蚊抗药性相关糜蛋白酶基因的表达、纯化及活性分析  

Expression,purification and enzymatic activity assay of recombinant protein,NYD-Ch associated with insecticide resistance from Culex pipiens pallens

作　　者：孙艳[1] 马磊[1] 钱瑾[1] 孙静[1] 孙立新[1] 

机构地区：[1]南京医科大学基础医学院病原生物学系,江苏南京210029

出　　处：《中国病原生物学杂志》2006年第6期412-415,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金资助项目(No.30371255);江苏省自然科学基金资助项目(No.BK2004147)

摘　　要：目的表达、纯化淡色库蚊溴氰菊酯抗性相关糜蛋白酶（NYD-Ch）基因并进行酶活性分析。方法应用PCR和基因重组技术，构建原核表达载体，表达目的蛋白；用酶促底物法测定表达蛋白NYDCh的酶学活性和最适pH值。结果成功构建重组表达载体pET32a（＋）／NYD-Ch，并转化到感受态菌E．coli BL21（DE3）中。SDS-PAGE和Western blot显示，目的蛋白分子质量与理论值相符。NYD-Ch最大活力通过S（Ala）2ProPhe-pNA获得。NYD-Ch活力随pH的增高而增大，在pH8．01～11．0的范围内较高，pH10．0时达最高。抑制剂PMSF、SBTI、TPCK对NYD-Ch酶活力均有明显的抑制作用。结论成功表达了NYD-Ch蛋白，并分析了其酶学活性。本结果为深入研究NYD-Ch与杀虫剂抗性关系奠定基础。Objective To perform prokaryotic expression, purification and motrypsin associated with insecticide resistance from Culex pilpiens pallens. enzymatic activity assay of NYD-Ch, a chy Methods By PCR and gene reconstructed technology, the recombinant plasmid pET-32a（＋）/NYD-Ch was constructed and expressed. Then the expressed protein was purified through Ni^＋ column chromatography. Enzymatic activity was assayed by different substrates and pH. Re-sults The recombinant plasmid was transformed in E. coli BL21 and expressed NYD-Ch protein. A tape of protein about 48.5 ku could be viewed according to SDS-PAGE, which was coincident with the designed protein. The result was confirmed by Western blot. Enzymatic activity assay demonstrated that the expressed product had the common features of chymotrypsin： NYD-Ch was able to hydrolyze the synthetic substrate S（Ala）2 ProPhe-pNA and it was strongly inhibited by PMSF, SBTI and TPCK. The activity of NYD-Ch had strongly alkaline pH optima, range from pH 8.0 to 11.0. Optimal activity was observed at pH 10.0. Conclusion Successful prokaryotic expression, purification and enzymatic activity assay of NYD-Ch should greatly facilitate attempts to learn its function of insecticide metabolism.

关 键 词：糜蛋白酶 原核表达 纯化 酶活性 

分 类 号：R384.1[医药卫生—医学寄生虫学]