表达肿瘤抗原HPV-58E7的HLA-A^＊0201阳性宫颈癌细胞模型的构建和鉴定

Establishment and characterization of a HLA-A* 0201 human cervical cancer cell model expressed tumor antigen HPV-58 E7

为建立内源性表达58型人乳头瘤病毒(Huamn papillomavirus type 58,HPV-58)重要癌抗原E7蛋白的HLA-A*0201阳性人宫颈癌细胞模型.以HPV-58全基因组为模板,采用PCR法扩增E7蛋白的编码基因,构建真核表达载体p EGFP-N2-HPV58E7;转化大肠杆菌DH5α,通过DNA测序鉴定质粒载体;经除内毒素纯化后,将大量制备的重组质粒瞬时转染293T细胞,采用RT-PCR和细胞免疫荧光方法研究重组质粒在人源细胞中的表达情况;最后,选择HPV检出阴性的HLA-A*0201阳性人宫颈癌C33A细胞株为宿主细胞,采用脂质体转染、G418抗性加压筛选和细胞克隆化培养技术,建立能够稳定表达HPV-58 E7的恒定转染细胞株.采用RT-PCR、Western-blot和细胞增殖测定等方法从转录、翻译和表型等3个层次对所建细胞株进行鉴定研究.研究结果显示:克隆制备了HPV-58 E7蛋白的编码基因;构建获得了能够在人源细胞中表达HPV58E7-EGFP融合蛋白的真核表达质粒;并成功建立了能够稳定表达HPV-58 E7的HLA-A*0201阳性人宫颈癌细胞模型;核酸和蛋白水平的鉴定结果表明所建模型细胞能够表达HPV-58 E7癌抗原,宿主细胞本身表达的Ⅰ类组织相容性抗原HLA-A*0201有望通过内源性抗原加工途径将E7抗原的细胞毒性T细胞(Cytotoxic lyphocyte,CTL)表位提呈到细胞表面;另外,细胞增殖试验结果亦表明该细胞模型体现出E7蛋白显著的诱导细胞增殖的生物学特征.总之,能够稳定表达HPV-58 E7癌蛋白的HLA-A*0201阳性人宫颈癌细胞模型的建立为后续即将展开的以HPV-58 E7为靶标的宫颈癌治疗性疫苗和药物的寻找和研究奠定基础.

To meet the requirement of research and development of therapeutic vaccine againsthuman cervical cancer caused by continuous infection of human papillomavirus type 58（ HPV- 58）,a novel HLA-A^＊0201positive human cervical cancer cell model which could expressed HPV- 58 oncoprotein E7 was established and characterized.In the first place,the encoding gene of E7 was amplified from the whole-genome of HPV-58 by using PCR,and was further cloned into the vector of p EGFP- N2 to build the recombinant expression plasmid p EGFP-N2-HPV58E7.After being transformed into E. coli DH5α and verified by DNA sequencing,the isolated endotoxin-free plasmid was transformed into human cell line HEK-293 T to test its capability of HPV-58 E7 expression by using RT-PCR and immunofluorescence. Furthermore,the E7-negative human cervical cancer cell line C33 A which initially expresses human HLA-A^＊0201 molecular was chose as a host cell to build the target cell model by plasmid transfection,G418 selection and clonal cultivation. Finally,the biological characteristics of cell model,including the correct transcription and expression of HPV- 58 E7 and its relatedbiological phenotypes,were evaluated by using RT-PCR,Western-blot,and cell viability assay.As a result,the encoding gene of HPV-58 E7 was correctly cloned and successfully used to build expression vector,leading the effective expression of HPV- 58 oncoprotein E7 in human cells in a fused form. Moreover,those results of further experimental verification suggested that the target cancer cell model C33 A / p EGFP- N2- HPV58E7 was successfully established.The oncoprotein of HPV-58 E7 was not onlycorrectly expressed in this cell model,but it also could be effectively presented by initially expresses HLA- A＊0201 molecular for specific recognition of active cytotoxic T lymphocyte.In addition,the viability analysis also revealed that the expressed oncoprotein could effectively stimulate the growth of model cells. In conclusion,the generation of this stably-transfected cell lines would provide a wide range of applications for future research and development of HLA-A^＊0201 restricted vaccine against HPV-58 associated cervical cancer.