Antigens of Clonorchis sinensis and their applications

Abstract

This invention discloses an antigen of Clonorchis sinensis and its application, belonging to the field of biodetection technology. To provide a Clonorchis sinensis-specific antigen protein and solve the technical problem of low specificity and sensitivity in the diagnosis of clonorchiasis in existing technologies, this invention provides an antigen of Clonorchis sinensis, wherein the antigen is composed of 1-4 repeating conserved amino acid sequences; the sequence of the conserved amino acid sequence is shown in SEQ ID NO.1. The test strip of this invention has high specificity and sensitivity, is easy to operate, and the test results can be read quickly, realizing on-site visual detection of human clonorchiasis, providing technical support for the screening, diagnosis, and prevention of this disease.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the antigens of Clonorchis sinensis and their applications. Background Technology

[0002] Clonorchis sinensis is an important foodborne zoonotic parasite that primarily infests the hepatobiliary ducts and gallbladder of various mammals, including humans, dogs, and cats, causing clonorchiasis. Reports of clonorchiasis have been found in 18 provinces, municipalities, and autonomous regions of my country. Therefore, this disease has become a significant public health issue in my country. Furthermore, clonorchiasis is closely associated with hepatobiliary diseases such as cholangiocarcinoma and liver fibrosis. In 2009, the WHO classified Clonorchis sinensis as a Group 1 biological carcinogen, highlighting the crucial importance of controlling this disease.

[0003] Currently, fecal testing remains the gold standard for diagnosing Clonorchis sinensis, but it is highly prone to missed diagnoses and misdiagnoses, and it cannot overcome the drawbacks of early diagnosis. Clinically, there is an urgent need for sensitive, accurate, and real-time visual detection methods for the prevention and control of this disease. With in-depth research on the pathogen, the emergence of novel nanomaterials, and the integration of multidisciplinary research, immunological detection technologies for pathogen infection have developed rapidly, becoming a novel analytical technique that integrates materials science, nanotechnology, and genetic engineering.

[0004] To facilitate rapid diagnosis, timely treatment, and effective control of clonorchiasis, specific, sensitive, and convenient methods are needed.

[0005] The test population was tested for Clonorchis sinensis infection. Summary of the Invention

[0006] The purpose of this invention is to provide a Clonorchis sinensis-specific antigen protein to solve the technical problem of low specificity and sensitivity in the diagnosis of Clonorchis sinensis in the prior art.

[0007] The present invention provides an antigen of Clonorchis sinensis, the antigen being composed of 1-4 repeating conserved amino acid sequences; the sequence of the conserved amino acid sequence is shown in SEQ ID NO.1.

[0008] Further specifying, the sequence of the antigen is as shown in SEQ ID NO.2.

[0009] The present invention provides the encoding gene of the above-mentioned antigen.

[0010] The present invention provides a recombinant vector containing the above-described recombinant vector.

[0011] The present invention provides a recombinant microbial cell carrying the above-mentioned genes or expressing the above-mentioned antigens.

[0012] The present invention provides a vaccine containing the above-mentioned antigen.

[0013] This invention provides a kit for human clonorchiasis, the kit containing the aforementioned antigen.

[0014] Further specifying, the kit includes a sample buffer, a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad; the sample buffer consists of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescent microsphere-labeled rabbit IgG antibody, and 0.4% fluorescent microsphere-labeled Clonorchis sinensis CSTR1 antigen.

[0015] Further specified, the coupling ratio of fluorescent microspheres to goat anti-rabbit IgG antibody is 1:2, and the coupling ratio of fluorescent microspheres to CSTR1 antigen is 1:20; the detection line coated on the nitrocellulose membrane is mouse anti-human IgG antibody, and the control line is coated with rabbit IgG antibody; the coating concentration is 5 μL / mm.

[0016] The present invention provides the use of the above-mentioned antigenic peptide, the above-mentioned encoding gene, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of a kit for diagnosing or detecting Clonorchis sinensis or antibodies against Clonorchis sinensis, or in the preparation of a vaccine for preventing Clonorchis sinensis infection.

