绵羊eIF3h基因克隆、原核表达及蛋白鉴定

摘要

[Objective] To clone gene of ovis aries eukaryotic translation initiation factor 3 subunit H and construct its prokaryotic expression vector,induce the expression and identify target protein in vitro.[Method]Firstly,the ovis aries gene of eIF3h would be cloned from Ovis aries cDNA library using the primers based on eIF3h sequence.The prokaryotic expression vectors were transformed into expression host strain BL21,then after,IPTG was added to induce expression.SDS-PAGE and Western blotting assays were used to detect the expression of target protein.[Result]It successfully cloned eIF3h gene sequences of CDS.The length of the CDS was 1,059 bp.The Escherichia coli containing recombinant vector expressed inclusion body proteins of 40kD after induction by IPTG at 37°C.[Conclusion]The construction of recombinant plasmid is successful,which has provided the basis for further research of eIF3h molecule.%[目的]克隆绵羊真核翻译起始因子eIF3h,构建原核表达载体,并诱导外源蛋白表达,检测、鉴定带有His标签的目的蛋白.[方法]根据Genbank中绵羊eIF3h基因CDS序列设计引物,PCR特异扩增DNA片段,酶切、连接至pET28α(+)载体,将重组质粒转入大肠杆菌E.coli BL21(DE3)株中诱导eIF3h蛋白表达.采用SDS-PAGE电泳分析目的蛋白表达情况,利用Western blot技术检测、鉴定目的蛋白.[结果]正确、完整的克隆到绵羊eIF3h基因CDS区序列,片段全长1 059 bp.将重组质粒转入大肠杆菌E.coli BL21(DE3)株后,在37℃,1 mmol/L浓度的IPTG诱导3.5 h后获得以包涵体形式存在的目的蛋白,分子量大小约为40 kD.[结论]获得了完整、正解的绵羊eIF3h基因CDS区序列片段,构建了该基因的原核表达载体,成功诱导了绵羊eIF3h基因外源表达的目的蛋白,为绵羊eIF3h基因的后续研究提供了科学数据.