Suppression subtractive hybridization for cloning of genes transactivated by XTP3 protein
- VernacularTitle:应用抑制性消减杂交技术筛选XTP3的反式激活基因
- Author:
Chunhua WANG
;
Jun CHENG
;
Huiping YAN
- Publication Type:Journal Article
- Keywords:
suppression subtractive hybridization;
clone;
XTP3 protein;
transactivation
- From:
Medical Journal of Chinese People's Liberation Army
1983;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a subtractive cDNA library of genes transactivated by XTP3 protein with suppression subtractive hybridization technique in order to clone genes associated with transactivation. Methods 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 subcloned 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. Results The amplified library contained 30 positive clones. Colony PCR showed that 23 clones contained 200-1 000bp 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. Conclusion 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.
