大鼠GAD65基因慢病毒载体的构建及其在MSCs中的表达

Construction of lentiviral vectors carrying GAD65 gene and its expression in bone marrow mesenchymal stem cells

目的构建谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)重组慢病毒表达载体,感染间充质干细胞(mesenchymal stem cells,MSCs)并进行鉴定。方法 PCR法扩增GAD65基因,构建LV5-GFP-GAD65慢病毒载体;与包装质粒共转染293T细胞包装病毒;将慢病毒感染大鼠MSCs,荧光显微镜鉴定转染率,Western blot检测GAD65的表达。结果双酶切及测序结果表明LV5-GFP-GAD65慢病毒载体构建成功;包装病毒产生的病毒液滴度为5×107TU/ml;慢病毒感染大鼠MSCs的转染率高于90%,Western blot结果显示GAD65蛋白表达比对照组明显升高。结论 GAD65重组慢病毒载体构建成功,包装得到高浓度病毒液,感染大鼠MSCs能稳定过表达GAD65蛋白,为进一步探索侧脑室注射基因化的MSCs治疗癫痫奠定实验基础。

Objective To construct recombinant lentiviral vectors carrying GAD65 gene and examine their expres- sion in bone marrow mesenchymal stem cells（MSCs）. Methods GAD65 gene was amplified by PCR to construct the re- combinant plasmid LVS-GFP-GAD65 together with lentiviral package plasmid to produce the lentiviral partieles;MSCs was infected with the lentivirus and the transfection efficiency was assessed under fluorescent microscope using western Western blot to examine the expression of the GAD65. Results The results of double enzyme digestion and sequence proved that the recombinant lentiviral vector was correctly constructed;The virus in the supernatant reached a titer of 5 x 107 TU/ml and transfection efficiency of the infected MSCs by virus exceeded 90% , Western blot analysis showed that the expression of GAD65 protein was significantly higher than that of the control group. Conclusion The lentiviral vector for GAD65 has been successfully constructed with a high yield of lentivirus. Infected MSCs has been stably overexpressed the GAD65 pro- tein, which laid the experimental foundation to further explore the genes of intraeerebroventricular injection of mesenchymal stem cells in epilepsy treatments.