细菌内快速构建两种用于肝纤维化基因治疗的重组复制缺陷型腺病毒  

Rapid construction of 2 kinds of replication-deficient recombinant adenoviruses in bacteria for hepatic fibrosis gene therapy

作　　者：林勇[1] 谢渭芬[1] 张忠兵[1] 陈伟忠[1] 张新 曾欣[1] 陈岳祥[1] 

机构地区：[1]第二军医大学长征医院消化内科,上海200003 [2]国家人类基因组南方研究中心

出　　处：《第二军医大学学报》2003年第6期654-657,共4页Academic Journal of Second Military Medical University

基　　金：上海市青年科技启明星计划项目( 0 0 QB14 0 5 4)

摘　　要：目的 :在大肠杆菌内快速构建两种分别含有人肝细胞生长因子 (hepatic growth factor,HGF)和非分泌型人尿激酶型纤溶酶原激活剂 (urokinase- type plasm inogen activator,u PA) c DNA片段的重组复制缺陷型腺病毒。方法 :分别将人 HGF和 u PA c DNA片段与穿梭载体 p Track- CMV连接 ,线性化后与骨架载体 Ad Easy- 1在大肠杆菌 BJ5 183内同源重组获得腺病毒载体 p Ad HGF和 p Adu PA,经 2 93细胞包装后得到复制缺陷型重组腺病毒 Ad HGF和 Adu PA;将 Ad HGF和 Adu PA分别体外感染肝细胞株 L0 2 ,以 Northern印迹法和 Western印迹法检测两种病毒在肝细胞中的表达。 结果 :连接、重组后通过酶切和测序法筛选出病毒质粒 p Ad HGF和 p Adu PA;经 2 93细胞包装 ,3d后均观察到绿色荧光蛋白明显表达 ,Cs Cl梯度离心纯化最终分别获得 4× 10 1 0 efu/ m l滴度的重组病毒 Ad HGF和 6× 10 1 0 efu/ ml Adu PA;两种病毒体外感染肝细胞 3d后 ,HGF和u PA表达均明显增加。结论 :细菌内同源重组制备复制缺陷型腺病毒 ,纯化过程简单 ,重组腺病毒在肝细胞中均高效表达 。Objective:To construct 2 kinds of replication deficient recombinant adenoviruses in bacteria rapidly:AdHGF inserted with human hepatocyte growth factor (HGF) cDNA;AduPA inserted with non secreted human urokinase type plasminogen activator (uPA) cDNA.Methods:The HGF and uPA cDNA were obtained from the plasmids by digestion,and the shuttle plasmids pAdTrack CMV HGF and pAdTrack CMV uPA were established by ligation respectively.Then the linearized shuttle plasmids were co transformed into E.coli BJ5183 with backbone vector AdEasy 1 to obtain the recombinant adenoviral plasmids pAdHGF and pAduPA by homologous recombination severally.After packed in 293 cells,the recombinant adenoviruses AdHGF and AduPA were generated.The expression of HGF or uPA in hepatic cell line L02 was detected by Northern blot and Western blot on the third day after infection.Results:The plasmids pAdHGF and pAduPA were established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing.Green fluorescent protein (GFP) expression could be observed on the third day after packing of the linearized pAdHGF and pAduPA in 293 cells and 4×10 10 efu/ml titer of AdHGF and 6×10 10 efu/ml titer of AduPA were obtained by CsCl gradient purification respectively.When the L02 cells were infected by the viruses for 3 d,the expression of HGF and uPA in hepatocytes increased significantly.Conclusion:These results reveal that the replication deficient recombinant adenovirus can be generated simply and rapidly and express with high efficiency in hepatocyte, which can be used for hepatic fibrosis gene therapy.These 2 kinds of adenovirus may serve as an ideal tool for hepatocyte transplantation and gene therapy in hepatic fibrosis.

关 键 词：肝纤维化 基因治疗 细菌内快速构建 重组复制缺陷型腺病毒 治疗 

分 类 号：R575.2[医药卫生—消化系统]