摘要
目的应用噬菌体展示技术筛选HBV前-S1蛋白反式激活基因1(PS1TP1)的启动子DNA结合蛋白,进一步阐述PS1TP1在慢性乙型肝炎发病机制中的作用。方法根据GenBank中的PS1TP1启动子DNA序列设计引物,运用PCR技术以人基因组DNA为模板,扩增获得PS1TP1启动子的DNA片段,并通过亚克隆方法构建真核报告载体pCAT3-PS1TP1p后转染HepG2细胞系,采用酶联免疫吸附试验(ELISA)检测转染细胞中氯霉素乙酰转移酶(CAT)的表达活性,以验证所获得的DNA片段具有启动子活性;将PS1TP1启动子DNA片段进行固相化,对噬菌体人肝细胞cDNA文库进行筛选,将所获得的噬斑裂解液进行PCR扩增,测序后进行DNA序列分析和同源性生物信息学搜索。结果 pCAT3-PS1TP1p瞬时转染的HepG2细胞中CAT表达活性是对照组(pCAT3-Basic空载体)的32.9倍,是pCAT3-promoter的4.2倍,提示所获得的DNA片段具有启动子活性;经4轮筛选共得到阳性克隆18个,经PCR扩增、测序,并经生物信息学分析后筛选出PS1TP1的启动子DNA结合蛋白编码基因共15个。结论 PCR扩增后所获得的PS1TP1启动子DNA片段具有顺式激活下游基因表达的作用,为研究PS1TP1启动子功能奠定了基础;噬菌体展示筛选结果提示PS1TP1可能参与了细胞周期调节、MAPK信号转导通路,并且有可能在辅助性T细胞亚群的发育和B细胞分化过程中发挥作用。
Objective To select proteins binding to pre-S1 transactived protein 1 （PS1TP1） promoter and to clarify the regulation mechanism of PS1TP1 gene in pathogenesis of chronic hepatitis B. Methods The PCR primer set was designed based on the PS1TP1 promoter sequence registrated in GenBank, the template was HepG2 genomic DNA. PCR technology was applied to amplify the promoter of PS1TP1. The eukaryotic reporter plasmid pCA33-PS1TPlp was constructed by routine subcloning method and transfected into HepG2 cell lines mediated by liposome. The choloraphenical acetyltransferase （CAT） activity of transfected cell was determined by ELISA to verify the promoter activity of pcArI3-PS1TPlp. Phage display technique was employed to screen the promoter DNA-binding protein of PS1TP1 gene, after four rounds of screening, clones exhibiting high DNA-binding activity were selected and amplified. Results The level of CAT in HepG2 cells transfected with pCAT3-PS1TPlp was detected by ELISA, which was 4.2 times higher than that of pCArI3-promoter. Sequence analysis was performed in 18 positive plaque and the full length sequences were obtained with bioinformatics method, a total of 15 coding sequences were obtained, which consisted of 13 known and 2 unknown ones. Conclusions The PCR fragment of PS1TP1 gene promoter has transcriptional activity on its down-stream genes, which could be used for further study of PS1TP1 promoter. Phage display screening suggests that PS1TP1 might be involved in cell cycle regulation, MAPK signal pathway and immune regulation of helper T cells and B cells lymphocyte genes.
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2012年第4期1-4,共4页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
基金
国家自然科学基金(No.30700713)
北京自然科学基金(No.7122177)
