Sheep hepatic fascioliasis early-stage specific diagnosis antigen FhCatL6 as well as preparation method and application thereof
By screening and optimizing the liver fluke-specific antigen FhCatL6, combined with the ELISA method, the problems of accuracy and sensitivity in the diagnosis of liver fluke disease have been solved, enabling early identification and efficient prevention and control.
Patent Information
- Application Number
- CN202511288991.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2025-12-02
AI Technical Summary
Current technologies lack efficient diagnostic methods for liver fluke disease, resulting in inaccurate diagnosis and difficulty in early identification and control of the disease's spread.
FhCatL6, a candidate diagnostic antigen specific to liver fluke, was screened out. The target gene was amplified and recombinant protein was constructed using overlap extension PCR technology. Diagnosis was performed using ELISA. The signal peptide and codon were optimized to improve specificity and sensitivity.
It improves the diagnostic accuracy and sensitivity of liver fluke disease, enabling positive results to be detected at high dilutions, reducing production costs, and minimizing confusion with other diseases.
Smart Images
Figure CN121049525A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of immunological detection, and in particular to a specific diagnostic antigen for early stage of sheep liver fluke disease, FhCatL6, its preparation method, and its application. Background Technology
[0002] Fasciola hepatica is a zoonotic parasitic disease caused by the liver fluke, which primarily parasitizes the liver and bile ducts of various ruminants, including cattle and sheep, as well as humans. In livestock farming, ruminants such as cattle and sheep are important farmed species, and fascioliasis severely disrupts their normal physiological functions. In cattle, infection disrupts the normal metabolism and physiological functions of the liver and bile ducts, affecting important production indicators such as growth rate, milk production, and meat quality. Sheep also experience similar symptoms, including stunted growth and decreased reproductive capacity. These combined effects ultimately have a serious negative impact on the overall development of my country's livestock industry. From a human health perspective, fascioliasis also poses a threat. Humans can become infected through contact with contaminated water or food, or through close contact with infected animals. Human patients infected with liver flukes may experience a variety of symptoms such as abdominal pain, bloating, diarrhea, and anemia, severely impacting their quality of life and health. Currently, preventative measures against liver flukes are inadequate because a highly effective vaccine has not yet been developed. Therefore, timely diagnosis before widespread transmission of liver flukes becomes crucial for disease control. Early diagnosis allows for appropriate treatment or isolation measures, preventing further spread. In the development of diagnostic methods for liver flukes, screening for liver fluke-specific antigen genes is of paramount importance. These specific antigen genes provide the foundation for developing more sensitive and specific diagnostic methods for liver flukes. They help diagnostic methods accurately identify liver flukes, avoiding confusion with other similar diseases, thereby improving diagnostic accuracy and reliability. Summary of the Invention
[0003] Based on the above technical challenges, this study conducted a series of investigations. First, through proteomics data analysis, candidate diagnostic antigens specific to *Fasciola hepatica* were screened. Simultaneously, overlap extension PCR technology was used to amplify the target gene during gene construction, providing a reference for future fusion gene construction.
[0004] This study established an indirect ELISA diagnostic method for fascioliasis. ELISA (enzyme-linked immunosorbent assay) is a widely used technique in biomedical research and clinical diagnosis, possessing advantages such as high sensitivity, strong specificity, and ease of operation. The establishment of this indirect ELISA diagnostic method provides a new and effective means for the diagnosis of fascioliasis. Finally, the established indirect ELISA diagnostic method was used to test clinical serum samples to verify the accuracy and practicality of this diagnostic method. The ultimate goal of this study is to provide specific candidate antigens for the diagnosis of fascioliasis and to support the future construction of new fusion genes as diagnostic antigens, thereby contributing to the prevention and control of fascioliasis.
[0005] Specifically as follows: A specific diagnostic antigen for early stage of sheep liver fluke disease, FhCatL6, is disclosed. The diagnostic antigen FhCatL6 is based on the expression of the BAA23743-1 gene. The diagnostic antigen FhCatL6 is a recombinant protein FhCatL6, the amino acid sequence of which is shown in SEQ ID NO.1. The amino acid sequence of BAA23743-1 is shown in SEQ ID NO.2.
[0006] Furthermore, after removing the signal peptide from BAA23743-1, codon optimization was performed to prepare the diagnostic antigen FhCatL6; the nucleotide sequence of the optimized codon is shown in SEQ ID NO.3.
