Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

Abstract

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-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-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 DH5alpha. 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 beta-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 positive clones. 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 transduction and cell apoptosis. This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

Figure 1

A: Products of pcDNA3.1(-)-pre-S2 PCR and restriction enzyme cleaved were eletrophoresed in 2.0% agarose gel. Lane 1: EcoRV/BamHI cleaved, lane 2: PstI cleaved, lane 3: production of plasmid PCR, M: DNA marker (2 000 bp). B: Structure of expression plasmid pcDNA3.1(-)-pre-S2.

Figure 2

RT-PCR products were eletrophoresed in 0.9% agarose gel. Lane 1:negative control, lanes 2-4: mRNA was isolated from pcDNA3.1(-)-pre-S2 and RT-PCR was performed by three different Oligod T, lane 5: blank control, lane 6: positive control, M: DNA marker (2 000 bp).

Figure 3

Result of b-galactosidase enzyme analysis. The numbers represent mean±SD of 7 separate transfections.

Figure 4

Reduction of G3PDH abundance by PCR-select subtraction. PCR was performed on unsubtracted (lanes1-4) or subtracted (lanes 5-8) secondary PCR products with the G3PDH 5’ and 3’ primers. Lanes 1, 5: 18 cycles; lanes 2, 6: 23 cycles; lanes 3, 7: 28 cycles; lanes 4, 8: 33 cycles. Lane M: DNA marker (2 000 bp).

Figure 5

Results of PCR amplification of part clones (17-32). M: DNA marker (2 000 bp).