中国东北地区人乳头瘤病毒16型全基因组克隆及HPV16.HaCaT重组细胞模型的构建及初步鉴定  

Cloning of the Complete Genome of Human Papillomavirus Type 16 from Patient in Northeast China,and Construction and Preliminary Identification of the Recombinant Cell Model HPV16.HaCaT

作　　者：刘淼[1] 吴盈盈 路一平 吴思[1] 王爽[1] 崔畅婉 于淼[1] 孙峥嵘[1] LIU Miao;WU Yingying;LU Yiping;WU Si;WANG Shuang;CUI Changwan;YU Miao;SUN Zhengrong(Biobank of Shengjing Hospital Affiliated to China Medical University,Shenyang 110000,China)

机构地区：[1]中国医科大学附属盛京医院生物样本库,沈阳110000

出　　处：《病毒学报》2021年第5期1051-1059,共9页Chinese Journal of Virology

基　　金：辽宁省重点研发计划(项目号:2018225009),题目:人乳头瘤病毒16型E7蛋白通过选择性调控干扰素刺激基因群ISGs表达抵抗β干扰素抗病毒作用;辽宁省高等学校基本科研项目(项目号:LFWK201710),题目:分析STAT1在抗人乳头瘤病毒反应中的作用。

摘　　要：为构建含东北地区人乳头瘤病毒16型(HPV16)全基因组的HPV16.HaCaT细胞模型,收集中国东北地区HPV16单一感染患者宫颈脱落细胞,提取DNA,将HPV16全基因组分成4个区段,通过4对特异性引物对HPV16全基因组进行分段扩增,测序后进行序列拼接及核酸序列分析,克隆HPV16全基因组序列;通过细胞转染,构建含HPV16全基因组的HPV16.HaCaT重组细胞模型;利用聚合酶链式反应(PCR)和细胞免疫荧光法检测重组细胞内HPV16早期基因的表达。成功克隆出中国东北地区HPV16全基因组序列(GenBank登录号:MW320358);构建了东北地区HPV16全基因组的重组质粒及HPV16.HaCaT重组细胞模型;证明了HPV16早期基因E1^E4、E5、E6和E7在重组细胞模型内均有表达,从而获得中国东北地区HPV16全基因组序列及含有HPV16全基因组的HPV16.HaCaT重组细胞模型。To construct a Human papillomavirus16(HPV16).HaCaT cell model containing the complete genome of human papillomavirus(HPV)16 from patient in Northeast China,cervical exfoliated cells from patients with HPV16 infection in Northeast China were collected and DNA was extracted.The whole genome of HPV16 was divided into four sections and amplified by four pairs of specific primers,resulting in cloning of the whole genome of HPV16.The sequences were spliced and nucleic acid sequence was analyzed after sequencing.A recombinant cell model of HPV16.HaCaT containing the whole genome of HPV16 was constructed by cell transfection.Expression of HPV16 early genes in recombinant cells were detected by Polymerase Chain Reaction(PCR)and immunofluorescence.The whole genome sequence of HPV16 from Northeast China was successfully cloned(GenBank accession number:MW320358).The whole genome recombinant plasmid of HPV16 was constructed and HPV16.HaCaT.E1^E4,E5,E6 and E7 mRNA were expressed in the recombinant cell model.Consequently,the complete genome sequence of HPV16 from Northeast China and the recombinant cell model of HPV16.HaCaT containing the complete genome of HPV16 were obtained.

关 键 词：人乳头瘤病毒16型全基因组(HPV16) 表达载体构建 重组细胞模型 聚合酶链式反应(PCR) 

分 类 号：R373.9[医药卫生—病原生物学]