Cloning and screening of transregulated genes of NS5ABP37 by SSH

Objective: To screen and identify human genes transactivated by NS5ABP37 by constructing a cDNA 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 cDNA 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 200-1000 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.