具有抑制肿瘤前景基因-人MCPIP基因的克隆及细胞定位

Cloning and cell localization of a new potential anti-tumor gene-human MCPIP gene

目的构建人MCPIP基因的真核表达载体,观察其在人胚肾细胞系(HEK293T)细胞中的表达及其定位特征。方法佛波酯(PMA)刺激单核细胞THP-1转化为巨噬细胞,提取细胞mRNA,反转录为cDNA作为模板,扩增MCPIP基因编码区序列,克隆入真核细胞载体中,酶切鉴定目的基因。将重组质粒转染HEK293T细胞,Westernblot检测MCPIP蛋白质表达状况,激光共聚焦显微镜下直接观察基因定位情况。结果酶切鉴定和测序证明,成功构建MCPIP真核表达载体。Westernblot检测表明MCPIP真核表达载体转染293T细胞表达大小约为70kD的蛋白质。激光共聚焦显微镜下直接观察RFP-MCPIP表达可见在293T细胞中主要为细胞质中点状分布。结论本研究成功构建了MCPIP真核表达载体,检测到MCPIP在巨噬细胞中高表达,并成功证实其细胞质点状定位的特征。

Objective To constrcuct eukaryotie expression vector of human MCPIP gene,and to detect its expression and localization in HEK293T cell lines. Methods The coding sequence of MCPIP gene was amplified from THP-1 cell lines treated with PMA for 3 days. The fragment was inserted into eukaryotic expression vector,and verified by digestion and DNA sequencing. The expression and localization of MCPIP gene in HEK293T cells was assayed by western blot and confocal microscope,respectively. Results Digestion and DNA sequencing proved that the recombinant plasmids of MCPIP were correct,and western blot showed that MCPIP expressed a protein of about 70kD. RFP-MCPIP distributed mainly in the cytoplasm in HEK293T cells observed by the confocal microscope. Conclusions The recombinant eukaryotic expression vector of MCPIP was suscessfully constructed and expressed in HEK293T cell hnes. Moreover,we examined that MCPIP expressed high in macrophage cells,and distributed in cytoplasm in MCF-7 cells.