过表达HSPC238对Bel7402细胞周期的影响

Effect of HSPC238 overexpression on cell cycle of human hepatoma carcinoma cell line Bel7402

目的:构建pcDNA3.1(-)/HSPC238真核表达载体,研究过表达HSPC238基因对人肝癌细胞株Bel7402细胞周期的影响。方法:采用PCR法从pET28b/HSPC238重组质粒中克隆得到HSPC238 cDNA全长序列,并将该片段亚克隆到真核表达载体pcDNA3.1(-)中,构建pcDNA3.1(-)/HSPC238真核表达质粒。经酶切和测序鉴定后,采用脂质体法将该重组质粒转染Bel7402细胞,随后经G418筛选4周,得到阳性克隆细胞株。用RT-PCR和Western blot技术分别检测HSPC238基因在Bel7402中的mRNA和蛋白水平的表达。随后用饥饿法使各组细胞周期同步化,再用流式细胞仪分析HSPC238过表达对细胞周期的影响。结果:pcDNA3.1(-)/HSPC238经酶切鉴定及DNA测序证实,目的基因HSPC238的序列完全正确;RT-PCR和Western blot检测显示,与转染pcDNA3.1(-)组和未转染组相比较,转染pcDNA3.1(-)/HSPC238组的mRNA和蛋白表达水平均明显增高,但前两者间没有差异。用饥饿法同步化后,流式细胞仪检测显示过表达HSPC238可延缓Bel7402细胞株从G1期进入S期。结论:成功构建了pcDNA3.1(-)/HSPC238真核表达载体,并在Bel7402中表达。初步发现过表达HSPC238可延缓肝癌细胞的细胞周期,为更进一步研究HSPC238蛋白的功能奠定了基础。

Objective：To construct the recombinant eukaryotic expressing vector haboring human gene HSPC238,and to study the effect of HSPC238 overexpression on the cell cycle of Bel7402.Methods：The cDNA of HSPC238 was amplified from the recombinant plasmid pET28b/HSPC238 by PCR method.After sequencing,the target gene frame of HSPC238 cDNA was subcloned into eukaryotic expression vector pcDNA3.1（-）.The recombinant plasmid pcDNA3.1（-）/HSPC238 was transfected into Bel7402 cell line by lipofectamine 2000.Positive clones were screened by G418 for 4 weeks.Then,the mRNA and protein expression levels of HSPC238 in positive clones were detected by RTPCR and Western blot,respectively.Finally,cell cycle was analyzed by flow cytometry after synchronization of cell cycle by starvation method.Results：The construction of the recombinant plasmid pcDNA3.1（-）/HSPC238 was proved by restriction enzyme digestion analysis and DNA sequencing.Compared with pcDNA3.1（-） transfected group and untransfected group,the expression of HSPC238 in pcDNA3.1（-）/HSPC238 transfected group was significantly increased both in mRNA level and in protein level.Flow cytometry suggested that HSPC238 could postpone the velocity of HSPC238-transfected Bel7402 cell from phase G1 into phase S.Conclusion：The eukaryotic expression plasmid pcDNA3.1（-）/HSPC238 has been successfully constructed and HSPC238 is overexpressed in Bel7402 cell line.Our research suggests that overexpression of HSPC238 could postpone the cell cycle of Bel7402.This will benefit further studies on the function of human gene HSPC238.