新疆多浪羊IL-1β基因原核表达载体的构建与表达  

Construction and expression of prokaryotic expression vector of IL-1β from Duolang sheep in Xinjiang

作　　者：曾国航 徐宏伟[1] 李莲瑞[2,3] ZENG Guohang XU Hongwei LI Lianrui(College of Life Science College of Animal Science, Tarim University ,Alaer, Xinjiang 843300, China Key Laboratory of Tarim Husbandry Science and Technology, Xinjiang Production ＆ Construction Group ,Alaer, Xinjiang 843300 ,China)

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

出　　处：《西北农林科技大学学报（自然科学版）》2016年第9期17-21,34,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目"棉酚对新疆多浪羊白细胞介素表达的影响研究"(30960277);创新群体项目"南疆地区羊标准化养殖的群发病防控及预警响应机制研究"(TDZKCX201401)

摘　　要：【目的】构建新疆多浪羊IL-1β基因编码区的原核表达载体,在大肠杆菌中进行诱导表达,为进一步研究IL-1β蛋白的结构功能奠定基础。【方法】从含质粒pMD-18T-IL-1β的大肠杆菌DH5α中获取IL-1β基因,与pET-28b质粒DNA连接,构建原核表达载体pET-28b-IL-1β。先将其转化到克隆载体E.coli DH5α感受态细胞中大量拷贝,经菌液PCR和EcoRⅠ、XhoⅠ双酶切鉴定后,提取pET-28b-IL-1β质粒转化到表达载体E.coli BL21(DE3)中,再次进行菌液PCR和EcoRⅠ、XhoⅠ双酶切鉴定。将阳性单克隆接种于LB液体培养基,以终浓度为1mmol/L IPTG进行诱导,用SDS-PAGE电泳检测IL-1β蛋白的表达及存在形式,通过Western blotting验证表达产物是否为目的蛋白。【结果】成功构建了新疆多浪羊IL-1β基因的原核表达载体pET-28b-IL-1β,该载体经诱导后表达出融合蛋白,分子质量为32.4ku,主要以包涵体的形式表达;经Western blotting检测,带有6×His标签的融合蛋白有很好的反应原性。【结论】成功构建了新疆多浪羊IL-1β基因的原核表达载体pET-28b-IL-1β,其在大肠杆菌BL21(DE3)中经诱导后表达出分子质量约为32.4ku的IL-1β融合蛋白。【Objective】The prokaryotic expression vector of IL-1βgene encoding area of Xinjiang Duolang sheep was constructed and induced to express in E.coli to lay foundation for further study on its structure and function.【Method】The prokaryotic expression vector pET-28b-IL-1βwas constructed by connecting IL-1βgene from plasmid pMD-18T-IL-1βof E.coli DH5αand pET-28 bof E.coli BL21（DE3）.Large copies of competent cells were obtained by transforming it into cloning vector E.coli DH5α.After identification using bacteria PCR and double enzyme digestion with EcoRⅠ and XhoⅠ,pET-28b-IL-1βplasmid was extracted and transformed into E.coli BL21（DE3）expression vector.Then,it was identified again using PCR and double enzyme digestion.The positive monoclonal antibody was inoculated into LB culture medium.With a final concentration of 1mmol/L IPTG induction,the IL-1βprotein expression and its form was detected by electrophoresis SDS-PAGE and checked by Western blotting test.【Result】The recombinant prokaryotic expression vector pET-28b-IL-1βof Xinjiang Duolang sheep was constructed suc-cessfully.After induction,fusion protein was expressed with molecular weight of 32.4ku.It mainly expressed in the form of inclusion body.Western blotting detection found that the fusion protein with 6×His tag had good immunogenicity.【Conclusion】The prokaryotic expression vector pET-28b-IL-1βof IL-1βgene from Xinjiang Duolang sheep was successfully constructed and the obtained fusion protein in Escherichia coli BL21（DE3）had molecular weight of 32.4ku.

关 键 词：多浪羊 IL-1β基因 原核表达 

分 类 号：S826[农业科学—畜牧学]