抑制性消减杂交技术筛选重组IFNβ下调HepG2细胞靶基因

目的利用抑制性消减杂交（SSH）技术构建重组干扰素β（IFNβ）刺激人肝癌细胞系HepG2差异表达基因的cDNA消减文库，筛选IFNβ下凋HepG2相关基因。方法重组IFNβ2000U／ml刺激对数生长期HepG2细胞，以生理盐水作用的HepG2细胞为阴性对照；制备细胞裂解液，从中提取mRNA并逆转录为cDNA，经Rsa Ⅰ酶切后将实验组cDNA分成两份，分别与两种不同的接头连接，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，并转染大肠埃希菌进行文库扩增，随机挑选克隆PCR后进行测序及同源性分析。结果成功构建重组IFNβ刺激HepG2细胞差异表达基因的cDNA消减文库。文库扩增后，得到58个阳性克隆，进行菌落PCR分析，均得到200～1000bp插入片段。随机挑选其中35个插入片段测序，并通过生物信息学分析，结果共获得12种编码基因。结论应用SSH技术成功构建了IFNβ刺激HepG2细胞差异表达基因的cDNA消减文库，为进一步了解IFNβ在肝细胞内的免疫调节机制提供了依据。

Screening and cloning of the down-regulation gene by recombinant interferon-β using suppression subtractive hybridization technique

BACKGROUND: To construct a subtractive cDNA library of target genes down-regulated in human hepatocarcinoma cell line HepG2 cells treated with IFNB, and clone genes of the down-regulation by IFNB using suppression subtractive hybridization (SSH) technology and bioinformatics techniques. METHODS: The mRNA was isolated from HepG2 cells induced by recombinant interferon-B and 0.9 percent sodium chloride, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two portions and each was ligated to the specific cDNA adaptor 1 and adaptor 2 respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the DNA fragment was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS: The subtractive library of genes down-regulation in HepG2 cells treated with recombination interferon-B was constructed successfully. The amplified library contained 58 positive clones. Colony PCR and sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method. Altogether 12 coding sequences were obtained. CONCLUSION: A subtractive cDNA library of genes down-regulation in HepG2 cells treated with IFNB using SSH technique was constructed successfully, which brings some new clues for studying the regulation mechenism of IFNB in liver cells.