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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。
Objective: To construct a lentiviral vector co-expressing human glucocorticoid receptor (GR) β and green fluorescent protein (GFP). METHODS: Human GRβ cDNA was obtained from human embryonic brain expression library by gene recombination technique. The target fragment was cloned into pGC-LV vector by double enzyme digestion, and the recombinant vector pGC-FU-GRβ was obtained. The correctly sequenced plasmid pGC-FU-GRβ was transfected into 293T cells by Lipofectamine 2000. The lentiviral expression vector was identified by fluorescence detection and Western Blot. 293T cells were transfected with the auxiliary packaging plasmids and the virus liquid was packaged. The titer was determined by Real-time PCR. Results: The human GRβ-GFP recombinant lentiviral vector was successfully constructed. After transfected into 293T cells, a large amount of green fluorescence was observed under fluorescence microscope. The expression of GRβ-GFP fusion protein was detected by Western Blot. Real-time PCR measurement of virus titer was 2.00E +8 TU / ml. CONCLUSION: The lentiviral expression vector co-expressing human GRβ and GFP gene has been successfully constructed. In order to explore the relationship between GRβ and hormone sensitivity and resistance in the treatment of chronic rhinosinusitis and other diseases with hormone as first choice, stable infection Cell carrier.