重组腺相关病毒介导的TIMP-1载体的构建及鉴定

Construction and identification of adeno-associated virus mediated gene transfer of TIMP-1

目的:构建及鉴定载入金属蛋白酶组织抑制因子1(TIMP鄄1)的重组腺相关病毒载体。方法:应用基因重组的方法构建含全长TIMP鄄1的重组腺相关病毒骨架质粒(pAAV鄄TIMP鄄1),利用磷酸钙沉淀法将腺相关病毒载体质粒共转染入HEK293细胞中,分别装配成rAAV鄄TIMP鄄1和rAAV鄄hrGFP,将rAAV鄄hrGFP转导HT1080细胞测定重组病毒的滴度,Westernblot方法检测rAAV鄄TIMP鄄1在HT1080细胞中的表达情况。结果:成功构建rAAV鄄TIMP鄄1,病毒滴度达到109TU/ml,转导细胞后,转导组TIMP鄄1的表达水平高于对照组。结论:构建的rAAV鄄TIMP鄄1,为基因修饰胰岛移植提供了新的手段。

Objective: To construct and identify recombinant adeno-associated virus encoding rat tissue inhibitor of metalloproteinase-1(TIMP-1) full length cDNA. Methods: Using the technique of gene recombination, the main plasmid of pAAV-TIMP-1 was constructed. Recombinant AAV-TIMP-1 was obtained from HEK293 cells, which were cotransfected with AAV vector plasmids by calcium phosphate precipitation method. The titer of recombinant AAV was determined by counting positive HT1080 cells which were transduced with rAAV-GFP. Finally, Western blottings were performed to confirm the expression of rAAV-TIMP-1 in HT1080 cells. Results: The plasmid of pAAV-TIMP-1 was verified by restriction endonuclease digestion and DNA sequencing. The titer of rAAV viral stock could be reached up to 109 TU/ml. Western blotting showed rAAV-TIMP-1 expressed TIMP-1 proteins in HT1080 cells. Conclusion: The development of rAAV provided a tool for TIMP-1 gene altered islets for transplant.