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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。
OBJECTIVE: To screen differentially upregulated genes of HepG2 cells transfected with IFN-α eukaryotic expression plasmids by suppression subtractive hybridization (SSH). Methods: The recombinant eukaryotic expression vector pcDNA3.1 (-) - IFN - α was constructed and transfected into HepG2 cells. HepG2 cells transfected with empty vector pcDNA3.1 (-) as control; The cell lysate was extracted and reverse transcribed into cDNA. After digested with RsaI, the experimental group was divided into two groups, and each cDNA was ligated with two different linkers. Then the cDNA was digested and subtracted twice with the control group, The PCR products were ligated with T / A vector to construct a cDNA subtractive library, which was then transformed into E. coli for library amplification. Randomly selected clones were identified by PCR amplification, and sequenced and homology analysis was performed. Results: The pcDNA3.1 (-) - IFN-α eukaryotic expression plasmid was constructed and the cDNA subtractive library of differentially expressed genes was successfully constructed. A total of 200 white clones were obtained after amplification. 70 colonies were randomly selected for PCR analysis. The results showed that all the clones contained inserts, and 35 clones containing 200-1000 bp inserts were sequenced and analyzed by bioinformatics analysis. A known gene sequence and a chromosome sequence. Conclusion: cDNA subtractive library of differentially up-regulated genes in HepG2 cells transfected with pcDNA3.1 (-) - IFN-α was successfully constructed by SSH. The establishment of this library provides an important theoretical basis for further clarifying the molecular biological mechanism of IFN-α.