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目的构建含有增强型绿色荧光蛋白(EGFP)报告基因的睾丸特异性新基因T490超表达质粒pDT490-EGFP,检测其在Hela细胞中的表达情况。方法采用RT-PCR的方法从成年Balb/c小鼠睾丸组织中扩增T490基因,将该基因连接克隆到含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1上,再将T490和EGFP融合基因酶切下来,克隆到pcDNA3.1(-)真核超表达载体上,以构建重组质粒pDT490-EGFP。将该重组质粒通过阳性脂质体Lipofectamine 2000导入Hela细胞系中,在荧光显微镜下观察转染24h后融合基因的超表达情况,并用RT-PCR的方法进一步证实目的基因mRNA的表达水平。结果RT-PCR获取编码T490基因的全序列cDNA,大小为508bp;DNA测序和PCR方法证实T490和EGFP融合基因成功克隆到真核超表达载体上;在荧光显微镜下,转染24h后的Hela细胞具有很强的绿色荧光;RT-PCR显示转染后的Hela细胞T490基因mRNA的表达明显。结论重组质粒pDT490-EGFP构建成功,并在细胞内明显表达,此质粒可应用于研究睾丸特异性新基因T490的功能,为T490的生物学研究奠定基础。
Objective To construct a novel testis-specific gene T490 overexpression plasmid pDT490-EGFP containing enhanced green fluorescent protein (EGFP) reporter gene and detect its expression in Hela cells. Methods The T490 gene was amplified from adult Balb / c mouse testis by RT-PCR. The gene was cloned into the eukaryotic expression vector pEGFP-N1 containing the enhanced green fluorescent protein (EGFP) reporter gene T490 and EGFP fusion gene were digested and cloned into pcDNA3.1 (-) eukaryotic overexpression vector to construct recombinant plasmid pDT490-EGFP. The recombinant plasmids were transfected into Hela cell line by Lipofectamine 2000, and the overexpression of fusion gene was observed by fluorescence microscopy. The expression of target gene mRNA was further confirmed by RT-PCR. Results The full-length cDNA encoding T490 gene was obtained by RT-PCR and the size was 508bp. The fusion gene of T490 and EGFP was successfully cloned into eukaryotic overexpression vector by DNA sequencing and PCR. Under the fluorescence microscope, Hela cells transfected 24h With strong green fluorescence; RT-PCR showed that the transfected Hela cells T490 mRNA expression. Conclusion The recombinant plasmid pDT490-EGFP was successfully constructed and expressed in cells. This plasmid can be used to study the function of testicular-specific new gene T490 and lay the foundation for the biological study of T490.