[0007] On the other hand, the present invention provides a specific diagnostic kit for early-stage liver fluke disease in sheep, the kit comprising the diagnostic antigen FhCatL6 as described in claim 1 or 2; the method of using the kit includes the following steps: S1. Antigen coating: Dilute recombinant protein FhCatL6 with coating buffer, add to 96-well microplate, and incubate at 4°C for 8-12 hours; S2. Sealing: Add sealing liquid to each well and seal at 35-40℃; S3. Sample addition: Add diluted serum to each well and incubate at 35-40℃. S4. Add secondary antibody: Add diluted enzyme-labeled secondary antibody to each well and incubate at 35-40℃; S5. Color development and termination: Add TMB color development solution under light-protected conditions, incubate, and then add termination solution to terminate the reaction. S6. Result determination: Measure the OD450nm value and determine the positive or negative nature of the serum to be tested based on the cutoff value.
[0008] Furthermore, in step S1, after diluting the recombinant protein FhCatL6 with the coating solution, the concentration of the recombinant protein FhCatL6 is 0.1875 μg / 100 μL, and the dilution of the serum to be tested is 1:3200.
[0009] Moreover, in step S2, the sealing liquid is 5% skim milk.
[0010] Moreover, in step S2, the concentration of the diluted enzyme-labeled secondary antibody is 7000 times.
[0011] Moreover, in step S5, the color development time is 15 minutes.
[0012] Furthermore, in step S6, if OD450nm ≥ 0.299956, it is determined to be positive; if OD450nm < 0.251826, it is determined to be negative; if 0.251826 < OD450nm < 0.299956, it is determined to be suspected positive, and the sample is retested.
[0013] In another aspect, the present invention provides an application of the early specific diagnostic antigen FhCatL6 for sheep liver fluke disease, used to prepare an ELISA kit for the early specific diagnosis of sheep liver fluke disease.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention screened and identified the early-stage specific diagnostic antigen FhCatL6 for sheep liver fluke disease. This antigen is based on the expression of the BAA23743-1 gene and was prepared by removing the signal peptide and optimizing the codons of BAA23743-1. This improved the specificity and sensitivity of the antigen, enabling accurate identification of liver fluke disease and avoiding confusion with other similar diseases, thereby greatly improving the accuracy and reliability of diagnosis.
[0015] 2. Western blot analysis showed that the recombinant protein FhCatL6 could be specifically recognized by serum from sheep naturally infected with Fasciola hepatica, demonstrating its immunogenicity and diagnostic potential. Sensitivity tests showed that even with serum dilutions as high as 6400-fold, the ELISA method established in this invention could still detect positive results, demonstrating its high sensitivity. Furthermore, clinical test results showed that compared with commercially available kits, the ELISA method established in this invention had a higher positive rate and could more effectively identify sheep infected with Fasciola hepatica, providing strong support for clinical diagnosis and treatment.
[0016] 3. Through experimental optimization, this invention determined the optimal coating concentration of recombinant protein FhCatL6 to be 0.1875 μg / 100 μL, which is significantly lower than the conventional amount. At the same time, the serum detection dilution was increased to 1:3200. While ensuring high sensitivity and specificity, the amount of antigen and serum used was greatly reduced, thereby effectively reducing the production cost of the later development of ELISA kits. Attached Figure Description
[0017] Figure 1 The expression identification results of recombinant protein FhCatL6 in SDS-PAGE gel electrophoresis; Figure 2 SDS-PAGE analysis results of recombinant protein FhCatL6 purified by Ni column affinity chromatography; Figure 3 Presentation of Western Blot analysis results. Detailed Implementation
[0018] Example 1: Construction of prokaryotic expression vector and codon optimization 1.1 Codon Optimization After removing the signal peptide from BAA23743-1 (amino acid sequence as shown in SEQ ID NO.2), codon optimization was performed to prepare the diagnostic antigen FhCatL6; the nucleotide sequence of the optimized codon is shown in SEQ ID NO.3.
[0019] Based on the gene sequence of SEQ ID NO.3, specific primers were designed (see Table 1) to amplify the target gene fragment by overlap extension PCR.
[0020] 1.2 Carrier Construction pET-28a was selected as the prokaryotic expression vector. The optimized BAA23743-1 gene sequence was fused and cloned into the pET-28a vector using primer synthesis and overlap extension PCR. The specific steps are as follows: 1.2.1 Primer design: Based on the optimized gene sequence, specific primers with NcoI and XhoI restriction sites were designed and synthesized, as shown in Table 1.
[0021] Table 1 Primer Sequences
[0022] 1.2.2 PCR amplification: The optimized BAA23743-1 gene was amplified by PCR using the designed primers to obtain the target gene fragment with restriction sites.
