重组LacZ复制缺陷型腺病毒的构建和鉴定  

Construction and identification of recombinant adenovirus vector expressing LacZ gene

作　　者：裘秀春[1] 许彦鸣[2] 马保安[1] 周勇[1] 单乐群[1] 杨彤涛[1] 于翠娟[3] 孟艳玲[3] 杨安钢[3] 范清宇[1] 

机构地区：[1]第四军医大学唐都医院骨肿瘤研究所 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710038 [3]第四军医大学基础部免疫学教研室,陕西西安710038

出　　处：《医学研究生学报》2006年第11期963-966,I0009,共5页Journal of Medical Postgraduates

基　　金：国家杰出青年科学基金资助项目(批准号:399Z5036);国家自然科学基金资助项目(批准号:30471988);中国博士后基金资助项目(批准号:2005038259)

摘　　要：目的:构建表达LacZ基因的复制缺陷型腺病毒。方法:用AdEasy-1TM系统,在大肠杆菌中同源重组,构建表达LacZ基因的复制缺陷型腺病毒载体(Ad-LacZ)。重组腺病毒在HEK293细胞内包装并扩增,测定病毒滴度、电镜鉴定病毒存在、斑点杂交检测其复制缺陷性,然后感染HeLa细胞X-gal染色检测LacZ的表达情况。结果:成功构建了重组LacZ腺病毒表达载体,包装获得的重组腺病毒的滴度为1.58×109PFU/m l。在转染的细胞和感染的靶细胞内均可检测到LacZ的表达。电镜下可见感染的HEK293细胞核内含晶格状结构的腺病毒包函体。斑点杂交提示重组病毒为复制缺陷型,其基因转移百分率和感染强度和感染时间有一定的相关性。结论:成功构建了表达LacZ基因的复制缺陷型腺病毒。Objective：AIM To obtein a recombinant replication-deficient adenovirus expressing LacZ protein. Methods：The recombinant adenovirus vector engineered to express LacZ gene（Ad-LacZ） was constructed by recombination in E. coli-BJS183. The replication-deficient adenovirus was then packed in HEK293 cells after transfection with the construct and subsequently identified by X-gal staining, electron microscopic observation and dot blotting. The expression of LacZ in HeLa cells infected by Ad-LacZ could be detected by X-gal stain. Results：Recombinant adenovirus expression vector of LacZ was successfully reconstructed. And the titer of Ad-LacZ virus reached 1.58 ×10^9 pfu/ml. Under transmission electron microscope, adenoviral inclusions could be found in the nucleus of the infected HEK293 cells. Ad-LacZ virus was comfirmed to be replication-deficient by dot blotting. And the expression of LacZ was observed in the infected HeLa cells. The gene transfer capability of the recombinant virus was correlated with multiplicity of infection and the infection time. Conclusion：Recombinant adenovirus expression vector of LacZ was successfully reconstructed.

关 键 词：腺病毒表达载体 LACZ 

分 类 号：R373.1[医药卫生—病原生物学]