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目的构建GalR2基因真核表达质粒,并在HeLa细胞中表达。方法从C57BL∕6J小鼠海马组织中提取总RNA,RT-PCR法扩增GalR2基因,克隆至pEGFP-C1载体中,构建重组真核表达质粒pEGFP-GalR2。将重组表达质粒转染至HeLa细胞,荧光显微镜观察融合蛋白在细胞内的定位,RT-PCR和Western blot分别检测GalR2基因的转录及表达。结果从C57BL∕6J小鼠海马组织扩增出1 116 bp的GalR2基因;重组表达质粒pEGFP-GalR2经PCR、双酶切及测序鉴定证明构建正确;转染细胞融合基因的表达产物定位于细胞核,GalR2基因在mRNA及蛋白水平上均有表达。结论已成功构建了GalR2基因真核表达质粒pEGFP-GalR2,并在HeLa细胞中成功表达了GalR2蛋白,为进一步探讨GalR2的生物学活性奠定了基础,也为寻找抗抑郁药物的靶点提供了新的思路。
Objective To construct the eukaryotic expression plasmid of GalR2 gene and express it in HeLa cells. Methods Total RNA was extracted from the hippocampus of C57BL / 6J mice. The gene of GalR2 was amplified by RT-PCR and cloned into pEGFP-C1 vector to construct recombinant eukaryotic expression vector pEGFP-GalR2. The recombinant plasmid was transfected into HeLa cells. The localization of the fusion protein in HeLa cells was observed by fluorescence microscopy. The transcription and expression of GalR2 gene was detected by RT-PCR and Western blot respectively. Results The 1 116 bp GalR2 gene was amplified from the hippocampus of C57BL / 6J mice. The recombinant plasmid pEGFP-GalR2 was confirmed by PCR, double enzyme digestion and sequencing. The expression of the fusion gene was localized in the nucleus, GalR2 gene is expressed both at the mRNA and protein levels. Conclusion The GalR2 gene eukaryotic expression plasmid pEGFP-GalR2 was successfully constructed and the GalR2 protein was successfully expressed in HeLa cells, which laid a foundation for further exploring the biological activity of GalR2 and also provided a new target for finding anti-depressant drugs Train of thought.