重组人白细胞介素-10融合表达载体的构建及其在BL21(DE3)pLysE细菌细胞中的表达  

Construction of Recombinant Plasmid and Fusion Expression of rhIL-10 in BL21(DE3)pLysE Cells

作　　者：方爱平[1] 杨国成[1] YEShui-qing 夏腊菊[1] 孙凌聪[1] 刘少平[1] 

机构地区：[1]武汉大学医学院武汉430071 [2]美国霍普金斯大学医学院，Baltimore，MD21287-365

出　　处：《中国生物制品学杂志》2004年第2期75-77,共3页Chinese Journal of Biologicals

基　　金：湖北省科技攻关计划课题 (3 0 113 0 5 2 5 )

摘　　要：目的　构建重组人白细胞介素 10 (recombinanthumaninterleukin 10 ,rhIL 10 )融合蛋白的表达载体 ,并在大肠杆菌中表达。方法　应用RT PCR方法扩增IL 10基因 ,克隆PCR产物 ,构建PCRR○T7/NT TOPOR○ IL 10重组质粒 ,以AppiedBiosystems 370 0DNA分析仪进行分析。构建成功的重组质粒转化大肠杆菌BL2 1(DE3)pLysE细胞 ,经 12 %SDS PAGE鉴定融合表达蛋白。结果　PCRR○T7/NT TOPOR○ 质粒已载入rhIL 10基因 ,其序列与理论设计完全一致 ,表达质粒在BL2 1(DE3)pLysE中得到高效表达 ,产物主要以包涵体形式存在。结论　已成功构建重组PCRR○T7/NT TOPOR○ IL 10质粒载体 ,并在大肠杆菌BL2 1(DE3)Objective To construct the recombinant plasmid and express the fusion protein of rhIL 10 in E.coli. Methods Amplify IL 10 gene by RT PCR,clone the PCR product to construct recombinant plasmid PCRT7/NT TOPO IL 10,and identify by Appied Biosystems 3 700 DNA analyzer.Transform the constructed plasmid into E.coli BL21(DE3)pLysE cells and identify the expressed fusion protein by 12% SDS PAGE.Results rhIL 10 gene was successfully inserted into vector PCR T7/NT TOPO.The sequence of the insert was completely consistent with that desinged.The insert in the recombinant plasmid was highly expressed mainly in inclusion bodies in BL21(DE3)pLysE cells.Conclusion Recombinant plasmid PCR T7/NT TOPO IL 10 was successfully constructed and IL 10 fusion proteins highly expressed in BL21(DE3)pLysE cells.

关 键 词：RHIL-10 人重组白细胞介素-10 融合表达 包涵体 大肠杆菌 

分 类 号：R977.6[医药卫生—药品] Q78[医药卫生—药学]