AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridizat...AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, 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 adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.展开更多
文摘AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, 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 adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
