ABCE1基因稳定过表达肺腺癌细胞株的建立  

Establishment of lung adenocarcinoma cells over-expressing ABCE1 gene

作　　者：田野[1] 韩旭[1] 陈拥[1] 刘思洋[1] 姜文军[1] 田大力[1] 

机构地区：[1]中国医科大学附属第四医院胸外科,辽宁沈阳110032

出　　处：《生物医学工程与临床》2016年第2期188-193,共6页Biomedical Engineering and Clinical Medicine

基　　金：中国医科大学附属第四医院青年创新发展计划基金资助项目

摘　　要：目的构建能够携带绿色荧光蛋白ABCE1基因的表达载体p EGFP-C1-ABCE1,转染肺腺癌细胞株LTEP-a-2,筛选并建立过表达ABCE1基因的细胞株。方法逆转录-聚合酶链反应(RT-PCR)扩增ABCE1全长m RNA,通过酶切、连接ABCE1及表达载体p EGFP-C1,构建重组质粒p EGFP-C1-ABCE1。采用脂质体转染将重组质粒转染至LTEP-a-2细胞,并以G418筛选阳性克隆,建立起能稳定过表达ABCE1基因的细胞株。结果成功构建重组质粒载体p EGFP-C1-ABCE1,并通过酶切及基因测序验证。将重组质粒成功转染入LTEP-a-2细胞,通过G418筛选后(600μg/m L),获得的细胞株能够稳定持续地表达绿色荧光,其ABCE1基因表达明显高于野生型肺腺癌细胞。结论成功构建了ABCE1表达载体p EGFP-C1-ABCE1,建立稳定过表达ABCE1基因的肺腺癌细胞株LTEP-a-2,为进一步研究ABCE1在肺腺癌中的功能及机制打下了基础。Objective To construct the expression vector pEGFP-C1-ABCE1 which carry green fluorescent protein and Irans-feet LTEP-a-2 lung adenoearcinoma cells, and establish cell lines over-expressing ABCE1 gene. Methods The ABCE1 full- length mRNA was amplified by reverse transcription-polymeras chain reaction（RT-PCR）. The coding regions were confirmed hy electrophoresis and DNA sequencing. The recombinant plasmid pEGFP-C1-ABCE1 was constructed by enzyme digestion and linking of ABCE 1 and expression vector pEGFP-C1, and then transfected into LTEP-a-2 （：ells. Positive clone was screened by G418 to establish stable cell lines of over-expressing ABCE1 gene. Results ABCEI gene was demonstrated into eukaryotie expression vector pEGFP-C1 by DNA sequencing. LTEP-a-2 lung adenocarcinoma cells expressing pEGFP-C1-ABCE1 and pEGFP-C1 were screened and established by liposome transfection and G418（600μg/mL） screening. The cell lines could express green fluorescent continuously and stably, and their ABCE1 gene expression was significantly higher than that of wild-type lung adenocarcinoma cells. Conclusion It is demonstrated that the recombinant plasmid vector expressing ABCEl can be success- fully constructed and stable lung adenocareinoma cell lines over-expressing ABCEI gene can be established, which can provide foundation for function and mechanism study of ABCEI in lung adenocareinoma.

关 键 词：ABCE1基因 肺腺癌细胞株 绿色荧光蛋白 基因表达 

分 类 号：R734.2[医药卫生—肿瘤]