小鼠IDO基因重组腺病毒载体的构建表达和鉴定  

Construction,expression and identification of recombinant adenovirus vector of indoleamine 2,3-dioxygenase

作　　者：侯卫平[1] 黄钢[2] 翟志芳[3] 袁发焕[1] 

机构地区：[1]第三军医大学新桥医院肾内科,重庆400037 [2]第三军医大学基础部医学遗传教研室,重庆400038 [3]第三军医大学西南医院皮肤科,重庆400038

出　　处：《重庆医学》2007年第5期391-393,共3页Chongqing medicine

基　　金：重庆市自然科学基金资助项目(CSTC;2006BB5055)

摘　　要：目的构建携带小鼠IDO基因的重组腺病毒载体,以期进一步进行研究其免疫学效应。方法酶切含有小鼠全长IDOcDNA的PCOUS-2质粒,亚克隆至穿梭质粒pAdtrack-CMV上,在BJ5183细菌中和AdEasy-1进行同源重组,筛选阳性克隆,酶切、测序鉴定正确后,脂质体法转染AD-293细胞进行包装,扩增,氯化铯密度梯度离心纯化病毒并检测其滴度。RT-PCR和荧光显微镜鉴定和检测重组腺病毒转染AD-293细胞后IDO的表达,同时进行野生型腺病毒的检测。结果经酶切及测序证实IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,且无野生型腺病毒的产生。结论成功构建了含小鼠IDO基因的重组腺病毒载体,为探讨IDO的生物学功能奠定了基础。Objective To construct a recombinant adenovirus encoding for indoleamine 2,3-dioxygenase（IDO）for further experiments. Methods Full-length mouse derived IDO eDNA was subcloned into pAdtrack-CMV shuttle plasmid. The product was linearized to homologous recombination with AdEasy-1 vector in BJ5183 bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA transfected into AD-293 cells for packaging and amplification of AdIDO virus. Then the virus was purified by CsC1 density gradient eentrifugation. The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells. Results Construction of recombinant adenoviruses IDO was successful confirmed by restriction endonuclease digestion and sequencing. The specific expression of mouse IDO was identified by RT-PCR in AD-293 cell after infection with AdIDO, but not in the control AD-293. Transfection with adenovirus DNA caused cytopathic effect only in AD-293 cell but not in HeLa cell line, which confirmed only replication-defective adenovirus was produced. There was no evidence for production of wild type adenovirus. Conclusion A recombinant adenovirus AdIDO is successfully constructed. The virus will be useful to further investigate the biologic role of IDO.

关 键 词：IDO 腺病毒 色氨酸 

分 类 号：R346[医药卫生—基础医学]