肿瘤相关基因AMER1不同功能段表达载体的构建及其在人肺腺癌A549细胞中的表达

Construction of expression vector for various domains of tumorassociated gene AMER1 and expression in human lung adenocarcinoma A549 cells

目的构建APC膜结合蛋白1(APC membrane recruitment protein 1,AMER1)基因不同区域片段重组质粒,并检测其在人肺腺癌A549细胞中的表达。方法以人基因组DNA为模板,PCR扩增AMER1基因不同功能区域片段2-1135AA、2-327AA、2-785AA、327-785AA、785-1135AA及327-1135AA,扩增片段分别经T4 DNA酶与EGFP-N1载体连接,构建AMER1不同功能片段重组质粒,产物分别转化至感受态E.coli DH5α中,提取阳性克隆进行双酶切及测序鉴定;用脂质体2000介导各重组质粒转染肺腺癌A549细胞,同时设转染空载体EGFP-N1为对照组,q-PCR法检测AMER1不同功能区域片段m RNA转录水平。结果经双酶切及测序鉴定,AMER1不同功能区域片段重组质粒构建正确;与对照组比较,A549细胞中6个重组质粒AMER1基因m RNA转录水平均显著升高(P均<0.01)。结论成功构建了AMER1基因不同功能片段截短子重组质粒,并在A549细胞中高表达,为进一步研究AMER1不同功能区域在肺腺癌中的作用及相关分子机制提供了生物学工具。

Objective To construct the expression vectors for various domains of APC membrane recruitment protein 1(AMER1)gene and determine their expressions in lung adenocarcinoma A549 cells.Methods Various domains of AMER1 gene,2-1135 AA,2-327 AA,2-785 AA,327-785 AA,785-1135 AA and 327-1135 AA,were amplified by PCR using human genomic DNA as template,and cloned into EGFP-N1 vector with T4 DNA ligase respectively.The constructed recombinant plasmids were transformed to competent E.coli DH5α,from which the positive clones were identified by restriction analysis and sequencing,then transfected to A549 cells in mediation of lipidosome 2000,using the cells transfected with empty vector EGFP-N1 as control.The m RNA transcription levels of various domains of AMER1 were determined by q PCR.Results Restriction analysis and sequencing proved that the recombinant plasmids with various domains of AMER1 were constructed correctly.The transcription levels of AMER1 m RNAs in six recombinant plasmids in A549 cells were significantly higher than those in control group(each P<0.01).Conclusion Recombinant plasmids with various domains of AMER1 gene were successfully constructed and expressed in A549 cells,which provided a biological tool for further study on the roles of various domains of AMER1 gene in lung adenocarcinoma.