F10蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of F10 and preparation of its polyclonal antibody

目的:表达F10蛋白,并制备兔抗F10多克隆抗体。方法:利用PCR方法扩增F10基因片段,经BamH I和EcoR I酶切后连接入pET-GST原核表达载体,构建的pET-GST/F10融合重组表达质粒转化大肠杆菌BL21,经IPTG诱导表达蛋白,并以亲和层析的方法进行纯化。表达产物用SDS-PAGE电泳和Western blot进行分析鉴定。以纯化的F10蛋白免疫新西兰大白兔,制备兔抗F10的多克隆抗体,并以ELISA法检测抗体效价。结果:经酶切和核酸序列分析证实重组质粒包含有正确编码的F10读码框。SDS-PAGE电泳分析显示pET-GST/F10诱导后表达一相对分子质量(Mr)约为61 000的融合蛋白,与预期结果相符。目的蛋白纯化后的纯度达90%以上,Western blot证实该蛋白是GST/F10的融合蛋白。将纯化的GST/F10融合蛋白免疫家兔,得到的兔抗F10抗体效价达1∶20 000。结论:成功构建了人F10基因原核表达载体,并获得了高纯度的F10重组蛋白及兔抗F10抗体,为下一步研究F10基因功能奠定了实验基础。

AIM： To induce the expression of F10 in E. coil and prepare the rabbit polyclonal antibody against it. METHODS： The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E. coil BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA. RESULTS： Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDSPAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Westem blot confirmed the recombinant protein was GST/ F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1：20 000. CONCLUSION： F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high Uter and will facilitate the study of the biological function of F10.