摘要
目的:构建人乳头瘤病毒(HPV)16型衣壳蛋白L1与癌胚抗原(CEA)细胞毒T细胞(CTL)表位嵌合基因,探讨其在原核表达系统中的表达情况。方法:选择CEA CTL表位(CEA691 IMIGVLVGV),以HPV16阳性宫颈癌组织提取的DNA为模板,通过PCR方法扩增出HPV16 L1/CEA CTL表位嵌合基因,克隆入pET32a(+)表达载体,转化大肠杆菌BL21(DE3),经IPTG诱导表达融合蛋白,通过Ni-NAT离子交换柱层析纯化,进一步采用SDS-PAGE和Western-blot分析鉴定。结果:pET32a(+)/HPV16 L1/CEA CTL表位重组质粒构建成功,并在大肠杆菌中得到表达。SDS-PAGE分析表明表达产物分子质量(Mr)约为83×103,与预期相符,Western-blot方法进一步证实其为目的蛋白。结论:利用pET32a(+)表达载体能表达HPV16 L1/CEA CTL表位重组蛋白,为其进一步的免疫效应研究打下了基础。
Objective：To construct the chimeric gene of HPV16 capsid protein L1 and CEA CTL epitope and to explore the protein expression of it in Escherichia coll. Methods： The CEA CTL epitope （CEA691 IMIGVLVGV） was selected and the chimeric gene of HPV16 L1/CEA CTL epitope was amplified by PCR. The chimeric gene was cloned into the expression vector pET32a（＋） for forming the pET32a（＋）/HPV16 L1/CEA CTL epitope recombinant with which is transformed E.coli BL21. The pET32a（＋）/HPV16 L1/CEA CTL epitope fusion protein was expressed in E.coli, induced by IPTG, and purified by Ni-NTA agarose column ion-exchange chromatography. The protein was analyzed with SDS-PAGE and identified with Western-blot. Results： The pET32a（＋）/HPV16 L1/CEA-CTL epitope recombinant plasmid was successfully constructed and the fusion protein could be expressed in prokaryotic expression system, SDS-PAGE analysis showed the relative molecular mass of this fusion protein as 83×10^3, which was consistent with that of theoretically predicted, and the specificity of this fusion protein was comfirmed with Western-blot. Conclusion： The fusion protein of the HPV16 L1/CEA-CTL epitope chimeric gene can be successfully expressed in prokaryotic expression system, It lays the foundation for the detecting its immunity effectiveness.
出处
《温州医学院学报》
CAS
2008年第2期106-109,共4页
Journal of Wenzhou Medical College
基金
浙江省自然科学基金资助项目(Y204055)
关键词
乳头状瘤病毒
癌胚抗原
原核表达
T淋巴细胞
细胞毒性
papillomavirus
carcinoembryonic antigen
prokaryoti expression
T lymphocyte, lytotoxic
