论文部分内容阅读
目的构建丙型肝炎病毒(HCV)F蛋白反式激活相关基因差异表达的差异cDNA,克隆HCV F蛋白反式激活相关基因。方法以HCV F表达质粒pcDNA3.1(-)-F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性聚合酶链反应,将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆聚合酶链反应后进行测序及同源性分析。结果成功构建人HCV F蛋白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28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19种编码基因,其中2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 differentially expressed cDNAs for transactivation of hepatitis C virus (HCV) F protein and clone the transactivation related genes of HCV F protein. Methods HepG2 cells were transfected with HCV F expression plasmid pcDNA3.1 (-) - F and empty vector pcDNA3.1 (-) was used as a control. The transfected cell lysate was prepared and the mRNA was extracted and cDNA was synthesized. After RsaI enzyme After cleavage, the cDNAs were divided into two groups and ligated with two different linkers respectively. Two subtractive hybridization and two inhibitory PCR reactions were performed with cDNA of control group. The cDNA was ligated with T / A vector to construct cDNA The library was subtracted and transfected into E. coli for library amplification. Clones were randomly selected for sequencing and homology analysis. Results The cDNA of differentially expressed transactivation of HCV F protein was successfully constructed. After amplification, 56 clones with 200-1000 bp inserts were obtained and 28 of them were randomly selected for sequencing. The full-length gene sequences were obtained by bioinformatics analysis. As a result, 19 coding genes were obtained, of which 2 were 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.