miR-455慢病毒表达载体的构建与鉴定

Construction and Function Identification of Recombinant Lentivirus Vector Expressing has-miR-455

目的:构建人源microRNA-455(miR-455)慢病毒载体,并鉴定成熟has-miR-455在细胞内的表达水平。方法:提取SiHa细胞中的人基因组DNA,设计并合成人miR-455的上下游引物,PCR扩增目的基因,将其中表达miR-455的结构经酶切后插入慢病毒转移质粒pWPT-GFP,构建成pWPT-GFP-pri-miR-455,在293T细胞中与pMD2G、pSPAX2包装产生慢病毒,并用含慢病毒的上清感染SiHa细胞。结果:测序结果证明插入质粒载体中的miR-455前体序列完全正确,慢病毒载体构建成功并获得相应的慢病毒;重组慢病毒质粒pWPT-GFP-pri-miR-455感染SiHa细胞后上调miR-455的表达近40倍。结论:构建了miR-455的慢病毒载体,并能在293T细胞中表达,产生的慢病毒能成功感染SiHa细胞。为进一步研究miR-455的功能,以及利用慢病毒进行基因治疗奠定了基础。

Objective： To construct a lentivirus vector expressing microRNA-455（miR-455） and transfect the vector into SiHa cell line. Methods： Human miR-455 gene with its promoter was amplified by PCR from human SiHa cells genomic DNA. We constructed a lentivirus vector pWPT-GFP containing pri-miR-455 transcript. After having been conformed, the pWPT-GFP-pri-miR-455 plasmid was co-transfected with framework plasmid pMD2G, pSPAX2 into 293T cells to obtain the miR-455 lentivirus vector. After the lentivirus expression vector pWPT- GFP-pri-miR-455 was transduced into SiHa cells, the transduction efficiency was determined by real-time PCR. Results： The inserted pre-miR-455 sequence of plasmid vectors was absolutely correct confirmed by sequencing re- suits, miR-455 was notable up-regulation after transfecting SiHa cells with recombinant lentivirus vector pWPT- GFP-pri-miR-455. Conclusion： The recombinant lentivirus vector for miR-455 was successfully constructed which expressed in 293T cells, and then infected with SiHa cells. The study may lay a foundation on further researehing the function of miR-455 and gene therapy by lentivirus .