[0023] 1.2.3 Enzyme digestion and ligation: The PCR product and pET-28a vector were double-digested with NcoI and XhoI enzymes, respectively. Then, the target gene fragment was ligated into the vector using T4 DNA ligase to form the recombinant expression vector pET-28a-BAA23743-1.
[0024] 1.2.4 Transformation and Screening: The recombinant expression vector was transformed into Escherichia coli DH5α competent cells. Positive clones were screened by LB plates containing Kan antibiotic, and plasmids were extracted for enzyme digestion verification.
[0025] Example 2: Transformation of recombinant vector into Escherichia coli BL21-DE3 Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min. Heat shock at 42°C for 90 sec, then immediately place on ice for 5 min, and add 600 μL of ILB culture medium. Shake at 37°C and 220 rpm for 1 h, centrifuge, and spread the entire mixture onto LB agar plates containing 50 μg / mL Kans, and incubate upside down at 37°C overnight.
[0026] Example 3: Identification of IPTG-induced expression of recombinant bacterial fusion protein (1) Pick a single clone from the transformation plate and inoculate it into a test tube containing 4 mL LB medium containing 50 μg / mL Kan, and shake overnight at 37℃ and 220 r / min; (2) The next day, the bacteria were inoculated at a ratio of 1:100 into 100 mL of LB culture medium containing 50 μg / mL Kan, and shaken at 220 r / min at 37℃ until the bacterial cell OD reached the target value. 600 It is 0.5-0.8; (3) Take out 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS buffer; (4) Add IPTG to the remaining culture to a final concentration of 0.2 mM, shake at 220 r / min for 8-10 h at 30℃ to induce fusion protein expression; (5) Take 1 mL of culture, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in TBS; add PMSF to the resuspended solution to a final concentration of 1 mM, sonicate to disrupt, and then take the supernatant and pellet solution separately and resuspend them in TBS. (6) Perform 12% SDS-PAGE analysis and Coomassie brilliant blue staining for banding; (7) See the diagram for expression identification. Figure 1 This indicates that the protein is expressed in greater quantities in the precipitate, suggesting it is an inclusion body.
[0027] Example 4 Inclusion body denaturation (1) The bacterial cells identified as inclusion bodies in Example 3 were resuspended in 40 ml of TBS at pH 7.4; ultrasonically disrupted for 2 min at 15% power until completely broken down without particles; (2) Add 500 μl of 20% Triton (final concentration 0.25%) to the above liquid; invert the centrifuge tube, mix thoroughly, and let stand for 10 min; (3) Centrifugation: 4℃, 10000rpm, 5min; discard the supernatant; (4) The precipitate was resuspended in 4 ml TBS at pH 7.4 and sonicated for 2 min at 15% power until it was completely broken down into particles. (5) Add 16 ml of denaturing mother liquor TBS, 10 M urea, and pH 7.4 to the above liquid; (6) Place the centrifuge tubes in a rotary mixer and rotate for 2-3 hours to denature them; (7) Centrifugation: 4℃, 10000rpm, 5min; the supernatant is denatured protein.
[0028] Example 5 Ni column affinity chromatography purification (1) Purification was performed using a gravity column. The denaturing solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA binding-Buffer (TBS, 0 mM imidazole, 8 M urea, pH 7.4). (2) Rinse with 3-5 column volumes of Binding-Buffer to remove unbound proteins; (3) Elute the target protein with Ni-NTAElution-Buffer (TBS, 25mM imidazole, 8M urea, pH 7.4) and collect the eluent; (4) Elute the target protein with Ni-NTAElution-Buffer (TBS, 50mM imidazole, 8M urea, pH 7.4) and collect the eluent; (5) Elute the target protein with Ni-NTAElution-Buffer (TBS, 100mM imidazole, 8M urea, pH 7.4) and collect the eluent; (6) Elute the target protein with Ni-NTAElution-Buffer (TBS, 500mM imidazole, 8M urea, pH 7.4) and collect the eluent; (7) Perform SDS-PAGE analysis. The gel image is shown below. Figure 2 As shown, Ni-NTAElution-Buffer was used for elution, yielding relatively pure recombinant FhCatL6 protein.
[0029] Example 6: Refolding and Concentration After the collected protein solutions were identified and the target bands were confirmed, they were added to dialysis bags and refolded using gradient dialysis, i.e., TBS buffer containing 4M urea, 2M urea, and 0M urea at pH 8.0. Ultrafiltration was then used for concentration.
