应用抑制性消减杂交技术筛选HBeAg结合蛋白1反式激活基因

Screening genes trans-regulated by HBeAg-binding protein 1 with suppression subtractive hybridization technique

目的筛选与克隆HBeAg结合蛋白1(HBEBP1)新基因的反式激活基因,了解其可能存在的调节功能线索。方法应用抑制性消减杂交(SSH)技术及生物信息学(bioinformatics)技术筛选并克隆HBEBP1反式激活的新型靶基因。以HBEBP1表达质粒pcDNA3.1(-)HBEBP1转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,制备转染后的细胞裂解液,提取mRNA并逆转录为cDNA,经RsaI酶切后,将实验组cDNA分成两组,分别与2种不同的接头衔接,再与对照组cDNA进行2次消减杂交及2次抑制性聚合酶链反应(PCR),将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析。结果成功构建人HBEBP1反式激活基因差异表达的cDNA消减文库。文库扩增后得到85个阳性克隆,进行菌落PCR分析,均得到200～1000bp插入片段。对26个插入片段测序,并通过生物信息学分析获得其全长基因序列,结果共获得15种编码基因,包括14种已知基因和1种未知基因。结论筛选到的cDNA全长序列,包括一些与细胞生长调节、信号转导、肿瘤免疫发生及细胞凋亡密切相关的蛋白编码基因,推测了HBEBP1可能存在的调控机制的线索。

Objective To investigate the biological functions of HBeAg-binding protein 1(HBEBP1), suppression subtractive hybridization(SSH) technique was used to screen genes regulated by HBEBP1. Methods HBEBP1(GenBank number:AF529372) was screened and identified by yeast two-hybrid system 3 and co-immunoprecipitation technique. The HBEBP1 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell genome. The expressive vector of pcDNA3.1(-)-HBEBP1 was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1(-) and pcDNA3.1(-)-HBEBP1, respectively by using FuGENE6 transfection reagent, then the mRNA was isolated. SSH method was employed to analyze the differentially expressed DNA sequences between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNAs were hybridized with driver cDNAs twice and underwent polymerase chain reaction (PCR) twice, they were then subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNAs were sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes up-regulated by HBEBP1 was constructed successfully. The amplified library contained 85 positive clones. Colony PCR showed that these clones contained 200-1 000bp inserts. Sequence analysis was performed in 26 clones at random, and the full length sequences were obtained with bioinformatics method. Altogether 15 coding sequences were obtained. Conclusions The obtained sequences may be target genes up-regulated by HBEBP1, among which some genes coding proteins were involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some new clues for the study of the biological functions of HBEBP1 and HBeAg.