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目的构建重组质粒载体pc DNA3.1-RPL5,观察核糖体蛋白L5(RPL5)在293T、Hep G2和SMMC7721细胞株中的定位和表达情况。方法使用基因合成法合成RPL5基因,在其N端添加Kozak序列及His标签序列,扩增后的目的基因双酶切后连接至pc DNA3.1载体的Bam HⅠ和NotⅠ位点之间,从而构建重组质粒pc DNA3.1-RPL5。将后者转化DH5α大肠杆菌,培养后挑取阳性克隆子进行质粒抽提电泳、测序比对等鉴定。采用脂质体法将鉴定正确的重组质粒转染至293T、Hep G2和SMMC7721细胞株,激光共聚焦显微镜观察RPL5在此3种细胞中的表达和定位情况。结果重组质粒pc DNA3.1-RPL5经酶切电泳及DNA测序比对证实,目的基因RPL5的序列完全正确,重组质粒载体构建成功。激光共聚焦观察发现RPL5主要表达于293T、Hep G2和SMMC7721细胞的胞质中。结论成功构建pc DNA3.1-RPL5融合基因并在293T、Hep G2和SMMC7721细胞中表达,证实RPL5主要表达在真核细胞的细胞质中,为进一步探讨RPL5的生物学功能奠定了基础。
Objective To construct recombinant plasmid pcDNA3.1-RPL5 and investigate the localization and expression of ribosomal protein L5 (RPL5) in 293T, Hep G2 and SMMC7721 cell lines. Methods The RPL5 gene was synthesized by gene synthesis. The Kozak sequence and His-tag sequence were added to the N terminus. The amplified target gene was double-digested and ligated into the Bam HI and Not I sites of pcDNA3.1 vector to construct Recombinant plasmid pcDNA3.1-RPL5. The latter was transformed into E. coli DH5α, picked after positive clones for plasmid extraction and electrophoresis, DNA sequencing and identification. The recombinant plasmids were transfected into 293T, Hep G2 and SMMC7721 cell lines by lipofectamine. The expression and localization of RPL5 in these three kinds of cells were observed by laser confocal microscopy. Results The recombinant plasmid pcDNA3.1-RPL5 was confirmed by restriction enzyme digestion and DNA sequencing. The sequence of the target gene RPL5 was completely correct and the recombinant plasmid vector was successfully constructed. Confocal laser scanning revealed that RPL5 was mainly expressed in the cytoplasm of 293T, Hep G2 and SMMC7721 cells. Conclusion The pcDNA3.1-RPL5 fusion gene was successfully constructed and expressed in 293T, Hep G2 and SMMC7721 cells. It was confirmed that RPL5 mainly expressed in the cytoplasm of eukaryotic cells, which laid the foundation for further study on the biological function of RPL5.