猪塞尼卡谷病毒单克隆抗体的制备及鉴定

doi:10.16431/j.cnki.1671-7236.2022.01.033

Abstract

Abstract: [Objective] The aim of this study was to purify SVV-CH-HB2016 strain and prepare monoclonal antibodies of Seneca Valley virus (SVV) specific to the structural proteins VP1, VP2 and VP3. [Method] SVV-CH-HB2016 virus concentrate was purified using sucrose density gradient centrifugation and used as antigen to immunize BALB/c mice. Immune spleen cells and myeloma cells (SP2/0) were used in cell fusion experiment. Both indirect immunofluorescence assay (IFA) and indirect ELISA were conducted to screen positive cell lines which could secrete hybridoma cell lines specific to structural proteins. The reactivity of monoclonal antibody with recombinant and natural structural proteins was tested using Western blotting and IFA, the neutralization and adsorption effects of monoclonal antibody were also tested. Then plaque assay and Real-time PCR were used to explore the effect of neutralized monoclonal antibody on the absorption process of SVV-CH-HB2016 strain. Finally, the antibody addition test was used to analyze the epitope of the monoclonal antibody. [Result] Both the purity and concentration of SVV-CH-HB2016 strain structure protein could keep a higher level when sucrose gradient was between 5%-45% (W/V). The serum antibody titer of the immunized mice reached 1:12 800, and successfully prepared 17 hybridoma cell lines capable of stably secreting specific monoclonal antibody. It was found that 14 monoclonal antibody strains could react with recombinant structural proteins in Western blotting, and 17 monoclonal antibody strains could react with the virus in IFA. Strains 2G6, 4A3 and 4C11 not only had a significant neutralizing and protective effect on BHK-21 cells infected by SVV-CH-HB2016 strain but also could effectively inhibit the adsorption of SVV-CH-HB2016 strain on 293T cells. It was found that in addition to 1F5 and 2E1, 4B8 and 4F11, the other 10 monoclonal antibody strains were specific to different epitopes. [Conclusion] In this study, the purification method of SVV was preliminarily established, and 17 monoclonal antibody strains specific to SVV-CH-HB2016 strain were prepared, which laid a foundation for the further development of inactivated SVV vaccine, the establishment of ELISA detection method and the identification of protective antigen epitope in the future.

Key words: Seneca Valley virus (SVV); virus particles; monoclonal antibody; neutralization protection; antigenic epitope

CLC Number:

S852.65⁺1

MO Hongfang, JIE Kai, CHEN Xin, WEN Wei, LIU Wenqiang, CHEN Huanchun, QIAN Ping, LI Xiangmin. Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 307-317.

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Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus

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