重组大鼠TSHα亚基在大肠杆菌中表达的实验研究

Preparation of GST tagged recombinant rat TSHα Chain in E.coli

目的:通过构建重组大鼠TSHα链原核表达系统及表达产物的纯化工作,获得重组GST-rrTSHα融合蛋白,以备作为免疫原制备抗大鼠TSHα链的抗体。方法:提取大鼠垂体组织总RNA,RT-PCR扩增大鼠TSHα链编码序列,构建pGEX-3X/TSHα融合表达质粒转化入大肠杆菌中进行诱导表达。亲和层析纯化重组蛋白并以SDS-PAGE及Western Blot进行鉴定。结果:DNA序列分析表明重组pGEX-3X/TSHα质粒含有读码框正确的TSHα链编码序列,纯化产物的SDS-PAGE及Western Blot结果表明所获得的目的蛋白与预期分子量相符且可与抗GST抗体发生免疫识别。结论:成功获得重组GST-rrTSHα融合蛋白,为下一步的抗体制备奠定了基础。

Objective： To obtain recombinant GST-rrTSHoL protein by the construction of expression system of rat TSHα chain in Escherichia Coil （E.coli）. Methods： The cDNA sequence encoding rat TSHα chain was acquired by RT-PCR from the pituitary total RNA, and the expressing plasmid pGEX-3X/TSHα was constructed to be expressed in E.coli. The purification of the recombinant protein was also being accomplished via affinity chromatography. Results： Both the recombinant cloning plasmid pGEM-T/TSHα and the expressing plasmid pGEX-3X/TSHα were confirmed by DNA sequencing of containing correct TSHα open reading frame. SDS-PAGE and Western Blotting approved proper molecular weight of the recombinant GST tagged protein and its immune reactivity with anti-GST antibody. Conclusion： The recombinant protein GST-rrTSHα is successfully obtained and can be used in further experiment for generating anti rat TSHα antibody.