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

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。

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

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.