绿色荧光蛋白的真核表达及其单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.01.035

Abstract

Abstract: [Objective] The aim of the experiment was to construct a recombinant Swinepox virus (rSWPV) expressing green fluorescent protein (GFP) and prepare monoclonal antibody against GFP. [Method] Firstly, the enhanced green fluorescent protein (EGFP) sequence EGFP-His containing His-tag at 3'-end was synthesized and inserted into the basic plasmid vector pSW by double digestion to construct the recombinant transfer vector pSW-EGFP-His. The vector was homologously recombined with SWPV(SWPV-JX20G strain) by liposome transfection, and rSWPV-EGFP-His was obtained by plaque purification. The recombinant virus was identified by PCR and SDS-PAGE, and EGFP-His protein was amplified and purified to immunize BALB/c mice. The spleen cells of mice were fused with SP2/0 cells to screen hybridoma cells secreting anti-GFP specific antibodies, and ascites was prepared. The titer and specificity of anti-GFP monoclonal antibodies were identified. [Result] PCR and SDS-PAGE results showed that rSWPV-EGFP-His was successfully constructed and purified. The virus infected PK15 cells stably expressed EGFP-His protein, which was about 27 ku, and was soluble. The EGFP-His protein purified by Ni-agarose showed obvious green. The serum antibody titer of BALB/c mice immunized with EGFP-His protein was 1:256 000. Hybridoma techniques were employed to produce monoclonal antibodies. Nine hybridoma cell lines stably secreting monoclonal antibodies against EGFP-His were prepared by hybridoma technique. Indirect ELISA and Western blotting results showed that seven of the nine monoclonal antibodies were linear epitopes for GFP, and the other two were conformational epitopes. [Conclusion] In this study, EGFP-His protein and nine monoclonal antibodies against EGFP-His were successfully prepared, which provided necessary materials for the subsequent establishment of immunological detection methods for GFP.

Key words: green fluorescent protein (GFP); Swinepox virus vector; eukaryotic expression; monoclonal antibody

CLC Number:

S852.65⁺1

HU Huanyi, WANG Jianzhong, LIU Changjin, LIN Min, LIU Xiaolan, WEI Huangsiwu, DENG Shunzhou. Eukaryotic Expression of Green Fluorescent Protein and Preparation of Its Monoclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 328-337.

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Eukaryotic Expression of Green Fluorescent Protein and Preparation of Its Monoclonal Antibody

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