人乳头瘤病毒16 L1-CEA CTL表位融合蛋白原核表达探究.docVIP

人乳头瘤病毒16 L1/CEA CTL表位融合蛋白原核表达探究 　 作者：张德意 朱冠保，张丽芳，朱珊丽，陈俊 【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。 【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 Abstract: 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 coli. 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 SDSand 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, SDSanalysis showed the relative molecular mass of this fusion protein as 83×103，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. Key words: papillomavirus; carcinoembryonic antigen; prokaryoti expression; T lymphocyte, lytotoxic 癌胚抗原（carcinoembryonic antigen CEA）是一种重要的肿瘤相关抗原，在绝大多数结直肠癌和胃癌、50%乳腺癌及70%非小细胞肺癌中有较高的表达