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目的构建丙型肝炎病毒(HCV)F反式调节靶基因FTP2的反式激活基因的cDNA文库,克隆FTP2反式激活基因。方法以HCV FTP2表达质粒pcDNA3．1(-)-HCV FTP2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,从中提取mRNA并合成cDNA,经Rsa I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性聚合酶链反应(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,随机挑选克降PCR后进行测序及同源源性分析。结果成功构建人HCV FTP2反式激活相关基因差异表达的cDNA。扩增后得到56个200～1000 bp插入片段的克隆,随机挑选其中24个插入片段测序,并通过生物信息学分析获得其全长基因序列,结果共获得20种编码基因,其中1个为未知功能的新基因。结论筛选到的cDNA全长序列,包括一些与细胞生长调节、物质代谢和细胞凋亡密切相关的蛋白编码基因。
Objective To construct a cDNA library of transactivation gene of hepatitis C virus (HCV) F trans - regulatory target gene FTP2 and clone FTP2 transactivation gene. Methods HepG2 cells were transfected with HCV FTP2 expression vector pcDNA3.1 (-) - HCV FTP2 and empty vector pcDNA3.1 (-) was used as control. The transfected cell lysate was prepared and the mRNA was extracted and cDNA was synthesized. After Rsa I digested the experimental group cDNA into two groups. Respectively, with two different linkers, and then with control cDNA subtractive hybridization twice and two inhibitory polymerase chain reaction (PCR), the product was connected with the T / A vector to construct cDNA subtractive library, and transfected into the large intestine Escherichia coli library amplification, randomly selected g down PCR after sequencing and homologous analysis. Results The recombinant cDNA of human HCV FTP2 transactivation related gene was successfully constructed. After amplification, 56 clones with 200-1000 bp inserts were obtained and 24 inserts were randomly selected for sequencing. The full-length gene sequences were obtained by bioinformatics analysis. As a result, a total of 20 coding genes were obtained, of which 1 was unknown The new gene of function. Conclusion The full-length cDNA sequence was screened, including some protein-coding genes that are closely related to cell growth regulation, substance metabolism and apoptosis.