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目的应用抑制性消减杂交(SSH)技术构建丙型肝炎病毒非结构蛋白4A(HCVNS4A)转染细胞差异表达cDNA消减文库,克隆HCVNS4A蛋白反式激活相关基因。方法以HCVNS4A表达质粒pcDNA3.1(-)-NS4A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,提取mRNA并逆转录为cDNA,进行SSH分析。将富集的二次PCR产物与T/A载体连接,并转染大肠埃希菌进行文库扩增,随机挑取克隆,聚合酶链反应(PCR)扩增后进行测序及同源性分析。结果文库扩增后得到36个阳性克隆,经菌落PCR分析显示200~1000bp插入片段。对其中的25个片段测序,并进行同源性分析,显示18种已知基因编码蛋白和2种未知功能基因序列,包括一些与细胞周期、细胞凋亡、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因,可能是NS4A反式激活靶基因。结论成功构建了HCVNS4A反式激活基因差异表达的cDNA消减文库,为进一步阐明HCVNS4A反式调节的靶基因等提供了相关的平台。
Objective To construct a cDNA subtractive library of HCVNS4A transfected cells by Suppression Subtractive Hybridization (SSH) and clone the transactivation related gene of HCVNS4A protein. Methods HepG2 cells were transfected with HCV NS4A expression plasmid pcDNA3.1 (-) - NS4A. The empty vector pcDNA3.1 (-) was used as a control to prepare the cell lysate after transfection. MRNA was extracted and reverse transcribed into cDNA for SSH analysis . The enriched secondary PCR product was ligated to the T / A vector and transfected into Escherichia coli for library amplification. Random clones were picked and amplified by polymerase chain reaction (PCR) followed by sequencing and homology analysis. 36 positive clones were obtained after amplification of the results library and 200-1000 bp inserts were shown by colony PCR analysis. The 25 fragments were sequenced and analyzed by homology. The results showed that 18 kinds of known genes encoded proteins and 2 kinds of unknown functional gene sequences, including some related to cell cycle, apoptosis, signal transduction and tumorigenesis A closely related protein-coding gene may be the NS4A transactivator target gene. Conclusion The subtractive cDNA library of differentially expressed HCVNS4A transactivator gene was successfully constructed and provided a platform for further elucidating the target genes of HCVNS4A trans - regulation.