人乳头状瘤病毒E6和E7蛋白过表达慢病毒载体构建及稳定转染喉上皮细胞系的建立  

Construction of lentivirus vector with overexpression of human papillomavirus E6 and E7 protein and establishment of stable transfection into laryngeal epithelial cell line

作　　者：肖洋[1] 周思含 牛子捷 王军[1] XIAO Yang;ZHOU Sihan;NIU Zijie;WANG Jun(Department of Pharyngology,Beijing Tongren Hospital,Capital Medical University,Key Laboratory of Otolaryngology Head and Neck Surgery(Capital Medical University),Ministry of Education,Beijing,100730,China)

机构地区：[1]首都医科大学附属北京同仁医院咽喉科,耳鼻咽喉头颈科学教育部重点实验室(首都医科大学),北京100730

出　　处：《中国耳鼻咽喉头颈外科》2022年第12期790-794,共5页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：北京市医院管理局儿科学科协同发展中心儿科专项创新推广项目(XTCX201823)。

摘　　要：目的分别构建过表达人乳头状瘤病毒(human papillomavirus,HPV)6型和11型的E6、E7癌基因的慢病毒载体,建立HPV6-E6-E7和HPV11-E6-E7基因过表达的SNU-1076喉上皮细胞系模型。方法分别合成表达HPV6-E6-E7和HPV11-E6-E7基因的片段,构建重组慢病毒载体pHBLV-CMV-h-HPV6-E6-E7-3FLAG-ZsGreen-Puro和pHBLV-CMV-h-HPV11-E6-E7-3FLAG-ZsGreen-Puro。包装慢病毒后转染进入人喉上皮细胞系SNU-1076,经嘌呤霉素抗性筛选得到阳性克隆。采用实时定量PCR法分别检测HPV6-E6-E7和HPV11-E6-E7基因过表达的水平。结果通过测序验证质粒构建的成功。实时荧光定量PCR的结果显示,转染HPV6-E6-E7和HPV11-E6-E7基因的SNU-1076细胞系相较于转染对照空载体的细胞系出现了明显的扩增曲线。其中HPV6-E6-E7的过表达效果为41692倍,HPV11-E6-E7的过表达效果为124957倍。结论利用慢病毒法成功分别建立了过表达HPV6-E6-E7和HPV11-E6-E7的喉上皮SNU-1076细胞系。OBJECTIVE To construct lentiviral vectors overexpressing E6 and E7 oncogenes of human papilloma virus(HPV)types 6 and 11,respectively.And establish SNU-1076 laryngeal epithelial cell line models with HPV6-E6-E7 and HPV11-E6-E7 gene overexpression.METHODS The fragments expressing HPV6-E6-E7 and HPV11-E6-E7 genes were synthesized respectively,and the recombinant lentiviral vectors pHBLV-CMV-h-HPV6-E6-E7-3FLAG-ZsGreen-Puro and pHBLV-CMV-h-HPV11-E6-E7-3FLAG-ZsGreen-Puro were constructed.The packaged lentivirus was transfected into the human laryngeal epithelial cell line SNU-1076,and positive clones were obtained by puromycin resistance selection.The levels of HPV6-E6-E7 and HPV11-E6-E7 gene overexpression were detected by real-time quantitative PCR respectively.RESULTS The success of plasmid construction was verified by sequencing.The results of real-time PCR showed that the SNU-1076 cell line transfected with HPV6-E6-E7 and HPV11-E6-E7 genes showed a significant amplification curve compared with the cell line transfected with the control empty vector.Among them,the overexpression effect of HPV6-E6-E7 was 41692 times,and the overexpression effect of HPV11-E6-E7 was 124957 times.CONCLUSION Laryngeal epithelial SNU-1076 cell lines overexpressing HPV6-E6-E7 and HPV11-E6-E7 were successfully established by lentiviral method,respectively.

关 键 词：人乳头状瘤病毒6 人乳头状瘤病毒11 癌基因 慢病毒表达载体 稳定转染 喉上皮细胞系 

分 类 号：R739.65[医药卫生—肿瘤]