人乳头瘤病毒58型E6E7融合基因重组巨细胞病毒的构建及鉴定  

Construction and identification of recombinant cytomegalovirus with human papillomavirus 58 E6E7 fusion gene

作　　者：杜阳阳 张放 苗智颖 赵红 马迪 王静 李淑英 郑春阳 李劲涛[2] DU Yang Gyang;ZHANG Fang;MIAO Zhi-ying;ZHAO Hong;MA Di;WANG Jing;LI Shu-ying;ZHENG Chun-yang;LI Jin-tao(North China University of Science and Technology/Hebei Key Laboratory for Chronic Diseases,Tangshan Key Laboratory for Preclinical and Basic Researchon Chronic Diseases,Tangshan 063210,China;College of Life Science and Bio-engineering,Beijing University of Technology,Beijing 100124,China)

机构地区：[1]华北理工大学(河北省慢性疾病重点实验室,唐山市慢性病临床基础研究重点实验室),唐山063210 [2]北京工业大学,生命科学与生物工程学院,北京100124

出　　处：《中国人兽共患病学报》2020年第4期303-308,319,共7页Chinese Journal of Zoonoses

基　　金：河北省留学人员科技活动资助项目(No.CY201620);河北省卫计委项目(No.20170896)。

摘　　要：目的利用人乳头瘤病毒(Human papillomavirus,HPV)58突变型E6E7(Mutant type E6E7,mE6E7)融合基因为靶点,巨细胞病毒株(Towne株)细菌人工染色体(SW102 Towne bacterial artificial chromosome,SW102-T-BAC)为载体,构建携带HPV58 mE6E7融合基因的重组病毒,探讨HPV58mE6E7的转化活性。方法PCR扩增带有50 bp Towne开放读码框(open reading frame,ORF)75左右同源臂的GalK、mE6E7及野生型E6E7(Wild type E6E7,wE6E7)融合基因片段,切胶回收纯化;将GalK片段电转到(SW102-T-BAC)感受态细胞,经过同源重组、GalK及氯霉素抗性筛选,获得SW102-T-ORF75-Galk-BAC克隆;然后分别将纯化的mE6E7及wE6E7电转到SW102-T-ORF75-Galk-BAC感受态细胞,经脱GalK及氯霉素抗性筛选,获得SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC克隆;提取所得克隆质粒DNA,转染ARPE-19细胞(以转染SW102-T-BAC的细胞及未转染细胞为对照),逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达状况;观察重组病毒T-ORF75-mE6E7及T-ORF75-wE6E7对转染ARPE-19细胞的影响,通过软琼脂克隆分析mE6E7及wE6E7转化活性。结果获得了SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC的克隆。逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达正确。转染SW102-T-ORF75-wE6E7-BAC的细胞生长失去接触抑制,出现重叠生长现象,其形态由原来梭形变为变圆、肿胀、胞浆颗粒增多;并且在软琼脂中能够形成克隆;而转染SW102-T-ORF75-mE6E7-BAC、SW102-T-BAC及未转染细胞未出现上述细胞的特点。结论获得了T-ORF75-mE6E7及T-ORF75-wE6E7重组病毒,并且重组病毒T-ORF75-mE6E7的转化活性已消除,为HPV58型治疗性疫苗的研究奠定了基础。To investigate the conversion activity of Human papillomavirus58 mutant E6E7 fusion gene(HPV58 mE6E7) and establish Towne recombinant virus carrying HPV58 mE6E7 fusion gene, which used HPV58 mE6E7 as target and Towne cytomegalovirus bacterial artificial chromosome(SW102-T-BAC) as a vector. The GalK, mE6E7 and Wild type E6E7(wE6E7) gene fragments with about 50 bp Towne ORF75 homologous arms were amplified and purified. GalK was transferred to the competent cells of SW102-T-BAC. The clones of SW102-T-ORF75-GalK-BAC were obtained by homologous recombination, the selection from containing GalK and chloramphenicol medium. The fragments of mE6E7 and wE6E7 were transferred to the competent cells of SW102-T-ORF75-GalK-BAC. The clones of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were obtained by homologous recombination, the selection from replaced GalK and chloramphenicol medium, respectively. The plasmids of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were extracted, and transfected into ARPE-19 cells(ARPE-19 cells transfected with SW102-T-BAC and untransfected used as control). The expression of mE6E7 and wE6E7 in transfected cells were verified by reverse transcription PCR and sequencing. The effect of T-ORF75-mE6E7 and T-ORF75-wE6E7 on ARPE-19 cells were observed. The transformation activity of mE6E7 and wE6E7 were analyzed by soft agar cloning. The clones of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were obtained. The expression of mE6E7 and wE6E7 in transfected cells were verified by reverse transcription PCR and sequencing. ARPE-19 cells of transfected SW102-T-ORF75-wE6E7-BAC grow overlapping and lost contact inhibition, and the morphology of the cells changed from the original long fusiform to round, swelling, and full of cytoplasm particles, the cells can form clones in soft agar. The cells of transfected SW102-T-ORF-mE6E7-BAC, SW102-T-BAC, and untransfected do not show those characteristics of transfected SW102-T-ORF75-wE6E7-BAC cells. The recombinant virus of T-ORF75-mE6E7 and T-ORF

关 键 词：人乳头瘤病毒 E6E7融合基因 细菌人工染色体 同源重组 巨细胞病毒 

分 类 号：R373[医药卫生—病原生物学]