NFKBIA 3'UTR不同基因型载体的构建及其功能差异性比较  

Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR

作　　者：杨朔[1] 李嘉丽[1] 毕惠嫦[1] 周守宁 刘晓曼[1] 曾行[1] 胡冰芳[1] 黄民[1] 

机构地区：[1]中山大学药学院临床药理研究所,广东广州510006

出　　处：《药学学报》2016年第1期80-85,共6页Acta Pharmaceutica Sinica

基　　金："十二五"国家科技重大专项资助项目(2012ZX09506001-004);国家自然科学基金资助项目(81102515;81320108027;81173131);广东省重点实验室建设项目(2011A060901014);广东省重大科技专项资助项目(2011A080300001;2012A080202013)

摘　　要：验证NFKBIA基因3'非翻译区(3'UTR)两个SNP--rs8904C>T和rs696G>A的功能。以两个位点分别为CC纯合子和GA杂合子基因型的人全基因组DNA为模板,通过设计不同引物以扩增长度为503 bp的NFKBIA基因3'UTR片段,经测序验证后将该片段克隆至荧光素酶载体pGL3-promoter中,构建4种重组质粒pGL3-rs8904C/rs696G、pGL3-rs8904C/rs696A、pGL3-rs8904T/rs696G和pGL3-rs8904T/rs696A,并转染LS174T细胞,检测荧光素酶活性。与转染了pGL3-vector组(阴性对照)相比,转染重组双荧光素酶报告质粒的荧光素酶活性均显著下降(P<0.001)。对于rs696G>A,含等位基因A的两种重组质粒pGL3-rs8904C/rs696A和pGL3-rs8904T/rs696A的荧光素酶活性分别比含等位基因G的两种重组质粒pGL3-rs8904C/rs696G和pGL3-rs8904T/rs696G下降约45.1%(P<0.05)和56.1%(P<0.001)。对于rs8904C>T,含等位基因T的两种重组质粒分别与含等位基因C的两种重组质粒相比,荧光素酶活性无显著性差异。NFKBIA基因3'UTR双荧光素酶报告基因载体构建成功,并发现rs696G>A会减弱荧光素酶活性的表达,rs8904C>T对荧光素酶活性的表达无明显影响。This study aims to investigate the function of two SNPs（rs8904C T and rs696 G A） in 3＇ untranslated region（3＇UTR） of NFKBIA gene by constructing luciferase reporter gene. A patient＇s genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3＇UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696 G, pGL3-rs8904C/rs696 A, pGL3-rs8904T/rs696 G and pGL3-rs8904T/rs696 A. Then these plasmids were transfected into LS174 T cells and the luciferase activity was detected. Compared with pGL3- vector transfected cells（negative control）, the luciferase activity of the four kinds of recombinant plasmids was significantly decreased（P〈0.001）. For rs696 G 〉A, the luciferase activity of the recombinant plasmids containing A allele（pGL3-rs8904C/rs696 A and pGL3-rs8904T/rs696A） was about 45.1%（P〈0.05） and 56.1%（P〈0.001） lower than those containing G allele（pGL3-rs8904C/rs696 G and pGL3-rs8904T/rs696G）, respectively. For rs8904 C〉 T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3＇UTR were constructed successfully and rs696 G 〉A could decrease the luciferase activity while rs8904 C 〉T didn＇t have much effect on the luciferase activity.

关 键 词：NFKBIA 3'非翻译区 单核苷酸多态性 荧光素酶报告基因载体 rs696 rs8904 

分 类 号：R966[医药卫生—药理学]