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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 glutathione decarboxylase 65 (GAD65) recombinant lentiviral vector and infect and identify mesenchymal stem cells (MSCs). Methods The GAD65 gene was amplified by PCR, and the LV5-GFP-GAD65 lentiviral vector was constructed. The recombinant plasmid was co-transfected with 293T cell packaging virus. The lentiviral vector was used to infect MSCs. The transfection efficiency was identified by fluorescence microscopy. The expression of GAD65 was detected by Western blot . Results The results of double enzyme digestion and sequencing showed that the construction of LV5-GFP-GAD65 lentiviral vector was successful. The virus titer of packaged virus was 5 × 107TU / ml. The transfection rate of MSCs infected by lentivirus was higher than 90% The results of blot showed that GAD65 protein expression was significantly higher than the control group. Conclusions The recombinant lentiviral vector of GAD65 was successfully constructed and packaged into high concentration virus solution. Infected rat MSCs stably overexpressed GAD65 protein, which laid the experimental foundation for the further exploration of intracerebroventricular injection of genetically modified MSCs for the treatment of epilepsy.