Objective To construct human cholesterol ester hydrolase (hCEH) eukaryotic expression vector pEGFPN1 -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.%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-02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-Nl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1.7kb的片段,与预期片段大小相符;以pEGFP-Nl为载体,成功构建重组表达质粒pEGFP-Nl-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-Nl-hCEH大量表达.
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