid sequence is shown in SEQ ID NO: 4.

[0046] SEQ ID NO: 3

[0047] >T_Cs3P(in T Easy Vector,228bp):

[0048] TCAGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGC

[0049] ACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGTCAGCTGGCGGCAAGGCACCACCTCCTGAGT

[0050] CAGCTGCTCAACGACCGTTTAGTCACTGGATCGCTGGTTGGATCTTGGTTCTCCTGGCAGT CAAGGCCT CCGAT

[0051] CACTTC

[0052] SEQ ID NO: 4

[0053] Ser-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro- Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Ala-Gln-Arg-Pro-Phe-Ser-His-Trp-Ile-Ala-Gly-Trp-Ile-Leu-Val-Leu-Leu-Ala-Val-Lys-Ala-Ser-Asp-His-Phe.

[0054] In short:

[0055] SGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAAQRPFSHWIAGWILVLLAVKASDHF

[0056] The sequence of the Cs3P gene is basically consistent with the mature peptide sequence of contig1018.4380 assembled by transcriptome sequencing analysis. Compared with contig1018.4380, the number of tandem repeat sequence units changes from 8 to 5. That is, its deduced amino acid sequence contains only 5 tandem repeat polypeptide units.

[0057] Example 7: Synthesis of Cs3P polypeptide

[0058] Shanghai Yingji Biotechnology Co., Ltd. was commissioned to synthesize a polypeptide as shown in SEQ ID NO: 5, which is a repeating tandem polypeptide (4 units in series) consisting of 10 amino acids (GGKAPPPESA) unique to Cs3P protein. After purification, the synthesized Cs3P polypeptide was analyzed and identified using liquid chromatography-mass spectrometry (LC-MS). The mass spectrometry identification results showed that the molecular weight of the polypeptide was consistent with the target product ( Figure 4 ), indicating that the peptide was synthesized correctly; high-pressure liquid analysis showed that the product purity reached more than 85% ( Figure 5 ). The polypeptide product is freeze-dried into powder,

[0059] Store at -20℃ until use.

[0060] SEQ ID NO: 5

[0061] Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala-Gly-Gly-Lys-Ala-Pro-Pro-Pro-Glu-Ser-Ala.

[0062] The abbreviation is: GGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESA.

[0063] Example 8: Preparation of colloidal gold immunochromatographic test strips for detecting Clonorchis sinensis-specific antibodies

[0064] A nitrocellulose membrane (NCM) is attached to the middle of a plastic base plate, and then a water-absorbing membrane and a reagent pad are attached to the longitudinal ends of the NCM, respectively. In the meantime, the water-absorbing membrane and the NCM, as well as the NCM and the reagent pad, are overlapped and connected at their junctions. Adhesive tape is attached to the junction of the NCM and the reagent pad as a flow control. Near the water-absorbing membrane of the NCM, Staphylococcus protein A (SPA) is coated horizontally in a linear shape as a quality control (Control, C) line. Near the reagent pad of the NCM, Cs3P polypeptide (concentration 0.5-0.7mg / ml) is coated horizontally in a linear shape as a test (Test, T) line. Cut into 4mm wide strips for standby use.

[0065] Colloidal gold was prepared according to the method in the document A model of protein-colloidal gold interaction (J Histochem Cytochem, 1987, 35: 1191-1198) written by Roe CD et al.; protein G-colloidal gold conjugate was prepared according to the method in the document Colloidal Gold, Volume 1: Principles, Methods and Applications (Colloidal Gold: Principles, Methods and Application) (Academic press. San Diego, USA 1989: 1-39) written by Hayat MA for the preparation of chromatography strips.

[0066] Place the colloidal gold immunochromatographic test strip on a flat surface, add 90μl PBS solution to the reagent pad of the test strip, and the former quickly enters the NCM. After the colloidal gold solution enters the NCM, add 10μl of the serum to be tested to the NCM at the end of the flow control tape. Observe (or scan and archive) the results within 15 minutes. Those with only C line color development are judged as negative; those with C and T lines double color development are judged as positive; those without C line are judged as invalid.

[0067] Using Cs3P polypeptide-colloidal gold immunochromatographic test strips, 12 sera from people infected with Clonorchis sinensis and 12 sera from healthy people were tested. The results are as follows Figure 6 As shown, the upper row in the figure are the serum results of positive infected persons, and the lower row in the figure are the serum results of healthy persons. The sera of 12 persons infected with Clonorchis sinensis in the upper row all showed positive results, and the sera of 12 persons in the lower row all showed negative results. This test strip can accurately distinguish between positive and negative sera.

[0068] Example 9: Preliminary evaluation of the diagnostic value of colloidal gold immunochromatographic test strips

[0069] Cs3P polypeptide-colloidal gold immunochromatographic strips were used to detect sera from patients with clonorchiasis (47 samples), schistosomiasis japonicum (15 samples), cysticercosis (15 samples), Paragonimiasis westermani (15 samples), fascioliasis (15 samples) and healthy control sera (35 samples) to evaluate its diagnostic value. The results are shown in Table 1. Of the 47 sera from patients with clonorchiasis, 40 were positive, and one serum from patients with Paragonimiasis westermani and one serum from healthy controls showed cross-reaction. The sensitivity of the detection method was 85.1%, the specificity was 97.9%, and the total compliance rate was 93.7%.

[0070] Table 1 Results of Cs3P peptide test strips in detecting serum from patients with parasitic diseases and healthy subjects

[0071] Tab.1 Detection results of the sera from different patients and healthy donors using the colloid gold labeled immunochromatographic strip ofCs3P peptide

[0072]

[0073] Colloidal gold immunochromatographic test strips are simple, rapid, and particularly suitable for use at the grassroots and on-site, providing a practical new technology for the diagnosis of clonorchiasis.

Claims

1. An isolated gene, characterized in that Its sequence is shown in SEQ ID NO.

3.

2. A Clonorchis sinensis antigen, characterized in that: Encoded by the isolated gene according to claim 1, its amino acid sequence is shown as SEQ ID NO.

4.

3. An antibody, characterized in that It specifically binds to the Clonorchis sinensis antigen described in claim 2.

4. A detection reagent or kit, characterized in that: Containing the gene according to claim 1, the antigen according to claim 2, or the antibody according to claim 3.

5. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

9.

6. A synthetic polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

5.

7. A colloidal gold immunochromatographic test strip, characterized in that: Contains the polypeptide according to claim 5 or the synthetic polypeptide according to claim 6 as an antigen.

8. An antibody, characterized in that It specifically binds to the polypeptide according to claim 5 or the synthetic polypeptide according to claim 6.

9. A detection reagent or kit, characterized in that: Containing the polypeptide according to claim 5 or the synthetic polypeptide according to claim 6, or the antibody according to claim 8.

10. Use of the antigen according to claim 2, the polypeptide according to claim 5, the synthetic polypeptide according to claim 6, the colloidal gold immunochromatographic test strip according to claim 6, or the detection reagent or kit according to claim 9 in the preparation of a product for detecting, diagnosing or preventing Clonorchiasis sinensis.

11. Use of the antibody according to claim 3 or claim 5 or claim 8 in the preparation of a product for detecting, diagnosing or treating clonorchiasis sinensis.

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