Rab7真核表达载体的构建及其在小鼠巨噬细胞RAW264.7细胞中的表达

Cloning and expression of small G protein Rab7 in RAW264.7 cells

目的构建Rab7的真核表达载体,研究其在小鼠巨噬细胞RAW264.7中的表达。方法 RT-PCR方法分析Rab7在组织和细胞中的表达。以RAW264.7细胞的cDNA为模板,根据GenBank公布的小鼠Rab7全长序列设计引物,扩增Rab7基因的开放阅读框。将其与真核表达载体pcDNA3.1(-)连接,构建pcDNA-Rab7重组质粒。用脂质体将Rab7真核表达载体瞬时转染RAW264.7细胞,RT-PCR和Western blot法鉴定Rab7的过表达。结果 Rab7重组载体测序结果与GenBank中报道的Rab7序列的同源性为100%。RT-PCR和Western blot结果显示Rab7真核表达载体瞬时转染RAW264.7细胞后,Rab7表达显著增高。结论成功构建了Rab7真核表达载体。为今后研究Rab7在巨噬细胞中的功能奠定了基础。

Objective To construct Rab7 eukaryotic expression plasmid and analyze its expression in murine macrophage cell line RAW264.Methods The expression of Rab7 in tissues and cells were detected by RT-PCR.Using the cDNA of RAW264.7 cells as template,the open reading frame of Rab7 was amplified by primers designed according to the full-length sequence of Rab7 published in Genbank.The target gene was connected with the eukaryotic expression vector pcDNA3.1 to get pcDNA-Rab7.The recombinant eukaryotic vector of Rab7-pcDNA were transiently transfected into Raw264.7 cells by liposome,and the over expression state of Rab7 were indentified by RT-PCR and Western blot.Results Sequence analysis showed that the Rab7-pcDNA plasmid was 100% homologous with the sequence of Rab7 in Genbank.The expression of Rab7 was increased significantly as verified by RT-PCR and Western blot.Conclusion The eukaryotic expression vector of Rab7-pcDNA is successfully constructed.This study may lay a foundation for further elucidating the function of Rab7 in macrophages.