免疫基因CD1d和绿色荧光蛋白融合基因慢病毒载体的构建

目的:构建人免疫基因CD1d与绿色荧光蛋白(GFP)融合基因慢病毒载体。方法:实时定量聚合酶链反应(RT-PCR)方法扩增获得CD1d的全外显子片段,使之克隆到带GFP荧光报告基因慢病毒表达载体质粒中,慢病毒包装质粒和穿梭质粒转染293T细胞,包装成功后收集上清,浓缩,鉴定。取浓缩纯化后的病毒上清感染293T细胞,荧光显微镜观察荧光表达。结果:电泳鉴定结果与目的基因表达条带完全吻合,克隆测序结果与NCBI收录的CD1d基因序列完全一致。重组慢病毒质粒可高效转染293T细胞,荧光显微镜下可观察到大量绿色荧光。结论:成功构建了CD1d与GFP融合基因慢病毒表达载体,为进一步研究CD1d基因的相关功能提供了适合的稳定转染载体。

Construction of lentivirus carrying immunogene CD1d and green fluorescent protein gene

Objective： To construct lentivirus carrying both human immunogene CD1d and green fluorescent protein （GFP） gene. Methods： The fragments containing all the exons of CD1d were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP. The lentivirus was packaged and was used to transfect 293T cells together with pShuttle. The super- natant of virus-producing cells was harvested, concentrated, identified, and was used to infect 293T cells, Fluorescent microscopy was used to observe the fluorescence in the 293T cells. Results： Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis. The lentivirus effectively transfected 293T cells. Strong green fluorescence was observed by fluorescent microscopy. The supernatant of lentivirus-transfected 293T cells effectively infected 293T cells. Conclusion： The lentivirus vector containing CD1d-GFP recombinant gene have been successfully constructed, which provides a further study of CD1d function.