定点整合型基因治疗腺病毒载体的构建

目的：构建定点整合型基因治疗腺病毒载体。方法：以逐步亚克隆法将腺病毒伴随病毒的两个末端倒转重复序列与neo基因表达盒克隆至pAdE1CMV，其中HCMV启动子控制下的neo基因表达盒位于腺病毒伴随病毒两个末端倒转重复序列之间，将得到的转移载体pAdE1CMVITREXneoDNA和pJM17DNA以脂质体法共转染293细胞，G418法连续筛选重组腺病毒。以病毒核酸酶切及感染293细胞的CPE（cytopathiceffect　）观察鉴定是否获得重组腺病毒。结果：获得有腺病毒伴随病毒的两个末端倒转重复序列和位于其间的neo基因表达盒的重组腺病毒vAd－AAV。结论：重组腺病毒vAd－AAV的构建成功将为基因治疗提供定点整合型腺病毒载体。

CONSTRUCTION OF ADENOVIRUS VECTOR FOR GENE THERAPY WHICH CAN INTEGRATE FOREIGN GENE INTO HOST CELL GENOME AT SPECIFIC SITE

Objective: To construct a novel adenovirus vector for gene therapy which can integrate foreign geneinto host cells genome at specific site. Methods: Transfer vector pAdE1CMVITREXneo was obtained first bysubcloning adeno-associated virus two ITRs between which neo expression box was controlled under HCMVpromoter into pAdE1CMV. Recombinant adenoviruses vAd-AAV were derived by cotransfecting DNAs ofpAdE1CMVITREXneo and pJM17 into 293 cells using liposome method developed in this paper by G418selecting. Positive clones resistant to G418 were obtained after transfecting pAdE1CMVITREXneo DNA intoHela cells using liposome method by G418 selecting for 18 days. Results: Analysis of restriction enzymedigestion of recombinant adenovirus DNAs and the fact that recombinant adenoviruses can replicate in 293 cellsin presence of G418 indicated that the adeno-associated virus two ITRs and neo expression box controlledunder HCMV promoter between the two ITRs were cloned into Ad5 dl3O9 genome. There was no remarkabledifference between recombinant adenovirus vAd-AAV and wild type adenovirus in titre. Obtaining of positiveHela cell clones resistant to G418 indicated partly the potential integrating function of the recombinantadenoviruses Conclusion: Success in constructing recombinant adenovirus vAd-AAV will present a noveladenovirus vector for gene therapy which can intngrate foreign gene into host cells genome at specific site.