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Anti-African swine fever virus p54 protein monoclonal antibody, preparation method and application
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A technology of African swine fever virus and monoclonal antibody, applied in the field of genetic engineering
Active Publication Date: 2022-07-12
河南中泽生物工程有限公司
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However, there is no specific monoclonal antibody against NTD in the prior art and reports
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Embodiment 1
[0048] Example 1 Selection and preparation of immunogens
[0049] The viral structural protein p54 is encoded by the E183L gene and is located in the inner membrane of the viral envelope. The N-terminus of p54 is relatively conserved among virus strains and contains a unique cysteine. It is used in the process of virus morphogenesis, activity, and invasion. have an important role. In the preliminary test, a peptide was synthesized based on the NTD sequence of the p54 protein to simulate NTD. The peptide has a good reaction with the serum of clinically recovered pigs infected with ASFV, which proves that the NTD region of the p54 protein contains antigenic determinants, which can induce the body to produce antibodies against NTD. Therefore, the present invention uses p54 protein NTD coupled to carrier protein BSA as an immunogen. Specifically, the preparation of the immunogen includes the following steps:
[0050] (1) With reference to the p54 protein sequence of the ASFV str...
Embodiment 2
[0053] Example 2 Preparation of monoclonal antibodies
[0054] 1. Animal Immunization
[0055] (1) Freund's complete adjuvant is added to the immunogen NTD-BSA, and it is used for the first immunization after being emulsified;
[0056] (2) 2 female BALB / c mice aged 4-8 weeks were immunized by subcutaneous injection at multiple points on the back, and the immunization dose was 10 μg / mice;
[0057] (3) BALB / c mice were boosted with the same method and dose after emulsification with incomplete Freund's adjuvant and immunizing antigen every 3 weeks;
[0058] (4) After three weeks, the tailvein blood was collected to measure the specific antibodytiter against NTD, and the mice with higher titer were selected ( figure 1 ), 3 to 4 days before cell fusion, BALB / c mice were super-immunized with immunogen without adjuvant by tailvein injection, and the immunization dose was 20 μg / mice.
[0078] (1) Take 5ml of monoclonal antibody ascites, add 5ml of PBS buffer, and then add 2.5ml of saturated ammoniumsulfate solution dropwise to make a final concentration of 20% ammoniumsulfate solution, stir while adding, fully mix, and let stand 30min.
[0079] (2) 8000r / min, centrifuge for 20min, discard the precipitate to remove fibrin.
[0080] (3) Add 12.5 ml of saturated ammonium sulfate solution to the supernatant, mix well, and let stand for 30 minutes.
[0081] (4) Centrifuge at 8000 r / min for 20 min, and discard the supernatant.
[0082] (5) Add 10 ml of PBS buffer to the precipitate to dissolve it, then add 5 ml of saturated ammonium sulfate solution to make it a 33% ammonium sulfate solution, mix well, and let it stand for 30 minutes.
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Abstract
The invention relates to an anti-African swine fevervirus p54 proteinmonoclonalantibody, a preparation method and an application. The invention designs and synthesizes a polypeptide capable of simulating the NTD of the conservative p54 protein, and couples it with BSA as an immunogen, and immunizes BALB / c mice by an immunological method to prepare an anti-ASFVp54 proteinmonoclonalantibody, and the antibody can specifically It recognizes and binds to the N-terminal region of p54 protein, and this recognition region (aa 1-29) is different from existing commercial monoclonal antibodies. The present invention provides the heavy chain variable region and light chain variable region gene sequences of the above-mentioned antibodies. On this basis, conventional genetic engineering or protein engineering methods can be used to obtain the monoclonal antibody of the present invention. The monoclonal antibody has strong specificity and high sensitivity, and can specifically react with ASFV HLJ / 18 strains of viruses, but does not react with porcine-derived viruses such as CSFV, PCV2, PRRSV, PEDV and PRV. The research on the pathogenesis and clinical detection of ASFV pathogens have laid the foundation.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-African swine fevervirus p54 protein monoclonal antibody, a preparation method and an application. Background technique [0002] African swine fevervirus (ASFV), a large and complex DNA virus, is the causative agent of African swine fever (ASF) and the only known member of the genus Asfarviridae. ASF is a highly contagious hemorrhagic disease in domestic pigs with up to 100% morbidity and mortality. ASFV was first discovered in Kenya in 1921, nearly a century ago. Since its spread to Georgia in 2007, ASFV has spread rapidly across multiple countries in Africa, Asia, and Europe, with recurring outbreaks in these regions. At the same time, ASFV has multiple natural hosts and reservoirs, which further increases the potential threat of ASF. Given the enormous risk and threat of this transboundary animal disease, it has been included in the Terrest...
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