家蝇金属硫蛋白基因的克隆、原核表达及活性检测  

Cloning,prokaryotic expression and activity detection of the metallothionein gene in Musca domestica(Diptera:Muscidae)

作　　者：张迪[1] 任国栋[1] 唐婷[1] 董晓寅[1] 柳峰松[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4期379-384,共6页Acta Entomologica Sinica

基　　金：河北省自然科学基金项目(C2008000596);国家自然科学基金重点项目(30630010)

摘　　要：金属硫蛋白是一类普遍存在于生物体内、富含半胱氨酸的小分子蛋白,能螯合多种金属离子。本研究根据EST序列信息,利用RACE技术克隆到1条家蝇Musca domestica金属硫蛋白基因MdMtn(GenBank登录号为GU289398)。序列分析表明,MdMtncDNA全长408bp,包含1个123bp的开放阅读框,编码40个氨基酸残基,其中半胱氨酸残基10个,呈-C-X-C-方式排列。此蛋白理论分子量为3.8kD,等电点为8.78。为了解家蝇金属硫蛋白对重金属的结合活性,构建了pET-DsbA-MT表达载体,并转化Escherichia coliBL21(DE3)宿主菌进行融合表达。研究发现MT重组菌对重金属镉的耐受性得到了明显加强,提示MdMtn基因可能在家蝇适应重金属环境中起到积极作用。Metallothioneins（MTs）are low molecular weight,metal-binding and cysteine rich proteins found in a variety of living organisms.In this study,a 408 bp cDNA encoding for a metallothionein was cloned from housefly（Musca domestica）by RACE based on EST information and named as MdMtn（GenBank accession no.GU289398）.Sequence analysis showed that MdMtn contains a 123 bp open reading frame（ORF）encoding a protein of 40 amino acid residues.The MdMtn peptide sequence includes 10 cysteine residues with a distribution pattern of-C-X-C-.The predicted molecular weight of encoding protein is 3.8 kD with the isoelectric point（pI）of 8.78.In order to detect the MdMtn activity in binding heavy metals,the target gene was cloned into a prokaryotic expression vector pET-DsbA,and then a fusion protein was expressed in Escherichia coli BL21（DE3）.It was found that in the presence of CdCl2,the expression of MdMtn significantly increased the bacteria tolerance to Cd2＋,suggesting that MdMtn may play an active role in housefly adaption to the environment with heavy metals.

关 键 词：家蝇 金属硫蛋白 基因克隆 原核表达 蛋白活性 

分 类 号：Q966[生物学—昆虫学]