The study on genes trans-regulated by XTP4 using suppression subtractive hybridization technique
- VernacularTitle:抑制性消减杂交技术筛选XTP4蛋白反式调节基因
- Author:
Ping HAN
;
Yan LIU
;
Jun CHENG
- Publication Type:Journal Article
- Keywords:
hepatitis B virus;
X protein;
transactivation;
suppression subtractive hybridization
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the influence of XTP4 on genomic expression profile of hepatocyte through screening and cloning of genes trans-regulated by XTP4-expressing plasmid. Methods The expressive vector pcDNA3.1(-)-XTP4 was constructed by routine molecular biological methods, and suppression subtractive hybridization (SSH) method was employed to detect the mRNA differentially expressed by the HepG2 cells transfected with pcDNA3.1(-)-XTP4 and pcDNA3.1(-), respectively, using lipofectamine. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out in E. coli strain JM109. The screened cDNA were sequenced and analyzed in GenBank with Blast search after PCR. Results The amplified subtractive library containd 21 positive clones. Colony PCR analysis showed that there were 16 clones containing 200-1000bp inserts. Sequence analysis was performed and 9 kinds of encoding sequences were achieved. These genes trans-regulated by XTP4 protein involved in hepatic fibrogenesis, tumorgenesis, mitochondrial function, and cell growth regulation. Conclusions The findings obtained by SSH provide significant data for a preliminary understanding of the biological function of a new identified gene-XTP4. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HBxAg and the development of new therapy for chronic hepatitis B.
