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Anti-African swine fever virus p54 protein monoclonal antibody and preparation method and application thereof

A technology of African swine fever virus and monoclonal antibody, applied in the field of genetic engineering

Active Publication Date: 2021-03-26
河南中泽生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no specific monoclonal antibody against NTD in the prior art and reports

Method used

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  • Anti-African swine fever virus p54 protein monoclonal antibody and preparation method and application thereof
  • Anti-African swine fever virus p54 protein monoclonal antibody and preparation method and application thereof
  • Anti-African swine fever virus p54 protein monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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 previous experiment, a peptide was synthesized based on the NTD sequence of the p54 protein to simulate NTD. The peptide had a good reaction with the serum of ASFV-infected clinically recovered pigs, which proved that the NTD region of the p54 protein contained antigenic determinants, which could 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 ASFV strain...

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 tail vein blood was collected to measure the specific antibody titer 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 tail vein injection, and the immunization dose was 20 μg / mice.

[0059] 2. Cell fusion and monoclonal antibody preparation

[0060] The spleen cells of the im...

Embodiment 3

[0076] Example 3 Purification and identification of antibodies

[0077] 1. Purify antibody by saturated ammonium sulfate precipitation method, the operation method is as follows:

[0078] (1) Take 5ml of monoclonal antibody ascites, add 5ml of PBS buffer, and then add 2.5ml of saturated ammonium sulfate solution dropwise to make a final concentration of 20% ammonium sulfate solution, stir while adding, mix well, 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.

[0083] (6) Centrifuge at 8000 r / min for 20 mi...

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Abstract

The invention relates to an anti-African swine fever virus p54 protein monoclonal antibody and a preparation method and application thereof. According to the invention, polypeptide capable of simulating conservative p54 protein NTD is designed and synthesized, and the polypeptide is coupled with BSA to serve as immunogen, an anti-ASFVp54 protein monoclonal antibody is prepared by immunizing a BALB / c mouse through an immunological method, the obtained antibody can specifically identify and bind to an N-terminal region of p54 protein, and the recognition region (aa 1-29) is different from the existing commercial monoclonal antibody. The invention provides gene sequences of a heavy chain variable region and a light chain variable region of the antibody. On this basis, the monoclonal antibodyof the invention can be obtained by adopting a conventional genetic engineering method or a protein engineering method. The monoclonal antibody is high in specificity and high in sensitivity, capableof specifically reacting with the ASFV HLJ / 18 virus strain, without reaction with CSFV, PCV2, PRRSV, PEDV, PRV and other swine viruses, thus laying a foundation for the research of ASFV etiology and pathogenesis and the clinical detection research of ASFV pathogens.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-African swine fever virus p54 protein monoclonal antibody, a preparation method and an application. Background technique [0002] African swine fever virus (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 morbidity and mortality of up to 100%. ASFV was first discovered in Kenya in 1921, almost a century ago. Since spreading to Georgia in 2007, ASFV has rapidly spread throughout several countries in Africa, Asia, and Europe, where it has recurred outbreaks. At the same time, ASFV has multiple natural hosts and reservoirs, which further increases the potential threat of ASF. In view of the great risk and threat of this transboundary animal disease, it has been included in the Terrestria...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13G01N33/577G01N33/569
CPCC07K16/081G01N33/577G01N33/56983C07K2317/565C07K2317/56C07K2317/33G01N2333/01G01N2469/10Y02A50/30
Inventor 陈玉梅王爱萍蒋敏赵建国冯景石海宁赵孟孟
Owner 河南中泽生物工程有限公司
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