中华鲟半胱氨酸蛋白酶抑制剂C基因的原核表达

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(Acipenser sinensis)cystatin C cDNA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。

Expression of Acipenser sinensis cystatin C in prokaryote

In order to explore the functions of f ish cystatin and the potential values in the f ish 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 cystat in 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....g- 1.