[0017] Beneficial effects: The test strip of this invention has high specificity and sensitivity, overcoming the shortcomings of diagnostic methods such as ELISA and PCR, which cannot be performed on-site and have low accuracy. At the same time, this method is simple and quick to operate, requiring no professional personnel, and the test results can be read rapidly, enabling on-site visual detection of human clonorchiasis and providing technical support for the screening, diagnosis, and prevention of this disease. Attached Figure Description

[0018] Figure 1 Identification and reactivity analysis of CsTR1 synthetic peptides. Figure 1 A represents SDS-PAGE identification, M represents protein markers, and 1 represents the protein loading amount of 10 μg. Figure 1 B represents the Western blot identification of the CsTR1 synthetic polypeptide; M represents the protein marker; 1 represents human Clonorchis sinensis positive serum; and 2 represents human negative serum.

[0019] Figure 2 A schematic diagram of the structure of the time-resolved fluorescence immunochromatographic test strip for Clonorchiasis of the present invention, wherein 1 is a PVC base plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is a detection line; 5 is a control line; and 6 is an absorbent pad.

[0020] Figure 3This diagram illustrates the determination of test results for the test strip of the present invention. Wherein, T represents the test line; C represents the control line; a represents a strong positive result; b represents a weak positive result; and c represents a negative result.

[0021] Figure 4 This study focuses on the specificity analysis of the test strips of this invention. Wherein, T represents the detection line; C represents the control line.

[0022] Figure 5 This is a sensitivity analysis of the test strip of the present invention. Where T represents the detection line; C represents the control line.

[0023] Figure 6 These are the clinical sample test results for the test strip of this invention. Where T represents the test line; C represents the control line. Detailed Implementation

[0024] Example 1. Construction of CsTR1 peptide and verification of its synthesis and reactivity.

[0025] 1. Constructing the CsTR1 peptide:

[0026] The CsTR1 polypeptide sequence was sent to the company for synthesis (Sangon Biotech (Shanghai) Co., Ltd.), and the synthesized polypeptide sequence is shown in SEQ ID NO: 2 (or the target protein can be obtained by constructing a vector containing the coding gene of the polypeptide shown in SEQ ID NO: 2 and expressing the protein).

[0027] 2. Synthesis and reaction-probability verification of CsTR1 peptide:

[0028] Take 10 μg of the synthetically produced CsTR1 peptide and perform SDS-PAGE electrophoresis. Run at 80 V for 30 min, then at 110 V for 1 h. Remove the bromophenol blue dye from the bottom layer of the stacking gel and separating gel. Soak in Coomassie Brilliant Blue for 15 min, then destain with destaining buffer until the background is clear (e.g., ...). Figure 1 (As shown in A).

[0029] Western blot experiments were performed using a synthetically produced CsTR1 peptide. Human negative serum and human Clonorchis sinensis positive serum were used as primary antibodies, and HRP-labeled rabbit anti-human IgG (Abcam, catalog number ab6759) was used as the secondary antibody. The reactivity of the synthetic CsTR1 peptide to human serum was detected. The specific experiments are as follows:

[0030] Take 10 μg of the synthetically produced CsTR1 peptide for SDS-PAGE electrophoresis, first at 80 V for 30 min, then at 110 V for 1 h. Remove the bromophenol blue dye portion from the bottom layer of the stacking gel and separating gel, and soak them in water for later use. Cut filter paper and cellulose acetate membrane slightly larger than the protein gel, and immerse them in transfer buffer for 1 min.

[0031] Use a semi-dry transfer instrument to transfer the membrane, placing it in the order of filter paper, membrane, protein gel, and filter paper to avoid air bubbles. Set the temperature to 15 V and 20 μm.

[0032] Blocking. After the transfer was completed, the membrane was blocked with 5% skim milk at 4°C for 12 h, and then rinsed three times with PBST for 5 min each time.