[0030] Example 7 Immunogenicity Identification of Recombinant Protein Western Blot analysis: Using recombinant protein BAA23743-1 as the antigen, Western Blot analysis was performed using primary antibodies from sheep serum infected with Fasciola hepatica and negative serum from uninfected sheep, with rabbit anti-sheep IgG-HRP as the secondary antibody. Results are shown below. Figure 3 (Note: 1. Protein marker; 2. Recombinant BAA23743-1 protein) The results showed that a specific band appeared in the infected serum group at a relative molecular weight of 36 kDa, while no band was detected in the negative control, indicating that the recombinant BAA23743-1 protein can be recognized by sheep serum naturally infected with liver fluke and has immunogenicity.
[0031] Using recombinant BAA23743-1 protein as the antigen, sheep negative serum uninfected with Fasciola hepatica was used as the primary antibody, rabbit anti-sheep IgG-HRP was used as the secondary antibody, and no band was detected in the negative control.
[0032] Example 8 ELISA test (1) Dilute and coat the recombinant protein BAA23743-1 with coating buffer, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C; (2) Shake off the liquid in the 96-well microplate and pat dry on absorbent paper. Add 180 μL of PBST buffer to each well and vortex to wash 3 times, 1 min each time; (3) Add 100 μL of blocking solution (5% skim milk) to each well, seal at 37°C for 2 hours, and then shake and wash 3 times; (4) Add 100 μL of diluted sheep negative and positive serum to each well, incubate at 37°C for 2 hours, and then shake and wash 3 times. (5) Add 100 μL of diluted secondary antibody to each well, incubate at 37°C for 30 min, and then shake and wash 3 times; (6) Under light-protected conditions, add TMB colorimetric solution and incubate for 15 min; (7) Add 100 μL of stop solution to each well to terminate the reaction and measure the value of OD450nm.
[0033] I. Determination of the optimal working concentrations of antigen and serum The recombinant protein was diluted with coating buffer to 3 ng / μl, 1.5 ng / μl, 0.375 ng / μl and 0.1875 ng / μl, 0.09375 ng / μl, respectively. Negative and positive sera were serially diluted from 1:100 to 1:3200, and the OD450nm values of negative and positive sera were measured using an ELISA reader (Table 2) to determine the optimal working concentrations of antigen and serum: after diluting the recombinant protein FhCatL6 with coating buffer, the concentration of recombinant protein FhCatL6 was 0.1875 μg / 100 μL, and the dilution of the serum to be tested was 1:3200.
[0034] Table 2 Determination of ADP09371.1 antigen and serum dilution
[0035] II. Optimization of Antigen Incubation Time The recombinant protein was coated at 37°C for 1 h, 37°C for 2 h, and 4°C overnight, and the OD450nm value was measured to determine the optimal incubation time for the antigen (Table 3): overnight at 4°C.
[0036] Table 3 Determination of antigen incubation time
[0037] III. Determination of the Optimal Sealing Solution Under the optimal screening conditions described above, 1% BSA, 10% skim milk, and 5% skim milk were used as blocking solutions for blocking. The blocks were sealed at 37°C for 2 hours, and the OD450nm value was measured (Table 4), thus determining the optimal blocking solution as 5% skim milk.
[0038] Table 4 Determination of the Type of Sealing Agent
[0039] IV. Determination of the optimal incubation concentration of enzyme-labeled secondary antibody Under the optimal screening conditions described above, enzyme-labeled secondary antibody concentrations were set at dilutions of 3000, 4000, 5000, 6000, 7000, and 8000 times, and the OD450nm value was measured (Table 5) to determine the optimal incubation concentration of enzyme-labeled secondary antibody: 7000 times.
[0040] Table 5 Determination of Secondary Antibody Dilution
[0041] V. Determining the Optimal Color Development Time Under the optimal conditions of the above screening, the color development time was set to 10 min, 15 min, and 20 min respectively, and the OD450nm value was measured to determine the optimal color development time: 15 min.
[0042] Table 6 Determination of color development time
[0043] VI. Determination of Critical Values After optimization, indirect ELISA was performed on 21 sheep serum samples that were known to be negative for liver fluke infection. The calculated average absorbance (OD450nm) was 0.203696, and the standard deviation (SD) was 0.024065. The calculated value of X + 3SD was 0.276309, and the cutoff value for protein indirect ELISA was 0.275891. To avoid false positives, a difference of one standard deviation was added to or subtracted from the cutoff value to define the doubtful range. That is, assuming both negative and positive controls are valid, based on the indirect ELISA method, an OD450nm value ≥ 0.299956 is considered positive; OD450nm < 0.251826 is considered negative; and 0.251826 < OD450nm < 0.299956 is considered a suspected positive, requiring retesting.
