Screening and Cloning of the Target Genes Down-regulated by β-interferon plasmid Using Suppression Subtractive Hybridization Technique
Abstract: The suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes down-regulated byβ-interferon in HepG2. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)IFNβand pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA from pcDNA3.1(-) empty vector transfected cell 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 products were subcloned into T/A 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 Blast search. The subtractive library of genes down-regulated byβ-interferon was constructed successfully. The amplified library contained 50 clones, of which 37 clones had 200-1000 bp inserts. Sequence analysis indicated that 22 clones containing the coding sequences , of which 19 had homology in the GenBank and 3 were unknown. The obtained sequences may be target genes regulated by β-interferon, and some genes encode proteins involved in cell cycle regulation, metabolism, and cell apoptosis.