应用抑制性消减杂交技术克隆乙型肝炎病毒全S蛋白反式激活基因

目的应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)全S蛋白反式激活基因差异表达的cDNA消减文库,克隆HBV全S蛋白反式激活相关基因.方法以HBV全S表达质粒pcDNA3.1(-)-全S转染HepG2细胞,以空载体pcDNA3.1(-)为对照;制备转染后的细胞裂解液,从中提取mRNA并逆转录为cDNA,经RsaI酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性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HBV全S蛋白反式激活基因差异表达的cD-NA消减文库.文库扩增后得到86个白色克隆,进行菌落PCR分析,均得到100-1000 bp插入片段.挑取35个含有插入片段的阳性克隆测序分析,获得33个已知基因序列,和2个未知基因,通过生物信息学分析获得其全长序列,其中之一命名为全S蛋白反式激活基因1(CSTP1),已在GenBank中注册,注册号:AY553877.未知基因的功能还正在研究中.结论应用SSH技术成功构建了HBV全S反式激活基因差异表达的cDNA消减文库.该文库的建立为进一步阐明HBV全S反式调节的靶基因及致肝病发生的分子生物学机制提供理论依据.

Cloning of genes transactivated by hepatitis B virus complete S protein by suppression subtractive hybridization technique

Objective To construct a subtractive cDNA library of genes transactivated by hepatitis B virus (HBV) complete surface protein using suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivating function.Methods The mRNA was isolated from HepG2 cells transfected by pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sizes cDNA were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice, underwent nested polymerase chain reaction (PCR) twice, and then was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109.Results The cDNA was sequenced and analyzed. Genes transactivated by HBV complete S was constructed successfully. The amplified library contained 86 positive clones. Colony PCR showed that these clones contained 100-1000 bp inserts. Thirty-five clones were analyzed by sequencing and bioinformatics. Thirty-three known genes and two genes with unknown function were obtained. Conclusions A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique was constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protin, which brought some new clues for studying the biological functions of HBV complete S protin.