顶体素结合蛋白真核表达载体的构建及其在肝癌细胞株的表达  

Construction of eukaryotic expression vector encoding ACRBP and its expression in hepatocarcinoma cells

作　　者：罗彬[1] 云翔[2] 林永达[1] 肖绍文[2] 闫光华[1] 何少健[1] 贺菽嘉[3] 陈芳[1] 谢小薰[4] 

机构地区：[1]广西医科大学组织胚胎学教研室,广西南宁530021 [2]广西医科大学第一附属医院,广西南宁530021 [3]广西医科大学生化教研室,广西南宁530021 [4]广西医科大学

出　　处：《细胞与分子免疫学杂志》2011年第10期1072-1074,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30760055,81060207);广西高发疾病研究创新性团队基金资助项目(桂教人[2007-71]号);广西自然科学基金(桂科自0832144);广西教育厅立项项目(201010LX029),广西教育厅研究生创新计划项目(2009105981001M190);广西医学科学实验中心开放项目(KFJJ2010-33);广西大型仪器网协助项目(6912008104)

摘　　要：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。AIM： To construct the eukaryotic expression vector pEGFP-N1/ACRBP and stably express ACRBP in human hepatocarcinoma cells, providing functional clues for ACRBP. METHODS： A recombinant plasmid pMAL-C2/ACRBP was used as a template to amplify ACRBP cDNA. The PCR product was ligated into an eukaryotic expression vector pEGFP-N1 to construct a recombinant plasmid pEGFP-N1/ACRBP. Then the pEGFP-N1/ACRBP was transfected by Fugene HD into ACRBP-negative HepG2 cells. The stably transfected clones were selected by G418. RT-PCR and immunohistochemistry were used to detect the expression of ACRBP in HepG2 cells. RESULTS： The eukaryotic expression vector pEGFP-N1/ACRBP was constructed and confirmed by sequencing. The stably transfected HepG2 cells expressed ACRBP. CONCLUSION： The eukaryotic expression vector pEGFP-N1/ACRBP has been successfully constructed and transfected into HepG2 cells, resulting in stable expression of ACRBP.

关 键 词：ACRBP 基因克隆 转染 

分 类 号：R392.11[医药卫生—免疫学]