2005•Europe PMC (PubMed Central)Requires access
Suppression subtractive hybridization for cloning of genes transactivated by XTP3 protein
Chunhua Wang, Jun Cheng, Huiping Yan, Jie Yan, Ying Shi, Shengyu Cheng
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Abstract
mRNA was isolated from HepG2 cells transfected by pcDNA3.1(-)-XTP3 and pcDNA3.1(-) empty vector respectively, and then cDNA was synthesized. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively. After being hybridized with driver cDNA twice and underwent two times of nested PCR, tester cDNA was subloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. Coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 30 positive clones. Colony PCR showed that 23 clones contained 200--1000bp inserts. Sequence analysis was performed also. It was found that 20 kinds of known and 2 kinds of novel cDNA sequences may be target genes transactivated by XTP3 protein. The subtractive library of genes transactivated by XTP3 protein was constructed successfully. It provides a foundation for the further research on pathogenesis of the viral proteins.