葡萄糖调节蛋白75基因突变体及其真核表达载体构建.pdfVIP

生理学报 Acta Physiologica Sinica, December 25, 2009, 61 (6): 533-538 533 研究论文 葡萄糖调节蛋白75 基因突变体及其真核表达载体构建 郭纬纬，杨玲，刘晓宇，刘 雯，左伋* 复旦大学上海医学院细胞与遗传医学系，上海 200032 摘 要：葡萄糖调节蛋白75 (glucose-regulated protein 75, 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 (gene splicing 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1 mg/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0 h 、3 h 、9 h 、18 h 和36 h ，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。 关键词：葡萄糖调节蛋白7 5 ；缺失突变；真核表达载体；细胞活力 中图分类号：Q 2 -3 3 Construction of eukaryotic expression vector of glucose-regulated protein 75 gene deletion mutant and its expression in PC12 cells GUO Wei-Wei, YANG Ling, LIU Xiao-Yu, LIU Wen, ZUO Ji* Department of Cellular and Genetic Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China Abstract: 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(Δ253-282), was id