也木勒羊抑制素α亚基基因克隆及其真核表达

为了克隆也木勒白羊抑制素α(inhibin α,INHα)亚基基因和表达其重组蛋白质,本试验采集发情期也木勒白羊卵巢组织,并从中快速抽提总RNA,以此作为模板并根据白羊INHα亚基基因编码区序列(GenBank登录号:XM_004004955.1)设计合成1对引物,经反转录扩增获得了也木勒白羊INHα亚基基因编码区全长序列.并将扩增产物克隆到表达载体(pEGFP-N1)的Scal Ⅰ和EcoR Ⅰ酶切位点之间,构建重组质粒pEGFP-INHα并转化大肠杆菌DH5α感受态细胞,后进行鉴定、测序,证明成功构建了INHα亚基基因的真核表达载体.将也木勒白羊INHα亚基基因与人、大猩猩、牛等动物的INHα亚基基因序列进行同源性比对分析,相似度都在50%以上,说明了抑制素基因具有高度保守性.用RT-PCR和Western blotting方法验证了INHα亚基基因的真核表达载体在BHK细胞中的成功表达及蛋白表达.

Cloning and Eukaryotic Expression of Inhibin alpha Gene in Yemule Aries

To clone the Yemule aries inhibin α (INHα) subunit gene and express its recombinant protein,total RNA was extracted from Yemule aries ovary tissue by Trizol,and a pair of primer was designed according to the published aries INHα subunit gene sequence (GenBank No.XM_004004955.1). INHα subunit gene coding sequence was amplified by RT-PCR.The mature peptide cDNA was further amplified and cloned into the Scal Ⅰ and EcoR Ⅰ sites of the pEGFP-N1 expressing vector to generate the recombinant plasmid pEGFP-INHα.The plasmid was transformed into DH5α,after appraisal,sequencing,proves that successful build INHα subunit gene eukaryotic expression vector.The sequence result was compared with homologous sequences from animals including human,gorilla,cow and other animals alpha subunit of INHα subunit gene sequence homology comparison analysis.Sequence alignment showed that ranks of their similarities were above 50%,proved that INHα subunit gene was highly conservative.By RT-PCR and Western blotting method verified the eukaryotic expression vector of statin INHα subunit gene expression and protein expression of success in BHK cells.