Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique.
OBJECTIVE
To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.


METHODS
pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.


RESULTS
The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of beta-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877).


CONCLUSIONS
The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.
Department of Obstetrics and Gynecology of First Hospital, Xi'an Jiaotong University, Jiankang Road 1, Xi'an 710061, Shaanxi Province, China.
Gui-Qin
GQ
Bai
Yan
Y
Liu
Jun
J
Cheng
Shu-Lin
SL
Zhang
Ya-Fei
YF
Yue
Yan-Ping
YP
Huang
Li-Ying
LY
Zhang
D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015723:Gene Library; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000502:physiology*; D014764:Viral Proteins / Q000502:physiology*
D014764:Viral Proteins