绿色荧光蛋白的真核表达及其单克隆抗体制备

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

【目的】构建表达绿色荧光蛋白(green fluorescent protein,GFP)的重组猪痘病毒(recombinant Swinepox virus,rSWPV),制备抗GFP单克隆抗体。【方法】首先合成3′-端含His标签的增强型绿色荧光蛋白(EGFP)序列EGFP-His,用双酶切方法将其插入到基础质粒载体pSW中,构建重组转移载体pSW-EGFP-His;采用脂质体转染的方法使该载体与SWPV(SWPV-JX20G株)同源重组,经蚀斑纯化获得rSWPV-EGFP-His。重组病毒进行PCR和SDS-PAGE鉴定,扩增并纯化EGFP-His蛋白免疫BALB/c小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选分泌抗GFP特异性抗体的杂交瘤细胞,制备腹水,对抗GFP单克隆抗体进行效价及特异性鉴定。【结果】PCR和SDS-PAGE结果显示,成功构建并纯化到rSWPV-EGFP-His,该病毒感染PK15细胞可稳定表达EGFP-His蛋白,蛋白大小约27 ku,为可溶性表达,镍柱纯化的EGFP-His蛋白溶液呈明显的绿色。EGFP-His蛋白免疫BALB/c小鼠,免疫小鼠血清抗体效价高达1∶256000。采用杂交瘤技术制备了9株能稳定分泌抗EGFP-His单克隆抗体的杂交瘤细胞株,间接ELISA和Western blotting结果显示,9株单克隆抗体中的7株针对GFP的线性表位,另外2株单克隆抗体针对GFP的构象表位。【结论】本研究成功制备了EGFP-His蛋白和9株抗EGFP-His的单克隆抗体,为后续建立GFP的免疫学检测方法提供了必备材料。

【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∶256000.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.