鲑鱼降钙素基因在大肠杆菌中的克隆与表达

The Cloning and Expression of Salmon Calcitonin in E.coli

以鲑鱼基因组ＤＮＡ为模板，采用ＰＣＲ获得ｓＣＴ基因，并为ＤＮＡ序列分析所证实．以ｐＧＥＸ－３Ｘ为表达载体，利用体外定点突变技术成功地构建了融合蛋白ＧＳＴ－ｓＣＴ的重组表达质粒ｐＧＥＸ－３Ｘ－ｓＣＴ，在大肠杆菌中得到高效表达，其表达量约为菌体总蛋白的３０％；利用亲合层析法对融合蛋白ＧＳＴ－ｓＣＴ进行纯化，再经Ｆａｃ－ｔｏｒΧａ酶切后获得了重组ｓＣＴ，并对其进行活性检测．初步实验证明。

The sCT gene was amplied by PCR in which Salmon genomic DNA was used as the template.After sequence analysis,the sCT gene was cloned into the expression vector pGEX 3X,and the expression system pGEX 3X sCT was constructed by deleting with altered sites in vitro mutagenesis system.Gly extended sCT was produced in recombinant E.coli as part of a fusion with glutathione S transferase(GST).The produced soluble fusion protein(GST sCT) which accounts for 30% of total bacterial protein was purified to homogeneity by affinity chromatography.After the fusion protein (GST sCT) was cleaved,the recombinant sCT was obtained and then tested on rats,the results showed that the sCT had the biological activity.