[0033] Incubate with primary antibody. Dilute human Clonorchis sinensis positive serum with PBST at a ratio of 1:200, with human negative serum as a control. Incubate at 37°C for 1 hour, then wash three times with PBST for 5 minutes each time.

[0034] Incubate with secondary antibody. Dilute HRP-labeled rabbit anti-human IgG (secondary antibody) with PBST at a ratio of 1:10,000, incubate at 37°C for 1 h, and rinse three times.

[0035] Develop the color. Prepare the working color developing solution according to the DAB instructions, immerse the membrane in the solution, and incubate at 37°C in the dark for 10 minutes.

[0036] The results showed that the CSTR1 peptide could specifically recognize human Clonorchis sinensis-positive serum, but did not react with human negative serum (e.g., Figure 1 As shown in B).

[0037] Example 2. Preparation of test strips

[0038] The test strip is composed of a PVC base plate (1), a sample pad (2), a nitrocellulose membrane (3), a detection line (4), a quality control line (5), and an absorbent pad (6). Figure 2 (As shown).

[0039] The sample buffer consisted of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescently labeled goat anti-rabbit IgG antibody, and 0.4% fluorescently labeled Clonorchis sinensis CSTR1 antigen.

[0040] Coupled with antigens and antibodies to fluorescent microspheres. Time-resolved fluorescent microspheres were labeled with 1 mg / mL CSTR1 antigen and 2 mg / mL goat anti-rabbit IgG antibody. The coupling ratio of microspheres to antigen was 1:20, and the coupling ratio of microspheres to antibody was 1:2. The specific method is as follows: 100 μg of fluorescent microspheres were resuspended in 100 μL of 0.05 mol / L ethanesulfonic acid (MES) solution, centrifuged at 14000 g for 20 min, and then 100 μL of MES, 1 μL of carbodiimide (EDC), and 1.51 μL of hydroxysuccinimide (NHS) were added. After mixing, the mixture was incubated at room temperature in the dark for 0.5 h. Centrifuge the mixed solution at 14000 g for 20 min, add 200 μL of PB solution (0.39% NaH2PO4, 0.895% Na2HPO4·12H2O, pH 7.2), sonicate for 6 min, then add the required amounts of mouse IgG antibody and CSTR1 antigen, and incubate at room temperature in the dark for 3 h. Centrifuge at 14000 g for 20 min to remove the supernatant, resuspend in 200 μL of blocking buffer (0.1M glycine, 1% BSA), and incubate at room temperature in the dark for 2 h. Centrifuge at 14000 g for 20 min to remove the supernatant, resuspend in 200 μL of preservation buffer (25 mM Tris-base, 0.15M NaCl, 1% BSA, 5% trehalose, 0.05% Tween-20), sonicate for 6 min, and store sealed at 4℃ in the dark.

[0041] Preparation of nitrocellulose membrane: 2.5 mg / mL mouse anti-human IgG was sprayed onto the test line at a coating amount of 1 μL / cm, and 1 mg / mL rabbit IgG antibody was sprayed onto the control line at a coating amount of 1 μL / cm. The membranes were then dried in an oven at 45±1℃ and ≤35% humidity for 1 h.

[0042] Assembly of components: Attach the sample pad, nitrocellulose membrane, and absorbent pad to the PVC base plate in sequence. Connect the end of the sample pad to the beginning of the nitrocellulose membrane, and the end of the nitrocellulose membrane to the beginning of the absorbent pad. Align the beginning of the sample pad with the beginning of the PVC base plate, and align the end of the absorbent pad with the end of the PVC base plate. Cut the test strip into 4mm wide strips, place them in a specially made plastic case, and seal them in an aluminum foil bag at 4℃ to protect them from light.

[0043] Example 3. How to use the test strip

[0044] Take 5 μL of serum sample and add it to 95 μL of sample buffer. Mix well and drop the mixture onto the sample pad. Incubate at 37℃ for 10 min. Irradiate the test strip with a UV lamp and observe the results. If both the test line and the control line show color, the sample is positive; if only the control line shows color, the sample is negative (e.g., ...). Figure 3 (As shown).

