腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Transfection of adenovirus vector-mediated ptotein kinase B gene into the rat islets

目的观察携带外源基因的腺病毒在体外感染胰岛及外源基因在胰岛中的表达。方法构建表达Aktl的腺病毒载体（AdS—Akt1）。按病毒细胞比（MOI值）500感染新分离的胰岛。通过免疫印迹分析（Westernblot）测定胰岛培养上清液中Akt1的表达。结果（1）成功构建了携带Aktl基因的腺病毒载体AdS—Akt1，所获得病毒滴度为5．3×10^9pfu／ml；（2）表达增强型绿色荧光蛋白的腺病毒（AdS—EGFP）和Ad5-Akt1在体外可以感染胰岛，其中Ad5-EGFP转染率可达60％～70％。在Ad5-Akt1腺病毒感染的胰岛培养上清液中检测到了Akt1蛋白的表达。结论腺病毒在体外可以有效感染大鼠胰岛，且携带的外源基因可以在胰岛细胞中稳定表达。

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methotis The adenovirus vector,Ad5-Akt1 ,was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection （MOI） was 500）. The expression of Aktl in AdS-Aktl islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 10^9 pfu/ml. The transfection efficiency of AdS-EGFP was 60% -70%, and Aktl protein was detected in Ad5- Akt1 islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.