家蝇半胱氨酸蛋白酶抑制剂基因MdCPI的克隆、表达及活性检测  

Cloning,expression and activity detection of cysteine protease inhibitor gene MdCPI in Musca domestica

作　　者：柳峰松[1] 董晓寅[1] 张瑞英[1] 唐婷[1] 

机构地区：[1]河北大学生命科学学院,河北保定071002

出　　处：《昆虫学报》2010年第6期611-617,共7页Acta Entomologica Sinica

基　　金：河北省自然科学基金项目(C2008000596);河北大学自然科学基金项目(2010001)

摘　　要：半胱氨酸蛋白酶抑制剂是具有抑制半胱氨酸蛋白酶活性的一类蛋白超家族。本研究根据EST序列信息,通过RACE技术克隆得到1条家蝇Musca domestica半胱氨酸蛋白酶抑制剂基因MdCPI,该基因含有1个357bp开放阅读框,编码118个氨基酸残基,推导的多肽N端17个氨基酸残基为信号肽序列。同源分析表明,MdCPI氨基酸序列与红尾肉蝇Sarcophaga crassipalpis的CPI相似性最高(identity=51%)。以邻接法(NJ)构建的系统树表明,家蝇与其他双翅目昆虫CPI起源于共同的祖先,属于I25A型蛋白家族。为了解家蝇CPI对半胱氨酸蛋白酶的抑制活性,构建pET-17b-MdCPI表达载体,并转入大肠杆菌Escherichia coli BL21(DE3)进行重组表达。研究发现1μg重组家蝇CPI能够抑制约14μg木瓜蛋白酶的水解活性。结果表明MdCPI确属CPI家族,可能同其他家族成员具有相似的功能,参与免疫及生理调控。这些结果为研究MdCPI在家蝇体内作用机制奠定了基础。The superfamily of cysteine protease inhibitor consists of proteins which can inhibit the hydrolytic activity of cysteine proteinases.In this study,MdCPI,a gene of cysteine protease inhibitor was cloned from housefly （Musca domestica） by RACE based on dbEST information.It contains a 357 bp open reading frame encoding 118 amino acid residues.The deduced peptide contains a putative signal peptide of 17 amino acids.The amino acid sequence of MdCPI showed the highest homology with that of Sarcophaga crassipalpis （identity=51%） in the homologous assay.The phylogenetic tree indicates that the CPIs from housefly and other dipterans have descended from a single common ancestor and belong to I25A family.To understand the inhibitory activity of MdCPI to cysteine protease,a prokaryotic expression vector pET-17b-MdCPI was constructed and then expressed in Escherichia coli BL21.The results of activity detection showed that about 1 μg recombinant MdCPI could inhibit the activity of 14 μg papain.The results suggest that MdCPI is the member of the CPI supperfamily,and may have similar functions and be involved in immunological and physiological regulation.The successful cloning,expression and activity detection of MdCPI provided a basis for the further study on its function.

关 键 词：家蝇 半胱氨酸蛋白酶抑制剂 克隆 重组表达 抑制活性 

分 类 号：Q966[生物学—昆虫学]