[0045] Example 4. Test strip performance testing

[0046] Specificity of the test strips. The test strips were used to test positive serum samples from human Clonorchis sinensis, Schistosoma japonicum, Paragonimus westermani, Echinococcosis, Taenia solium, Trichuris trichiura, Hookworm, Ascaris lumbricoides, Toxoplasma gondii, and Trichinella spiralis, as well as negative human serum samples. The results showed that only Clonorchis sinensis positive serum was positive, while serum samples from other parasites were negative, indicating that the test strips of this invention have high specificity (e.g., ...). Figure 4 (As shown).

[0047] Test strip sensitivity testing. Standard positive serum was diluted 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240, 1:20480, and 1:40960, respectively. The results showed that the sensitivity of the test strip was 1:10240 (e.g., ...). Figure 5 (As shown).

[0048] Stability test of the test strips. The prepared test strips were placed in environments of 4℃, 16℃, 25℃, and 37℃ for accelerated testing to determine their stability. Human Clonorchis sinensis serum samples were tested weekly for four weeks. The results showed that the test strips could withstand environments from 4℃ to 37℃, indicating that the test strips provided by this invention have good stability.

[0049] Clinical sample testing. The test strip provided by this invention was used to test 14 human serum samples. Simultaneously, the corresponding samples were examined under a microscope. The results showed that 4 out of the 14 samples were positive, consistent with the results of stool microscopy, indicating that the test strip of this invention has high accuracy (e.g., ...). Figure 6 (As shown).

[0050] Example 5. Clinical Sample Testing

[0051] Twenty-six human serum samples and corresponding fecal samples from endemic areas of Clonorchiasis were selected. The serum samples were tested using the test strips provided in this invention, and the corresponding fecal samples were detected using microscopic examination. The results showed that the test strips of this invention detected 6 positive samples and 20 negative samples, with a positive rate of 23.1%. Microscopic examination of the fecal samples revealed clonorchiasis eggs in 7 samples, with a positive rate of 26.9%. The accuracy rate of the test strips provided in this invention was 96.15%, as shown in Table 1.

[0052] Fecal microscopy is the gold standard for detecting Clonorchis sinensis in the industry, but this method is time-consuming and complex. Furthermore, due to the high similarity of trematode eggs (such as *Phyllostachys orientalis*, *Opisthochae moschata*, and *Clonorchis sinensis*), professional interpretation of the results is required. The test strip of this invention is simple to operate and provides clear and direct results interpretation, making it more suitable for clonorchisinemia screening in primary care settings.

[0053] Table 1. Clinical Sample Testing

[0054]

[0055] SEQ ID NO.1: GGKAPPPESAGGKAPPPESA;

[0056] SEQ ID NO. 2: GGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESAGGKAPPPESA.

Claims

1. An antigen of Clonorchis sinensis, characterized in that, The sequence of the antigen is shown in SEQ ID NO.

2.

2. The encoding gene of the antigen according to claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the encoding gene as described in claim 2.

4. A recombinant microbial cell, characterized in that, Carrying the gene as described in claim 3 or expressing the antigen as described in claim 1.

5. A reagent kit for human clonorchiasis, characterized in that, The kit contains the antigen as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit includes a sample buffer, a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample buffer consists of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescently labeled rabbit IgG antibody, and 0.4% fluorescently labeled Clonorchis sinensis CSTR1 antigen. The sequence of the Clonorchis sinensis CSTR1 antigen is shown in SEQ ID NO.

2.

7. The reagent kit according to claim 6, characterized in that, The ratio of fluorescent microspheres to goat anti-rabbit IgG antibody was 1:2, and the ratio of fluorescent microspheres to CSTR1 antigen was 1:

20. The detection line on the nitrocellulose membrane was coated with mouse anti-human IgG antibody, and the control line was coated with rabbit IgG antibody. The coating concentration was 5 μL / mm.

8. The use of the antigen of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant microbial cell of claim 4 in the preparation of a diagnostic or detection kit for Clonorchis sinensis.

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