家蝇丝氨酸蛋白酶抑制剂Serpin15的克隆表达及重组蛋白的体外活性分析

Cloning and expression of Serpin15,a serine protease inhibitor of Musca domestica,and analysis its recombinant protein activity in vitro

为探明家蝇重组Serpin15蛋白的体外活性和酶稳定性,本文对家蝇Serpin15基因进行了生物信息学分析和克隆表达,并对纯化后的家蝇重组Serpin15蛋白进行了酶特性的研究。结果显示:家蝇Serpin15基因ORF框全长为1353 bp,编码450个氨基酸,理论分子量为51076.63 Da,有信号肽和一个标志抑制活性的功能结构域RCL反应环;成功构建原核表达载体pET-28a(+)-Serpin15,经诱导表达和纯化获得家蝇重组Serpin15蛋白;重组蛋白酶特性研究发现,家蝇重组Serpin15蛋白对胰蛋白酶有极显著的抑制作用,此外,将重组Serpin15蛋白经20~60℃热处理15 min,或经pH 6~10过夜处理,或经50℃和pH 8处理后室温存放15~90 min,重组蛋白的胰蛋白酶抑制活性均仍大于70%。研究结果为家蝇Serpin15蛋白的免疫功能研究奠定了重要实验基础,也为有害昆虫杀虫剂的研发提供思路。

In order to explore the in vitro activity and enzyme stability of recombinant Serpin15 protein in Musca domestica,bioinformatics analysis,cloning and expression of Serpin15 gene of M.domestica,as well as the enzyme characteristics of recombinant Serpin15 protein were carried out.The results showed that the total length of Open Reading Frame(ORF)was 1353 bp,encoding 450 amino acids with the theoretical molecular weight of 51076.63 Da,a signal peptide and a reactive center loop(Serpin functional domain).The recombinant expression plasmids pET-28a(+)-Serpin15 was constructed successfully,and the recombinant Serpin15 protein was obtained by inducing expression and purification.The study of recombinant protease properties found that the recombinant Serpin15 protein had a significant inhibitory effect on trypsin.In addition,after heat-treated at 20~60℃for 15 minutes,or overnight treatment at pH 6~10,or storage at room temperature for 15~90 min after treatment at 50℃and pH8,the trypsin inhibitory activity of recombinant Serpin15 protein remained greater than 70%.Overall,these results laid an important foundation for future in-depth analysis the immune system of M.domestica,and also provided ideas for the development of harmful insect pesticides.