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目的:应用抑制性消减杂交(SSH)技术构建新基因丙型肝炎病毒F蛋白结合蛋白2(HCVFBP2)反式激活基因差异表达的cDNA消减文库,克隆HCVFBP2反式激活相关基因。方法:以HCVFBP2表达质粒pcDNA3.1(-)FBP2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多聚酶链反应(PCR),将产物与pGEMTeasy载体连接,构建cDNA消减文库,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:成功构建人类新基因HCVFBP2反式激活基因差异表达的cDNA消减文库。文库扩增后选取48个克隆,进行菌落PCR分析,均得200～1000bp插入片断。挑取30个克隆进行测序,通过生物信息学分析获得28个已知功能基因序列和2个未知功能基因。结论:应用SSH技术成功构建了HCVFBP2反式激活基因差异表达的cDNA消减文库。该文库的建立为阐明HCVFBP2生物学功能提供理论依据。
OBJECTIVE: To construct a cDNA subtractive library of differentially expressed transactivator genes of hepatitis C virus F protein binding protein 2 (HCVFBP2) by SSH and to clone the transactivation related gene of HCVFBP2. Methods: HepG2 cells were transfected with HCVFBP2 expression plasmid pcDNA3.1 (-) FBP2 and HepG2 cells transfected with empty vector pcDNA3.1 (-) as control. The transfected cell lysate was prepared and the mRNA was extracted and reverse transcribed into cDNA was digested with RsaI, the experimental group cDNA was divided into two groups, respectively, with two different linkers, and then with the control group cDNA two subtractive hybridization and twice inhibition of polymerase chain reaction (PCR), the product and pGEMTeasy Vector, cDNA subtraction library was constructed, E. coli was transfected to amplify the library, randomly selected clones were amplified by PCR, sequenced and homology analysis. Results: cDNA subtractive library was successfully constructed for the differential expression of transactivated gene of human novel gene HCVFBP2. After amplification, 48 clones were selected for colony PCR analysis, and all obtained 200 ~ 1000bp inserts. Twenty clones were picked for sequencing and 28 known functional gene sequences and 2 unknown functional genes were obtained by bioinformatics analysis. Conclusion: The subtractive cDNA library of HCVFBP2 transactivator gene was successfully constructed using SSH. The establishment of this library provides a theoretical basis for clarifying the biological function of HCVFBP2.