抑制性消减杂交技术筛选干扰素α转染HepG2细胞中上调的表达基因

Suppression subtractive hybridization for screening of upregulated gene expressions in interferon-alpha transfected HepG2 cell line

目的:应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术筛选干扰素α(IFN-α) 真核表达质粒转染HepG2细胞后差异表达上调的基因。方法:首先构建IFN-α真核表达载体pcDNA3．1(-) -IFN-α,并转染HepG2细胞,以空载体pcDNA3．1(-)转染的HepG2细胞为对照;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2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次抑制多聚酶链反应(PCR),将产物与T／A载体连接,构建cDNA消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆PCR扩增鉴定,进行测序及同源性分析。结果:构建了pcDNA3．1(-)-IFN-α真核表达质粒,并成功构建了该质粒转染HepG2细胞后差异表达基因的cDNA消减文库。文库扩增后得到200个白色克隆,随机挑取70个进行菌落PCR分析,结果显示均含有插入片段,将含有200-1000 bp插入片段的35个克隆进行测序,并通过生物信息学分析获得17种已知基因序列和1个染色体序列。结论:应用SSH技术成功构建了pcDNA3．1(-)-IFN-α转染的HepG2细胞中差异上调表达基因的cDNA消减文库。该文库的建立为进一步阐明IFN-α作用的分子生物学机制提供了重要的理论依据。

AIM： To screen the upregulated genes in interferon-alpha（IFN-α） transfected HepG2 cells by suppression subtractive hybridization （SSH） technique with further investigation the molecular mechanism of IFN- α. METHODS： The mRNA was isolated from HepG2 cells transfected by pcDNA3.1 （-）-IFN-α and pcDNA3.1 （-） empty vector, and then eDNA was synthesized. After restriction enzyme Rsa I digestion, small sizes of eDNA were obtained. Then tester eDNA was subdivided into two portions and each was connected with different eDNA adaptor. After tester eDNA was hybridized with driver eDNA twice and underwent nested polymerase chain reaction （PCR） twice,the PCR products were subcloned into T/A plasmid vectors to set up the subtractive eDNA library. Amplification of the library, was carried out with E.Coli strain DH5α The eDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The subtractive eDNA library, of genes upregulated by IFN-α was constructed successfully. Colony PCR showed that these clones contained 200-1 000 bp inserts. Thirty-five clones were analyzed by sequencing and bioinfbrmatics. Seventeen known genes and one chromatic sequence were obtained. CONCLUSION： A subtractive eDNA library of genes upregulated in pcDNA3.1 （-）- IFN-α transfected HepG2 cells by SSH technique was constructed successfully,which may bring some new important theoretical clues for studing the biological functions of IFN-α.