大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

背景：体外克隆、表达热休克蛋白,尤其正常时少量或不表达,而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要。目的：构建热休克蛋白72基因真核表达载体并于COS7细胞内表达,为HSP72蛋白免疫调节功能的研究奠定基础。方法：采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1（＋）的相应酶切位点,并将重组质粒转染COS7细胞,进行基因表达鉴定。结果与结论：重组质粒插入基因序列检测证实为热休克蛋白72cDNA,并能在COS7细胞稳定表达。成功构建热休克蛋白72真核表达载体,并于COS7细胞中成功转录与表达。

Construction of eukaryotic expression vector carrying rat heat shock protein 72 gene and its expression in COS7 cells

BACKGROUND：In vitro cloning and expression of heat shock proteins （HSP）, especially HSP72, is important in the study of ischemia-reperfusion injuries. OBJECTIVE：To construct eukaryotic expression vector carrying HSP72 and study its transient expression in COS7 cells, and to establish the foundation for further research of the immune function of HSP72. METHODS：HSP72 cDNA was amplified from hepatocytes of BABL/C mice by RT-PCR, and correctly inserted into corresponding sites of eukaryotic expression vector pcDNA3.1（＋）after restriction endonuclease digestion, and the recombinant plasmid was transfected into COS7 cells and gene expression was determined by RT-PCR. RESULTS AND CONCLUSION：The gene fragment inserted into the vector pcDNA3.1（＋）was confirmed by nucleotide sequencing, and the recombinant plasmid mRNA was successfully expressed in COS7 cells.