牛蛙核糖核酸酶在大肠杆菌中的高效表达及其初步纯化

HIGH EXPRESSION AND PURIFICATION OF RANA CATESBEIANA RIBONUCLEASE IN E.COLI

目的:利用大肠杆菌系统表达重组RC蛳RNase融合蛋白,并对其表达产物进行初步纯化。方法:以PCR方法扩增出RC蛳RNase基因,插入到融合蛋白原核表达载体PET32a中,构建重组表达载体PET蛳RC蛳RNase,转化大肠杆菌E.coliBL21(DE3)plyS,利用异丙基硫代蛳β蛳D蛳半乳糖苷(IPTG)诱导表达。工程菌经超声碎菌,离心后,采用Ni亲合层析法在变性条件下进行初步纯化。结果:经IPTG诱导后,SDS蛳PAGE和免疫印迹分析显示,工程菌在相对分子质量为3.1×104处出现一条新生蛋白带,目的蛋白占菌体总蛋白的34%,主要以包涵体形式存在。所得包涵体经纯化,纯度可达90%以上。结论:重组融合蛋白RC蛳RNase在大肠杆菌中获得成功表达,纯化效果令人满意,为进一步研究其生物学特性和功能奠定了良好的基础。

Objective: To express the recombinant RC-RNase fusion protein in E.coli and purify its expression product. Methods: RC-RNase gene was amplified by PCR and ligated with prokaryotic expression vector PET32a. E.coli BL21(DE3)plyS transformed with the recombinant plasmid PET-RC-RNase was induced to express by IPTG. The E.coli was lysed by ultrasonication and contrifugation. The sediment of the lysate was purified by Ni-affinity chromatography under denature conditions. Results: SDS-PAGE and Western-blot analyses suggested that a new anticipated protein band appeared after induced by IPTG. Its molecular weight was 31KD and consisted of 34 % of the total bacterial proteins. The expression product existed mainly in a form of inclusion body. Purity of the recombinant RC- RNase fusion protein was above 90 % after being purified. Conclusion: The recombinant RC-RNase fusion protein was successfully expressed in E.coli. The effect of purification was satisfying.