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.
出处
《中国生物制品学杂志》
CAS
CSCD
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 )
