含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1-E7AS的构建及鉴定  

Construction and identification of the adeno-associated viral plasmid pUF1-E7AS containing E7 antisense gene of HPV16.

作　　者：邬素芳[1] 易诚青[2] 陈刚[1] 李辅军[1] 李静[1] 卢运萍[1] 廖国宁[1] 马丁[1] 

机构地区：[1]华中科技大学同济医院妇产科,武汉430030 [2]上海交通大学附属第一人民医院骨科

出　　处：《现代妇产科进展》2005年第2期119-122,共4页Progress in Obstetrics and Gynecology

基　　金：国家杰出青年基金 ( 30025017 );国家重点基础研究发展规划项目 ( 2002CB513100 );国家自然科学基金面上项目(30271358);湖北省科技厅攻关项目(2002AA301C54)

摘　　要：目的:构建含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1- E7AS并鉴定,探讨腺相关病毒介导的反义E7基因技术用于治疗早期宫颈癌的可能性。方法:使用RT -PCR法扩增全长HPV16型E7基因,利用基因重组法将目的片段反向插入腺相关病毒骨架质粒pUF1并酶切鉴定。结果:RT PCR法扩增全长315bp的HPV16型E7基因,装入pGEM -Teasy载体进行测序,经NCBI数据库blast检索为100%的符合,经基因重组获得含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS,AccⅠ和KpnⅠ双酶切鉴定pUF1 E7AS。结论:含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS可成功构建。Objective: To explore the potential of an adeno-associated viral antisense RNA transcript for gene therapy of cervical cancer.The antisense RNA transcript of E7 genes of human papillomavirus (HPV) type 16 was cloned into the adeno-associated viral vector pUF1 and pUF1-E7AS was identified.Methods:The full-length of HPV16 E7 cDNA in SiHa cells were cloned in pUF1 in reverse orientation.Molecular biological methods were performed to identified the pUF1-E7AS. Results:RT-PCR was performed to clone full-length 315bp HPV 16 E7 cDNA in SiHa cells into pGEM-Teasy vectors for sequencing,and the cDNA sequences were blasted in the data base of NCI,100% coincidence was found.pUF1-E7AS was obtained with gene-arrangement containing the antisense RNA transcript of E7 genes in reverse orientation,which was identified by restricted endonuclease reaction with AccⅠand KpnⅠ.Conclusion:The adenoassociated viral plasmide p UF1-E7AS can be cloned successfully.

关 键 词：宫颈肿瘤 乳头状瘤病毒 人 基因疗法 

分 类 号：R737.33[医药卫生—肿瘤]