犬表皮细胞生长因子的原核表达和纯化及其活性鉴定

Prokaryotic expression,purification and bioactivity identification of recombinant canine epidermal growth factor

为获得有生物活性的犬表皮生长因子(cEGF),设计了4条引物,用SOE-PCR合成cEGF基因,将cEGF基因克隆至表达载体pET-24a,构建融合表达质粒,转化至E.coli BL21,IPTG诱导表达,对表达得到的重组蛋白进行纯化,MTT法测定重组蛋白的生物活性。结果显示,SOE-PCR合成得到的cEGF基因大小为156bp,编码52个氨基酸。大部分表达为包涵体,通过变性、纯化、复性的过程,每1L菌液得到重组犬rcEGF 16.1mg,经MTT法检测,rcEGF具有生物活性。结果表明,获得了具有生物活性的rcEGF。

To prepare for canine epidermal growth factor（cEGF） with biological activities, a mers were designed,cEGF gene was amplified by struct a fusion expression plasmid pET-24a-cEGF cEGF was expressed by IPTG induction and purif sessed by MTT assay. The cEGF gene amplified pressed recombinant cEGF proteins were mainly i fication and renaturation, 16.1 mg rcEGF was obt SOE-PCR,and then cloned into pET-24a ,which was transformed into Escherichia c ied subsequently. The biological activity of was 156 bp n a form of ained from the rcEGF had biological activity. The results suggested that in length, encoding 52 am inclusion bodies. After den 1 L bacterium liquid. MTT cEGF with biological activi vect fter 4 pri or to con oli BL21. The cEGF was as- ino acids. The ex aturalization, puri- assay showed that ties was obtained.