人胆固醇酯水解酶真核表达载体的构建及U937细胞系的转染  

Construction of eukaryotic vector pEGFP-N1-hCEH and establishment of stable transfectant U937 cell line

作　　者：顾立学[1] 罗俊生[1] 王瑞[1] 冯旭[1] 关宁[1] 霍晓川[1] 

机构地区：[1]辽宁医学院附属第一医院,辽宁锦州121001

出　　处：《山东医药》2011年第26期14-16,共3页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(30971023);辽宁省自然科学基金资助项目(20092190);辽宁省教育厅高等学校科研项目(2009A481)

摘　　要：目的构建人胆固醇酯水解酶(hCEH)绿色荧光蛋白真核表达载体pEGFP-N1-hCEH,并观察其转染单核巨噬细胞株U937后,细胞中hCEH的表达情况。方法以人肝细胞L-O2总RNA为模板,采用RT-PCR技术扩增hCEH编码区序列,将扩增片段插入到pMD19-T载体中,回收、纯化目的片段后亚克隆到pEGFP-N1真核表达载体上,经双酶切、测序鉴定后,转染U937,观察U937中绿色荧光蛋白的表达情况。结果 RT-PCR扩增hCEH基因的编码区序列获得1条约1.7 kb的片段,与预期片段大小相符;以pEGFP-N1为载体,成功构建重组表达质粒pEGFP-N1-hCEH,该质粒可以在U937中表达。结论成功构建pEGFP-N1-hCEH,用其转染U937后,细胞中pEGFP-N1-hCEH大量表达。Objective To construct human cholesterol ester hydrolase（hCEH） eukaryotic expression vector pEGFP-N1-hCEH,and observe their monocyte-macrophage cell line U937 expression.Methods Human liver cell lines L-O2 total RNA as template,amplified by RT-PCR applications hCEH coding sequence,the amplified fragments were inserted into pMD19-T vector,recovery,purification fragment was subcloned into pEGFP-N1 eukaryotic expression vector by restriction enzyme digestion,DNA sequencing,the transfected monocyte-macrophage cell line U937,and observe its expression in U937 cells.Results RT-PCR amplification of gene coding sequence hCEH obtain a 1.7 kb fragment of the treaty,in line with the expected fragment size.Recombinant plasmid pEGFP-N1-hCEH was successfully constructed with pEGFP-N1 as carrier.Gene transfection experiments showed that the recombinant express in U937 cells.Conclusions The eukaryotic expression vector pEGFP-N1-hCEH is successfully constructed,and express in U937 cells after transfection.

关 键 词：人胆固醇酯水解酶 真核表达载体 单核巨噬细胞 U937细胞 转染 

分 类 号：R543.5[医药卫生—心血管疾病]