人糖皮质激素受体β与绿色荧光蛋白融合基因共表达慢病毒载体的构建

目的:构建人糖皮质激素受体(GR)β与绿色荧光蛋白(GFP)共表达的慢病毒载体。方法:采用基因重组技术从人胚脑表达文库内获得人GRβcDNA,双酶切法将目的片段克隆入pGC-LV载体中,获得重组载体pGC-FU-GRβ。测序正确的质粒pGC-FU-GRβ通过脂质体Lipofectamine 2000转染293T细胞。通过荧光检测和Western Blot检测鉴定慢病毒表达载体质粒。与辅助包装质粒转染293T细胞,包装后产生病毒液,Real-timePCR法测定其滴度。结果:成功构建人GRβ-GFP重组慢病毒表达载体,转染293T细胞后,荧光显微镜下可见大量绿色荧光,Western Blot检测到GRβ-GFP融合蛋白的表达。Real-time PCR测定病毒滴度为2.00E+8TU/ml。结论:成功构建了人GRβ与GFP基因共表达的慢病毒表达载体,为探讨以激素为首选药物的慢性鼻-鼻窦炎等疾病治疗中GRβ与激素治疗敏感及抵抗的关系,提供了稳定的感染细胞载体。

Construction of GRβ and GFP gene co-expressing lentivirus vector

Objective:To construct the co-expressing lentivirus vector of human glucocorticoid receptor(GR) β and green fluorescent protein(GFP).Method:cDNA encoding hGRβ obtained from the expression library of fetal brain using gene recombinant technology was cloned into pGC-LV by double digests technology.The constructed lentivirus vector of pGC-FU-GRβ that was confirmed the sequencing was correct,transfected 293T cells through lipofectamine 2000.The constructed lentivirus vector was identified by fluorescence detection and Western Blot method.The packed lentivirus vector was used to infect 293T cells.The titer of virus was tested by real-time quantitative PCR.Result:A recombinant lentivirus vector co-expressing hGRβ and GFP gene was constructed successfully.After transfection,a large number of 293T cells with green fluorescence were observed under fluorescent microscope,and the expression of GRβ and GFP fusion protein was detected by Western Blot.The virus titer was 2.00E+8 TU/ml tested by Real-time PCR.Conclusion:Successful construction of hGRβ and GFP co-expressing lentivirus vector provides a stable vector for investigating the relationship between GRβ and hormonal sensitivity or resistance in the therapy of chronic rhinosinusitis whose choice drug are glucocorticoids.