[Cloning of genes transactivated by hepatitis B virus X protein].
OBJECTIVE
To construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function.


METHODS
The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast.


RESULTS
The subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function.


CONCLUSIONS
A subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.
Liu
Yan
Y
Gene Therapy Research Center, Institute of Infectious Diseases, 302nd Hospital of People's Liberation Army, Beijing 100039, China.
Cheng
Jun
J
Lu
Yin-ying
YY
Wang
Gang
G
Mou
Jing-shong
JS
Li
Li
L
Zhang
Ling-xia
LX
Chen
Ju-mei
JM
D003001:Cloning, Molecular; D015723:Gene Library; D012333:RNA, Messenger / Q000032:analysis; D015534:Trans-Activators / Q000502:physiology*; D015533:Transcriptional Activation; D054334:Viral Regulatory and Accessory Proteins
D012333:RNA, Messenger; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein