葡萄糖调节蛋白75基因突变体及其真核表达载体构建

Construction of eukaryotic expression vector of glucose-regulated protein 75 gene deletion mutant and its expression in PC12 cells

葡萄糖调节蛋白75(glucose-regulated protein75,Grp75)与细胞内的p53结合抑制其核移位,起到了保护细胞的作用。为研究Grp75和p53的结合与否是如何影响细胞活力,现构建Grp75缺失突变基因的真核表达载体。以SOE-PCR(genesplicing by overlap extension)法得到Grp75缺失突变基因,与pcDNA3.0真核表达载体连接,构建Grp75缺失突变的特异性表达载体pcDNA3.0/Grp75(Δ253-282),经酶切、测序鉴定Grp75缺失突变蛋白表达载体成功构建。用脂质体将pcDNA3.0/Grp75(Δ253-282)转染到PC12细胞株,以1mg/mL浓度的G418筛选出稳定株,并命名为PC12/Grp75(Δ253-282)(+)。半定量RT-PCR和Western blot结果显示PC12/Grp75(Δ253-282)(+)细胞组内Grp75的mRNA和蛋白的表达水平较PC12细胞组增高。对PC12细胞组、PC12/Grp75(Δ253-282)(+)细胞组和PC12/Grp75(+)细胞组(pcDNA3/Grp75真核表达载体已构建)分别缺糖0h、3h、9h、18h和36h,MTT法检测各组细胞活力,Hoechst33324法检测细胞凋亡情况。结果显示PC12/Grp75(+)组细胞活力高于其它两组,PC12/Grp75(Δ253-282)(+)组高于PC12组,差异有统计学意义。Hoechst33324染色后,凋亡细胞的比率与MTT细胞活力检测结果基本一致。Western blot检测三种细胞内p53的表达量,PC12/Grp75(+)细胞内p53蛋白表达量低于另外两组,可能是由于Grp75蛋白量的增多部分抑制了p53的表达,提示Grp75对缺糖诱导细胞凋亡的抑制作用部分与p53的结合有关。以上结果表明Grp75基因的缺失突变在一定程度上降低了细胞的活力。

Glucose-regulated protein 75 （Grp75） binds to p53 and inhibits its nuclear translocation, and thus plays a role in cell protection. To investigate whether the binding of Grp75 and p53 would influence the viability of cells, we constructed the eukaryotic expression vector of Grp75 deletion mutant. The deletion mutant gene was obtained by SOE-PCR （gene splicing by overlap extension） and then linked to the pcDNA3.0 vector. The constructed specific expression vector, pcDNA3.0/Grp75（Delta253-282）, was identified by restriction enzymes and sequencing. Then we used liposome to transfect the specific vector into PC12 cells. The stable cell strain PC12/Grp75（Delta253-282）（＋） was selected by G418 （1 mg/mL）. Semi-quantitative RT-PCR and Western blot showed that Grp75 mRNA and protein expressions in PC12/Grp75（Delta253-282）（＋） cells were higher than those in PC12 cells. The viability of cells undergoing 0 h, 3 h, 9 h, 18 h and 36 h of glucose deprivation respectively was measured by MTT assay. The results showed that the cell viability of PC12/Grp75（＋） group was significantly higher than that of the other two groups, and the cell viability of PC12/Grp75（Delta253-282）（＋） group was significantly higher than that of the PC12 group （P〈0.05）. Hoechst33324 staining was employed to detect cell apoptosis and the results were consistent with the MTT assay results. Western blot results indicated that the expression of p53 in PC12/Grp75（＋） cells was lower than those in the other two groups, which might be due to the overexpression of Grp75. These results suggest that the protective role of Grp75 is partly associated with its binding to p53. The above results suggest that Grp75 deletion mutation could to some extent reduce the viability of cells.