[0044] VII. Evaluation of the Diagnostic Efficacy of Indirect ELISA 7.1 Sensitivity Test (1) Dilute and coat the recombinant protein BAA23743-1 with 0.1875 μg / 100 μl of coating buffer, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C; (2) Shake off the liquid in the 96-well microplate and pat dry on absorbent paper. Add 180 μL of PBST buffer to each well and vortex to wash 3 times, 1 min each time; (3) Add 100 μL of blocking solution (5% skim milk) to each well, seal at 37°C for 2 hours, and then shake and wash 3 times; (4) Add 100 μL of sheep negative and positive serum diluted (100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times, 6400 times, 12800 times) to each well, incubate at 37°C for 2 hours, and then shake and wash 3 times. (5) Add 100 μL of rabbit anti-sheep IgG-HRP diluted 7000 times to each well, incubate at 37℃ for 30 min, and then shake and wash 3 times. (6) Under light-protected conditions, add TMB colorimetric solution and incubate for 15 min; (7) Add 100 μL of stop solution to each well to terminate the reaction and measure the value of OD450nm. It can still be detected when the serum dilution factor is greater than 6400 times (Table 7).
[0045] Table 7 ELISA Sensitivity Results
[0046] 7.2 Clinical Testing Using a commercially available reagent kit (Indirect ELISA Antibody Detection Kit for Fasciola hepatica: INGENASA, Spain) and the ELISA method established in this invention, 38 sheep (numbered 1-38) from three different areas in Uxin Banner (Galutu Town, Sulide Sumu, and Tuke Town) were tested. The results showed that the ELISA method established in this invention had a higher positive rate. To avoid false positives, sheep (numbered 12, 13, 16, 22, 23, 26, and 28) with different positive results were tested again after half a month using both the commercially available reagent kit (Indirect ELISA Antibody Detection Kit for Fasciola hepatica: INGENASA, Spain) and the ELISA method established in this invention. All results were positive.
[0047] Table 8. Test results of 38 sheep
[0048] Table 9 Detailed data of 38 sheep
Claims
1. A specific diagnostic kit for early-stage liver fluke disease in sheep, characterized in that, The kit contains the diagnostic antigen FhCatL6 as described in claim 1 or 2; The diagnostic antigen FhCatL6 is based on the expression of the BAA23743-1 gene. The diagnostic antigen FhCatL6 is a recombinant protein FhCatL6, the amino acid sequence of which is shown in SEQ ID NO.
1. The amino acid sequence of BAA23743-1 is shown in SEQ ID NO.
2. After removing the signal peptide from BAA23743-1, codon optimization was performed to prepare the diagnostic antigen FhCatL6; the nucleotide sequence of the optimized codon is shown in SEQ ID NO.3; The method of using the kit includes the following steps: S1. Antigen coating: Dilute recombinant protein FhCatL6 with coating buffer, add to 96-well microplate, and incubate at 4°C for 8-12 hours; S2. Sealing: Add sealing liquid to each well and seal at 35-40℃; S3. Sample addition: Add diluted serum to each well and incubate at 35-40℃. S4. Add secondary antibody: Add diluted enzyme-labeled secondary antibody to each well and incubate at 35-40℃; S5. Color development and termination: Add TMB color development solution under light-protected conditions, and after incubation, add termination solution to terminate the reaction. S6. Result determination: Measure the OD450nm value and determine the positive or negative nature of the serum to be tested based on the cutoff value.
2. The specific diagnostic kit for early-stage liver fluke disease in sheep as described in claim 1, characterized in that, In step S1, the concentration of recombinant protein FhCatL6 after dilution with coating solution is 0.1875 μg / 100 μL; the dilution of the serum to be tested is 1:3200.
3. The early specific diagnostic kit for sheep liver fluke disease as described in claim 1, characterized in that, In step S2, the sealing liquid is 5% skim milk.
4. The specific diagnostic kit for early-stage liver fluke disease in sheep as described in claim 1, characterized in that, In step S4, the enzyme-labeled secondary antibody is diluted 7000 times.
5. The specific diagnostic kit for early-stage liver fluke disease in sheep as described in claim 1, characterized in that, In step S5, the color development time is 15 minutes.
6. The specific diagnostic kit for early-stage liver fluke disease in sheep as described in claim 1, characterized in that, In step S6, if OD450nm ≥ 0.299956, it is considered positive; if OD450nm ≤ 0.251826, it is considered negative; if 0.251826 < OD450nm < 0.299956, it is considered a suspected positive, and the sample is retested.
7. A specific diagnostic kit for early-stage liver fluke disease in sheep as described in any one of claims 1-6, characterized in that, Used for the early specific diagnosis of trematode disease.