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新疆多浪羊IL-1β基因原核表达载体的构建与表达
曾国航1, 徐宏伟1, 李莲瑞2,3
1.塔里木大学 生命科学学院;2.塔里木大学 动物科学学院;3.新疆兵团塔里木畜牧科技重点实验室
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1 mmol/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32.4 ku,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32.4 ku的IL-1β融合蛋白。
关键词:  多浪羊  IL-1β基因  原核表达
DOI:
分类号:
基金项目: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)
Construction and expression of prokaryotic expression vector of IL-1β from Duolang sheep in Xinjiang
ZENG Guohang,XU Hongwei,LI Lianrui
Abstract:
【Objective】The prokaryotic expression vector of IL-1β gene 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-1β was constructed by connecting IL-1β gene from plasmid pMD-18T-IL-1β of E.coli DH5α and pET-28b of 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-1β plasmid 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 1 mmol/L IPTG induction,the IL-1β protein 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-1β of Xinjiang Duolang sheep was constructed successfully.After induction,fusion protein was expressed with molecular weight of 32.4 ku.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-1β of IL-1β gene from Xinjiang Duolang sheep was successfully constructed and the obtained fusion protein in Escherichia coli BL21(DE3) had molecular weight of 32.4 ku.
Key words:  Duolang sheep  IL-1β gene  prokaryotic expression