Yang Qinggui, Zhou Dan, Sun Lixin, Zhang Donghui, Qian Jin, Xiong Chunrong, Sun Yan, Ma Lei, Zhu Changliang
Department of Pathogen Biology, Jiangsu Province Key Laboratory of Modern Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China.
Parasitol Res. 2008 Aug;103(3):507-16. doi: 10.1007/s00436-008-0997-1. Epub 2008 May 22.
Two deltamethrin resistance-associated serine protease genes (NYD-tr and NYD-ch) were isolated from Culex pipiens pallens in our previous study. To study the function of NYD-Tr and NYD-Ch in the metabolism of deltamethrin, we constructed the recombinant plasmid pET32a(+)/NYD-tr and pET32a(+)/NYD-ch with a 6x histidine tag. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses of the recombinant proteins revealed that the molecular weights of NYD-Tr and NYD-Ch are 42 and 50 kDa. Enzyme activity assay indicated that the recombinant NYD-Tr and NYD-Ch had the corresponding features of trypsin and chymotrypsin. Using BApNA as the substrate, NYD-Tr gave optimal activity between pH 9.0 and 10.5, while NYD-Ch was optimally active over the range of pH 8.0-11.0 using the S(Ala)(2)ProPhe-pNA as the substrate. Then, we investigated the metabolism of deltamethrin by NYD-Tr and NYD-Ch. Our results showed that NYD-Tr and NYD-Ch could hydrolyze deltamethrin. The acute oral toxicity of the metabolite to Wistar rats was much lower than deltamethrin.
在我们之前的研究中，从淡色库蚊中分离出了两个与溴氰菊酯抗性相关的丝氨酸蛋白酶基因（NYD-tr和NYD-ch）。为了研究NYD-Tr和NYD-Ch在溴氰菊酯代谢中的功能，我们构建了带有6x组氨酸标签的重组质粒pET32a(+)/NYD-tr和pET32a(+)/NYD-ch。对重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质免疫印迹分析显示，NYD-Tr和NYD-Ch的分子量分别为42 kDa和50 kDa。酶活性测定表明，重组NYD-Tr和NYD-Ch具有胰蛋白酶和糜蛋白酶的相应特征。以BApNA为底物时，NYD-Tr在pH 9.0至10.5之间具有最佳活性，而以S(Ala)(2)ProPhe-pNA为底物时，NYD-Ch在pH 8.0 - 11.0范围内具有最佳活性。然后，我们研究了NYD-Tr和NYD-Ch对溴氰菊酯的代谢情况。我们的结果表明，NYD-Tr和NYD-Ch可以水解溴氰菊酯。该代谢产物对Wistar大鼠的急性经口毒性远低于溴氰菊酯。
