Rho鸟苷酸解离抑制因子-2过表达载体的构建与慢病毒包装

Construction and package of a Rho GDI_2 lentiviral expression vector

目的:为研究β2肾上腺素能受体减敏的机制,构建Rho鸟苷酸解离抑制因子-2(Rho GDI2)过表达慢病毒载体。方法:GenBank搜索Rho-GDI2基因序列(Gene ID:14570,序列号:NM_008113),设计PCR引物扩增目的基因,双酶切后凝胶电泳回收酶切产物。质粒双酶切获得线性载体,将目的基因和线性载体进行琼脂糖凝胶电泳分离回收目的条带。再行目的基因和载体的连接,连接产物转化感受态细胞;菌液PCR阳性克隆鉴定并抽提质粒并测序。最后293T细胞内包装慢病毒质粒并测其病毒活力与滴度。结果:通过DNAStar SeqMan软件对测序结果进行分析,测序结果与目标序列一致。病毒质粒转染细胞效率较高,病毒活力较高,通过标准曲线测得病毒滴度pLenti-GDI慢病毒液:1.22×108vp/mL。结论:成功构建了Rho GDI2过表达慢病毒载体。

Objective： Construction of Rho GDI2 lentiviral expression vector in order to shed light on the mechanism of beta-2 adrenergic receptor desensitization. Methods： Rho guanine nucleotide dissociation-inhibitor-2（GDI2） gene sequence was obtained from GenBank （Gene ID： 14570,NM_008113）, and primers for the target gene were thereafter designed. PCR products following amplification were double-enzyme digested and purified by agarose gel electrophoresis. Also, the plasmid was linearized and subsequently enzymatically connected with the target genc. Then the vectors were transformed into compe- tent cells, and suffered to identification by PCR. The positive plasmid was extracted and sequenced. The constructed lentivi-ral vector was further packaged, and the vitality and titer of virus were tested. Results： Sequencing results were analyzed us-ing DNAStar SeqMan software, and showing a consistency with target sequence. The transfection efficiency of packaged virus was very high and the vitality was favorable. Viral titer was calculated up to 1.22×10^8 vp/mL through calculation from stan-dard curve. Conclusion： We are successful in construction and package of a Rho GDI2 lentiviral expression vector.