猪带绦虫丝氨酸蛋白酶抑制剂Tsserpin570的克隆表达与酶活性分析  

Cloning,expression and enzyme activity analysis on serine protease inhibitor 570 of Taenia solium

作　　者：毕研丽[1] 刘仲藜[1,2] 郭爱疆[1] 张少华[1] 王帅[1] 才学鹏[1,3] BI Yan-li;LIU Zhong-li;GUO Ai-jiang;ZHANG Shao-hua;WANG Shuai;CAI Xue-peng(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]中国兽医药品监察所,北京100081

出　　处：《中国兽医科学》2021年第5期545-551,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31802179,31772726);国家重点研发计划项目(2017YFD0501303)。

摘　　要：为阐明猪带绦虫(Taenia solium)丝氨酸蛋白酶抑制剂(Tsserpin570)对蛋白酶的抑制作用,通过RT-PCR从猪带绦虫成虫cDNA扩增获得Tsserpin570(TsM000807300)的完整CDS序列,并构建了pColdTsserpin570原核表达载体,利用发色底物法检测可溶性重组蛋白Tsserpin570对蛋白酶活性的抑制作用。结果显示:RT-PCR扩增获得的Tsserpin570基因片段长1 206 bp,其蛋白的分子质量为46 ku,含有serpin家族特有的反应中心环及DEEGAE和FIVDHPFLFFI家族保守序列。qRT-PCR结果显示,Tsserpin570基因在猪带绦虫成虫和囊尾蚴阶段均有表达,且在成虫的表达量显著高于囊尾蚴的。pCold-Tsserpin570原核表达载体经IPTG诱导表达后获得可溶性重组蛋白Tsserpin570,可被猪囊尾蚴阳性血清识别。基于发色底物法检测Tsserpin570对牛α-胰糜蛋白酶、猪胰弹性蛋白酶、猪胰蛋白酶、人白细胞组织蛋白酶G、人血浆凝血酶、猪胃蛋白酶和木瓜蛋白酶等蛋白酶的活性抑制作用发现,其对牛α-胰糜蛋白酶、胰蛋白酶及弹性蛋白酶的活性均有明显的抑制作用。以上结果提示,Tsserpin570对多种丝氨酸蛋白酶均具有较强的抑制作用。In order to clarify the protease inhibitory activity of the serine protease inhibitor of Taenia solium(Tsserpin570),the coding sequence of Tsserpin570 gene(Ts M000807300) was amplified from T.solium adults by RT-PCR for constructing the prokaryotic expression vector p Cold-Tsserpin570.Additionally,based on the chromogenic substrate method,the inhibitory effects of soluble fusion protein Tsserpin570 on serine proteases was detected.The results of RT-PCR showed that Tsserpin570 gene was1 206 bp in length and 46 ku in molecular weight of protein,which contains the serpin family specific reactive center loop and conservation sequences of DEEGAE and FIVDHPFLFFI.The results of q RT-PCR revealed that expression of Tsserpin570 in adult stage significantly higher than that in larval stage.After induced by IPTG,the soluble fusion protein Tsserpin570 was induced,and which could react with the positive serum from the pig infected with T.solium.By analysis the results of inhibitory effects of recombinant protein Tsserpin570 on alpha-chymotrypsin from bovine pancreas,elastase from porcine pancreas and trypsin from porcine pancreas,cathepsin G from human leukocytes,thrombin from human plasma,pepsin from porcine gastric mucosa and papain from papaya latex,the Tsserpin570 at 12 μg/m L showed significant inhibitory activity against α-chymotrypsin,trypsin and pancreatic elastase.Above all results suggest that Tsserpin570 shows strong inhibitory activity against multiple serine proteases.

关 键 词：猪带绦虫 丝氨酸蛋白酶抑制剂 Tsserpin570 原核表达 酶活性 

分 类 号：S852.734[农业科学—基础兽医学]