新疆卡拉库尔羊Fas基因原核表达载体的构建及表达  

Construction and Expression on Prokaryotic Expressing Vectors of Fas Gene from Xinjiang Karakul Sheep

作　　者：贾山岭[1,2,3] 潘伊微[1,2,3] 潘辉[1,2,3] 贺艳艳[1,2,3] 李莲瑞[1,2,3] 

机构地区：[1]塔里木大学生命科学学院,新疆阿拉尔843300 [2]新疆兵团塔里木畜牧科技重点实验室,新疆阿拉尔843300 [3]塔里木大学动物科学学院,新疆阿拉尔843300

出　　处：《西北农业学报》2013年第6期6-9,共4页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：新疆建设兵团应用基础项目(07GC07)

摘　　要：根据GenBank的羊Fas细胞凋亡因子序列设计1对特异性引物,将EcoRⅠ和XhoⅠ酶切位点引入到引物两端,同时加上保护性碱基。利用RT-PCR技术,从新疆卡拉库尔羊外周血淋巴细胞总RNA中扩增出特异性基因片段。将分离纯化后的PCR产物与pMD-18T载体进行连接,利用双酶切反应与菌液PCR对阳性重组克隆进行鉴定筛选,然后进行序列测定。使用原核表达载体pET28a来完成Fas的定向克隆,成功构建重组融合表达质粒pET-28a-Fas,然后将质粒转化至E.coli BL21感受态,设置不同IPTG浓度和诱导温度,选择最佳的表达条件,然后初步纯化重组融合蛋白。结果表明,在pET28a表达载体中,Fas基因的插入方向和读码框均正确;同时Fas基因也完成在E.coli BL21融合表达的目的,融合蛋白分子质量为39.7ku。Specific primers for Fas were designed through GenBank database,the protective bases and the EcoRⅠ and XhoⅠ restriction enzyme sites were added at the ends of the primer.Total RNA was extracted from peripheral blood lymphocyte of karakul sheep by Trizol reagent,than a specific sequence was amplified by reverse transcription-polymerase chain reaction（RT-PCR）.The sequence was ligated to pMD-18T vector after purified,the recombinant sequence was identified by double digests method and PCR.The Fas gene was linked into prokaryotic expression vector pET28a and the recombinant fusion expression plasmid pET28a-Fas was constructed.Then pET28a-Fas was transformed into E.coli BL21.The different concentration of IPTG and different temperature was set up,and the optimal conditions wwere defined,then the recombinant fusion protein was purified.The results showed that the insert direction sand reading frame of Fas gene were right.The pET28a-Fas gene had expressed in E.coli BL21,and the mass of molecule of the fusion protein was 39.7 ku.

关 键 词：卡拉库尔羊 FAS基因 原核表达 

分 类 号：S826[农业科学—畜牧学]