Studies On Hepatitis Type B Virus Promoters Rugulated By ApoA-Ⅰ

Aim: Screening transcriptional factors of hepatitis B virus (HBV) promotors and further studies on the biological functions and binding affinity properties between apolipoproteln A- I (apoA- I ), a candidate of hepatitis B virus (HBV) promotors' binding protein, and hepatitis B virus (HBV) promotorsMethods: Hepatitis B virus (HBV) promotors' binding proteins were fished by Biacore based on surface plasmon resonance (SPR) technique, and those binding proteins were identified by Short-gun Mass Spectrometry. Briefly, promotor genes conjugated with biotin were attached on SA chip via the strong affinity of biotin-avidin. The Nucleoprotein of HepG2.2.15 cells was then flowed through the chip slowing, and the proteins in the nucleus which could bind to the promotor sequence were captured on the chip. After eluting and recovering, the proteins were indentified by short-gun mass spectrum. Results of mass spectrum showed that apoA- I was one of those proteins which could bind to HBV core, S1, and S2 promotor genes. Thereafter, the binding affinity and biological function between apoA-I and those promotor genes were further studied by molecular clone technique, confocal microscope and western blotting. pcDNA3.1-apoA- I plasmid and recombinants HBV promotor gene plasmids with a CAT report gene , pCAT3-S1, pCAT3-S2 and pCAT3-X were constructed. pcDNA3.1-apoA- I and each promotor recombinant plasmid were co-transfected into HepG2 cell line, respectively. 48h later, protein was extracted from cells and CAT expression level was detected by Elisa. The CAT activities stand for the promotor transcription capacity. To determine the binding property between apoA-I and each promoter, the affinity analysis experiments were performed using Biacore, by anchoing apoA- I on CM5 chip and following with a flowing solution containing different concentration of promoter sequence DNA. The dynamics parameters were calculated by Biacore 3000 software.Results: ApoA-I mRNA is significant higher in HepG2.2.15 cell line,a cell model of cell model of HBV replication, derived from HepG2, than that in HepG2 by Real-time PCR.Meanwhile,immunostaning results showed that apoA- I is detectable in the nucleaer proteins of HepG2.2.15.While it is barely detected in HepG2 cell line,which indicated that apoA- I and HBV promotors are colocalized in the cell nucleus while HBV replication is processing. ApoA- I could promoter the transcription capacity of promotors of HBV core and X genes. However, Biacore SPR showed that apoA- I could binding to all the HBV promotor sequences with a highest KA of 7.98×10⁵ superscript with S2 promotor and a highest KD of 7.66×10^-4 superscript with S1 promotor.