XIAP基因3′非翻译区荧光素酶报告载体的构建及活性分析  

Construction of XIAP-3′UTR-luciferase reporter vector and its activity analysis

作　　者：董宁 葛高霞 张炜明 朱伟[3] 徐华国 

机构地区：[1]南京医科大学第一附属医院检验学部,江苏南京210029 [2]南京医科大学第一附属医院病理学部,江苏南京210029 [3]南京医科大学第一附属医院肿瘤科,江苏南京210029

出　　处：《国际检验医学杂志》2014年第9期1098-1099,1102,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金青年项目(81302531);江苏省自然科学基金青年基金项目(BK20131018)

摘　　要：目的构建重组X连锁凋亡抑制蛋白质(XIAP)基因3′非翻译区(3′UTR)荧光素酶报告载体,分析可能调控XIAP基因表达的微RNA(miRNA)。方法采用聚合酶链反应(PCR)从人cDNA中扩增XIAP-3′UTR序列,插入荧光素酶报告载体pGL3-Ctrl,获得重组载体pGL3-Ctrl/XIAP;采用Target Scan 6.2软件预测可能与XIAP-3′UTR结合的miRNA;将pGL3-Ctrl/XIAP重组质粒和miRNA共转染A549细胞,测定XIAP-3′UTR荧光素酶的活性。结果酶切及核酸测序证实,成功构建了XIAP-3′UTR序列的荧光素酶报告重组子;miRNA靶位点的预测显示,XIAP基因可能是miR-200b、miR-200c和miR-429的作用靶标;与miRNA mimic ctrl组比较,miR-200b、miR-200c和miR-429能明显降低pGL3-Ctrl/XIAP的荧光素酶活性,差异有统计学意义(P<0.05)。结论成功构建了XIAP-3′UTR荧光素酶报告载体,且miR-200b、miR-200c和miR-429可显著降低其荧光素酶的活性。Objective To construct the recombinant X-linked inhibitor of apoptosis protein（XIAP） gene 3′untranslational region （3′UTR）-luciferase reporter vector ,and analyze the microRNA（miRNA） which possibly regulate the expression of XIAP gene . Methods Polymerase chain reaction （PCR） was employed to amplify X IA P-3′UTR sequences from human cDNA ,in which luciferase reporter vector pGL3-Ctrl was inserted ,and the recombinant vector pGL3-Ctrl/XIAP was gained .Target Scan 6 .2 soft-ware was adopted to predict the miRNA which possibly combined with the X IA P-3′UTR .pGL3-Ctrl/XIAP recombinant plasmids and the miRNA were co-transfected into A549 cells ,and the X IA P-3′UTR-luciferase activity was measured .Results Confirmed by digestion and DNA sequencing ,the X IA P-3′UTR-luciferase reporter recombinant was successfully constructed .Prediction of miRNA target sites indicated that X IA P gene may be the target of miR-200b ,miR-200c and miR-429 .Compared with miRNA mim-ic ctrl group ,miR-200b ,miR-200c and miR-429 significantly reduced the luciferase activity of pGL 3-Ctrl/XIAP with statistically significant difference（P〈0 .05） .Conclusion X IA P-3′UTR-luciferase reporter vector is successfully constructed .miR-200b ,miR-200c and miR-429 can obviously decrease the luciferase activity .

关 键 词：X连锁凋亡抑制蛋白质 3′非翻译区 微小RNAS 荧光素酶类 细菌 载体 

分 类 号：R440[医药卫生—诊断学]