也木勒羊抑制素α亚基基因克隆及其真核表达

doi:10.16431/j.cnki.1671-7236.2015.06.003

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (6): 1340-1347.doi: 10.16431/j.cnki.1671-7236.2015.06.003

也木勒羊抑制素α亚基基因克隆及其真核表达

黄增文, 木尔扎提, 吾热力哈孜·哈孜汗

石河子大学动物科技学院, 石河子 832003

收稿日期:2014-10-09 出版日期:2015-06-20 发布日期:2015-07-23
通讯作者: 吾热力哈孜·哈孜汗 E-mail:1508217366@qq.com
作者简介:黄增文(1984-),男,重庆人,硕士,研究方向:动物遗传育种与繁殖机理,E-mail:xndaxue@126.com
基金资助:
石河子大学科学技术研究发展计划项目(gxjs2011-yz09)

Cloning and Eukaryotic Expression of Inhibin alpha Gene in Yemule Aries

HUANG Zeng-wen, Muerzhati, WureliHazi·Hazihan

College of Animal Science and Technology, Shihezi University, Shihezi 832003, China

Received:2014-10-09 Online:2015-06-20 Published:2015-07-23

摘要/Abstract

摘要： 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.

关键词: 也木勒白羊; 卵泡抑制素; 真核表达; 同源性分析

Abstract: 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.

Key words: Yemule aries; folliculostatin; eukaryotic expression; homology analysis

中图分类号:

黄增文, 木尔扎提, 吾热力哈孜·哈孜汗. 也木勒羊抑制素α亚基基因克隆及其真核表达[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(6): 1340-1347.

HUANG Zeng-wen, Muerzhati, WureliHazi·Hazihan. Cloning and Eukaryotic Expression of Inhibin alpha Gene in Yemule Aries[J]. , 2015, 42(6): 1340-1347.

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也木勒羊抑制素α亚基基因克隆及其真核表达

Cloning and Eukaryotic Expression of Inhibin alpha Gene in Yemule Aries

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