慢病毒表达双报告基因载体系统的构建及病毒制备

本研究旨在构建萤火虫荧光素酶(firefly luciferase,FLUC)和红色荧光蛋白(red fluorescent protein,RFP)双基因表达的慢病毒载体,并转染特定细胞系(HeLa),观察双基因的表达。采用PCR技术分别扩增FLUC和RFP报告基因,再将报告基因连接到慢病毒表达载体pLenti-Bi-cistronic中,获得具有双报告基因的慢病毒重组质粒pLenti-FLUC-RFP。然后,用脂质体将该慢病毒重组质粒转染293T细胞,收集纯化慢病毒颗粒。最后,测定病毒滴度,并用所获慢病毒感染HeLa细胞,采用荧光显微镜和荧光素酶报告基因检测试剂盒检测RFP和FLUC的表达。结果表明,酶切和测序证明RFP和FLUC基因成功连入目的载体,所构建的pLenti-FLUC-RFP慢病毒重组质粒序列符合预期。经纯化获得的慢病毒滴度为1×107TU/ml。将pLenti-FLUC-RFP转染HeLa细胞系后,在荧光显微镜下可见大量细胞表达RFP,荧光发光检测仪也检测到转染细胞具有较高的荧光素酶活性。结论:本研究成功构建了具有双报告基因的慢病毒重组质粒pLenti-Ⅲ-FLUC-RFP,经纯化获得了高滴度的慢病毒颗粒,该病毒颗粒具有很好的感染活性,为研究间充质干细胞在体内的动态分布奠定了基础。

Reconstruction and preparation of lentiviral vector system expressing dual-reporter genes

This study was aimed to construct,package and purify the recombinant lentivirus vector carrying the firefly luciferase gene(FLUC) and red fluorescent protein gene(RFP) and to transfect the recombinant lentivirus into HeLa cells,so as to observe the expression levels of these two genes.The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector(pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP.The viral particles were generated by cotransfection of 2...