家蝇金属硫蛋白基因的克隆、原核表达及活性检测

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，MdMtn cDNA全长408 bp，包含1个123 bp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3.8 kD，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 coli BL21（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。

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

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.