应用抑制性消减杂交技术筛选NS5ABP37的反式调节基因

Cloning and screening of transregulated genes of NS5ABP37 by SSH

目的应用抑制性消减杂交(SSH)技术构建NS5ABP37反式激活相关基因差异表达的cDNA文库,筛选NS5ABP37蛋白反式激活相关基因。方法以表达质粒pcDNA3.1(-)-NS5ABP37转染HepG2细胞,以空载体pcDNA3.1(-)为对照,从转染后的细胞裂解液中提取mRNA并合成cDNA,经RsaⅠ酶切消化后将实验组cDNA分成两组,分别衔接两种不同接头,再与对照组cDNA进行两次消减杂交及两次抑制性PCR反应;第2次PCR产物与pGEM-Teasy克隆载体连接,构建cDNA消减文库,并转化大肠杆菌进行文库扩增,随机挑选克隆经PCR鉴定后进行测序及同源性分析。结果成功构建了人NS5ABP37蛋白反式激活相关基因差异表达的cDNA文库。扩增后得到60个200～1000bp插入片段的克隆,随机挑选其中20个测序,并通过生物信息学分析获得其全长基因序列,结果共获得9种已知功能编码基因。结论NS5ABP37基因功能涉及细胞生长调节、信号转导和能量代谢。

Objective To screen and identify human genes transactivated by NS5ABP37 by constructing a eDNA subtractive library with suppression subtractive hybridization （SSH） technique. Methods SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by NS5ABP37 protein. After pcDNA3, 1（-）-NS5ABP37 and pcDNA3.1（-） empty vector transfected HepG2 cells, the mRNA was isolated from the cells. SSH method was employed to analyze the differentially expressed DNA sequence in the two groups. After restriction enzyme Rsa Ⅰ digestion, small-sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester eDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the second PCR production was subcloned into pGEM-T easy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blastn search after PCR. Results The subtractive library of genes transactivated by NS5ABP37 was constructed successfully. The amplified library contained 60 positive clones with 200-1 000 bp inserts. Sequence analysis was performed in 20 clones randomly, and the full-length sequences were obtained with bioinformatics method. Altogether 9 coding sequences were obtained. Conclusion The function of NS5ABP37 gene may be involved in cell growth regulation, signal transduction and metabolism.