中华鲟半胱氨酸蛋白酶抑制剂C基因的原核表达

Expression of Acipenser sinensis cystatin C in prokaryote

为了研究鱼类半胱氨酸蛋白酶抑制剂(cystatin)的功能并探索其在水产品加工和病害防治中的应用潜力,将改造后的中华鲟(Acipensersinensis)cystatinCcDNA亚克隆到原核表达载体pBV220,构建表达cystatinC的大肠杆菌基因工程菌。该工程菌经温度诱导、SDS PAGE检测,在约12.4kD处有一特异蛋白带,该特异蛋白的含量约为菌体总蛋白的25%。重组半胱氨酸蛋白酶抑制剂经洗涤、溶解、透析、复性后纯度为85%,实现了中华鲟cystatinC在大肠杆菌中的高效表达。木瓜蛋白酶活性抑制实验结果表明该重组cystatinC具有明显的酶活抑制作用。

In order to explore the functions of fish cystatin and the potential values in the fish disease prevention and cure, as well as seafood process industry, polymerase chain reaction (PCR) technique was used to modify the cDNA of Chinese sturgeon (Acipenser sinensis) cystatin mature peptide, The cDNA was subcloned into expression vector pBV220,then a cystatin recombinant expression bacteria was constructed. After temperature inducement and SDSPAGE analysis, the recombinant expression bacteria produced a special protein about 12.4 kD in molecular weight. The proportion of recombinant protein in total bacterial protein was about 25%.The purity of the recombinant cystatin, which was washed,dissolved, dialysed and renatured,was about 85%. The inhibitory activity of recombinant cystatin could be measured by inhibiting the protease activity of papain.The inhibitory activity of recombinant cystatin was 48U·μg-1.