噬菌体展示技术筛选诱导型NOS基因启动子DNA结合蛋白的研究

Screening of promoter DNA-binding protein of iNOS gene by phage display technique from human liver cDNA library

目的筛选诱导型一氧化氮合酶(iNOS)基因启动子DNA结合蛋白,探索iNOS基因的表达调控机制。方法应用生物信息学方法确定iNOS基因启动子序列,以PCR扩增iNOS基因启动子DNA片段,构建真核报告载体pCAT3iNOSp,并转染HepG2细胞系,用ELISA法检测氯霉素乙酰转移酶(CAT)的表达活性以明确所得到的DNA片段具有启动子活性;以iNOS基因启动子片段作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行4轮“吸附洗脱扩增”富集过程,噬斑裂解液PCR扩增后,进行DNA序列分析和蛋白质同源性生物信息学搜索。结果pCAT3iNOSp瞬时转染的HepG2细胞的CAT表达活性是pCAT3Basic空载体的4.2倍,说明所得到的DNA片段具有启动子活性;噬菌体经富集后,筛选出12个阳性克隆,成功获得了iNOS基因启动子的DNA结合蛋白编码序列。结论所构建的iNOS基因启动子具有顺式激活下游基因表达的作用;筛选得到的iNOS启动子DNA结合蛋白对于研究iNOS基因的转录调节机制具有重要意义。

Objective To screen promoter DNA-binding protein of inducible nitric oxide synthase gene by using phage display technique from human liver cDNA library, and to study the expression and regulation mechanism of iNOS gene. Methods The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame(ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase(CAT)activity was determined by enzyme linked immunosorbent assay(ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. Results The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. Conclusion The iNOS gene promoter identified in this study has shown to have transcription activity,and iNOS promoter DNA-binding proteins havs been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.