摘要
目的:构建尤文肉瘤EWS-FLI1基因原核表达质粒。方法:从人尤文肉瘤A673细胞中提取总RNA,以RT-PCR法扩增EWS-FLI1基因序列,目的基因两侧引入SacⅠ和HindⅢ酶切位点,克隆至pMD18-T载体中,转化大肠杆菌JM109,筛选阳性克隆作PCR鉴定、酶切鉴定及测序鉴定。再将EWS-FLI1基因亚克隆至原核表达载体pQE30中,构建重组原核表达质粒pQE30-EWS-FLI1,酶切及测序鉴定。所构建的重组质粒转化JM109,IPTG诱导表达,经SDS-PAGE电泳后转印到NC膜上,进行Western blot鉴定。结果:PCR结果显示扩增片段大小约为1.5kb,双酶切鉴定目的基因片段大小约为1.5kb,阳性克隆测序结果显示目的基因序列与预期相同。所构建重组质粒在大肠杆菌中表达出与预期大小相符的54kD蛋白,经鉴定系EWS-FLI1蛋白。结论:成功构建了pQE30-EWS-FLI1,并进行了原核表达,为进一步研究其生物学功能奠定了基础。
Objective：To construct Ewing＇s sarcoma EWS-FLI1 gene prokaryotic expression vector.Methods：The target gene of EWS-FLI1 was obtained by RT-PCR method after the total RNA was extracted from Ewing＇s sarcoma A673 cells.The site sequences of restrictive endonuclease SacⅠand Hind Ⅲ were introduced into the upstream and downstream of target gene respectively.The target gene fragment were cloned into pMD18-T and transformed into E.Coli JM109.Screened positive clones were confirmed by PCR,restrictive endonuclease digestion and DNA sequencing.The EWS-FLI1 gene was sequentially subcloned into prokaryotic expression vector pQE30,and the recombinant plasmid pQE30-EWS-FLI1 was confirmed by restrictive endonuclease digestion and DNA sequencing.The proteins,expressed in E.coli JM109 transformed with EWS-FLI1recombinant plasmid under IPTG induction,were characterized by SDS-PAGE and Western-blot.Results：PCR result indicated that an amplified DNA fragment was in size of 1.5 kb.Restrictive endonuclease digestion analysis indicated that the target gene was in size of 1.5 kb.DNA sequencing analysis demonstrated that sequence of target gene accorded with anticipated one.The EWS-FLI1 with a molecular weight of 54 kD was highly expressed in pQE30-EWS-FLI1.Western blot proved that the expressed product had the antigenicity of EWS-FLI1.Conclusion：The recombinant prokaryotic expression vector pQE30EWS-FLI1 is constructed successfully,which will contribute to the further research of EWS-FLI1.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2010年第5期645-648,共4页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:30760254)
