论文部分内容阅读
目的:构建ACRBP真核表达载体,建立稳定表达ACRBP的人肝癌细胞株,为后继研究该基因的功能奠定基础。方法:以pMAL-C2/ACRBP重组质粒作为模板进行PCR,扩增出ACRBP编码区cDNA,并与真核表达载体pEGFP-N1连接,构建pEGFP-N1/ACRBP重组质粒。通过Fugene HD将该重组质粒转染至ACRBP表达阴性的肝癌细胞株HepG2,经G418筛选阳性克隆获得稳定转染株。RT-PCR和免疫组织化学方法检测HepG2细胞中ACRBP的表达情况。结果:pEG-FP-N1/ACRBP真核表达载体经测序证实插入序列完全正确;ACRBP基因已经稳定转染至HepG2细胞中并获得表达。结论:成功构建了pEGFP-N1/ACRBP真核表达载体并将其转染HepG2后,能稳定地表达ACRBP。
OBJECTIVE: To construct the eukaryotic expression vector of ACRBP and to establish a human hepatoma cell line stably expressing ACRBP, which lays the foundation for further studies on the function of this gene. Methods: The recombinant plasmid pMAL-C2 / ACRBP was used as template to amplify the cDNA of ACRBP coding region and ligated with eukaryotic expression vector pEGFP-N1 to construct pEGFP-N1 / ACRBP recombinant plasmid. The recombinant plasmid was transfected into HepG2 hepatoma cell line with negative ACRBP expression by Fugene HD, and the positive clone was screened by G418 to obtain a stable transfectant. The expression of ACRBP in HepG2 cells was detected by RT-PCR and immunohistochemistry. Results: The eukaryotic expression vector pEG-FP-N1 / ACRBP was confirmed by sequencing. The inserted sequence of ACRBP gene was stably transfected into HepG2 cells for expression. Conclusion: The eukaryotic expression vector pEGFP-N1 / ACRBP was successfully constructed and transfected into HepG2 cells stably expressing ACRBP.