ANALYSIS OF THE TISSUE EXPRESSION PATTERN OF Rab5a AND CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR | Rab5a的组织表达分析及其真核表达载体的构建

中国人. 目的：观察小鼠各脏器中Rab5a表达情况,并构建Rab5a真核表达载体并验证其表达情况。方法：采用RT-PCR和Real-time PCR方法检测Rab5a在小鼠组织中的表达。以小鼠巨噬细胞RAW264.7的cDNA为模板,根据GenBank中公布的小鼠Rab5a全长序列设计引物,扩增Rab5a的开放阅读框。将目的基因与真核表达载体pcDNA3.1/Flag（-）B连接,转化大肠杆菌DH5α,挑取阳性克隆,酶切鉴定后测序。将Rab5a真核表达载体转染RAW264.7细胞,RT-PCR及Real time PCR检测Rab5a表达水平。结果：在小鼠的心、肝、肺、肾、脾、睾丸、小肠和结肠中均有Rab5a表达,在脾和小肠中的表达量最高。Rab5a真核表达载体测序的结果显示与GenBank中报道的序列的同源性为100%。用Rab5a真核表达载体转染RAW264.7后Rab5a的表达量显著高于对照质粒转染细胞。结论：Rab5a广泛表达于小鼠各组织。我们成功构建了Rab5a真核表达载体,并在RAW264.7中成功表达。

英语. Objective： To detect the tissue expression pattern of Rab5a,and construct Rab5a eukaryotic expression vector.Methods： Rab5a mRNA expression in murine tissues was detected by RT-PCR and Real-time PCR.Using the cDNA of RAW264.7 cells as templates,the open reading frame of Rab5a was amplified by primers designed according to the full-length sequence of Rab5a in Genbank.The target gene were connected with the eukaryotic expression vector pcDNA3.1,and transformed into E.coli DH5α.Positive clones were indentified by double restriction enzyme digestion and sequence analysis.Rab5a eukaryotic expression vector was transfected into RAW264.7 cells and the expression of Rab5a was verified RT-PCR and Real-time PCR.Result： Rab5a was widely expressed in murine heart,liver,lung,kidney,spleen,testis,small intestine and colon.The highest expression of Rab5a was found in the small intestine and spleen.Sequence analysis showed that the Rab5a-pcDNA plasmids were 100% homologous with the sequence of Rab5a in Genbank.Rab5a expression in transfected cells was significantly higher than the control cell group.Conclusion： Rab5a was widely expressed in murine tissues.Rab5a eukaryotic expression vector was successfully constructed and verified.