PENK基因过表达克隆载体构建及其功能意义

Construction of Recombinant Over Expression Vector of PENK Gene and the Function Study of PENK Gene

目的构建人前脑啡肽(homo sapiens proenkephalin,PENK)基因过表达克隆载体,研究其功能意义。方法用HIV载体质粒与PENK基因连接,构建成功后用双酶切及测序验证PENK克隆重组体的成功构建。用293Ta包装慢病毒后转染HT1080以测定慢病毒滴度。用PENK-HIV慢病毒颗粒转染PC12细胞,同步设立对照组(未转染PENK),对两组细胞转染后48h采集图片并经行细胞计数,收集细胞,用RT-PCR检测各组中PENK mRNA表达变化。结果 PENK克隆载体酶切及测序结果都验证了克隆构建成功。PENK-HIV慢病毒成功转染PC12细胞48h后,对照组细胞数为88.60±2.55,而PENK转染组细胞数为127.93±2.48,差异有统计学意义(P<0.01)。结论用HIV载体质粒成功构建的PENK克隆重组体可能会促进PC12细胞的生长。

Objective To construct recombinant over expression vector of Homo sapiens proenkephalin （PENK） gene and explore the function of PENK gene. Methods Fragment containing PENK ORF gene was inserted into vector plasmid HIV, then the recombinant over was confirmed by enzyme digestion and sequencing. Lentivirus containing the recombinant over expression vector was produced by virus packaging with 293Ta cell,and then the lentivirus was transfected into HT1080 cell and the virus titer was estimated. The PCl2 were tansfected with resulting lentivirus and un-transfected PC12 cells as control. The images of the PC12 cells were captured at 48 h post-transfection and the number of cells was also evaluated; the changes of PENK mRNA in transfection and control group were measured with RT-PCR. Results The constructed PENK ORF recombinant over expression vector was confirmed by enzyme digestion and sequencing. The number of PCl2 cells in transfection and control group at 48 h post-transfection was 127.93 ± 2.48 and 88. 60 ± 2. 55 respectively, and the statistical difference between them was observed （P〈0. 01）. Conclusion Recombinant over expression vector of PENK gene was successfully constructed and the PENK gene can promote the growth of PCl2.