PRP基因绿色荧光蛋白载体构建及其生物学功能初步研究

Construction of the eukaryotic expression vector of proliferin related protein and preliminary study on its biological activity in 293FT cells

目的:构建Prolifein related protein(PRP)基因荧光表达载体,并初步探讨其生物学功能。方法:提取小鼠睾丸RNA,RT-PCR扩增PRP基因编码区片段,酶切连入pEGFP-C1载体,构建含有PRP基因片段的绿色荧光表达载体PRP-pEGFP-C1。为了探讨PRP基因功能,PRP-pEGFP-C1经Lipofectamine 2000介导转染293FT细胞,MTT实验分析细胞增殖变化,流式细胞仪分析细胞周期分布。结果:质粒PRP-pEGFP-C1经测序验证,证实PRP基因已正确连入pEGFP-C1载体。免疫荧光染色显示PRP定位于293FT细胞胞浆。MTT结果表明,上调PRP基因引起293FT细胞增殖活性下降,细胞周期分布情况提示G1期细胞增加,S期细胞减少。结论:成功构建PRP-pEGFP-C1荧光表达载体,对其功能研究表明PRP具有抗人293FT细胞增殖,扰乱细胞增殖周期的功能。本研究为PRP在抗人肿瘤方面的研究提供基础。

Objective： To construct the eukaryotie expression vector of proliferin related protein, and to investigate its biological function. Methods：The coding region of proliferin related protein amplified by RT-PCR from mouse testis RNA was cloned into the pEGFP-C 1 vector to form the recombinant PRP-pEGFP-C1 plasmid. Then the recombinant plasmid was transfected into 293FF cells, and the expression of PRP was detected by RT-PCR and Western blotting. MTY and flow cytometry （ FCM ） analysis were employed to analysis the cell proliferation activity and the cell cycle, respectively. Results： PRP coding region was successfully cloned into the eukaryotic vector pEGFP-C1. The recombinant vector was transfected into 293FF, results of RT-PCR and Western blotting showed that the PRP could express in 293FF ceils. Overexpressing PRP gene decreased the cell proliferation activity as well as disturbing the cell cycle distribution of 293FF ceils in vitro. Conclusion： We have constructed a recombinant vector expressing PRP and eGFP fusion protein, which is in favour of the further study on the gene function of PRP.In addition, PRP has anti-proliferin activity in human-derived cells through disturbing the cell cycle, suggesting that PRP maybe act as a potential gene therapeutic candidate used in human tumor, not lust mouse tumor.