家蝇(Musca domestica)羧肽酶基因克隆及原核表达

Cloning and prokaryotic expression of Musca domestica carboxypeptidase gene

从构建的家蝇幼虫cDNA质粒文库中筛选到羧肽酶(Carboxypeptidase,CP)基因,以该基因的cDNA文库质粒为模板,通过PCR扩增,获得CP基因完整编码序列(登录号:KF939629.1)。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽和亚细胞定位等进行预测和分析。构建PEASY-E1-CP重组质粒,转化到大肠杆菌OrigamiB(DE3)中进行诱导表达。研究结果表明,CP基因ORF全长1 284 bp,编码428个氨基酸,理论分子量47.8ku,等电点为6.10,具有CP家族的蛋白保守结构域;重组原核质粒pEASY-E1-CP经IPTG诱导,蛋白在大肠杆菌中获得表达。

The complete coding sequence （registration number： KF939629.1） of carboxypeptidase （CP） gene screened from cDNA library plasmid of Musca domestica was obtained by PCR conducted with the CP cNA library plasmid as template. The basic physical and chemical properties of the gene and its coding protein, signal peptide and subcellular localization were predicted and analyzed by the bioinformatics methods. Recombinant plasmid PEAS^-El-CP was constructed and induced expression in E. coli OrigamiB（DE3）. The results showed that the CP-ORF was 1 284 bp in length encoding 428 amino acids with a predicted molecular mass of 47.8 ku, pI of 6.10 and conserved domain of carboxypeptidase family. The recombinant plasmid PEASY-E1-CP was induced by IPTG and the protein expressed in E. coll.