Isolation And Purification Of Antimicrobial Peptide HMGN2 And Investigation On Its Antiviral Activity Against Hepatitis B Virus

The high mobility group protein N2 (HMGN2) is one of the most abundant and well characterized nonhistone nuclear proteins which are found in the cell nucleus of vertebrate and invertebrate organisms. However, the biological role of this protein has not been fully defined. Recent work from our lab has shown that the human HMGN2 purified from IL-2-stimulated peripheral blood mononuclear leukocytes had antimicrobial activity against gram negative bacteria and HMGN2 may be a novel antibacterial effector molecule. To further understand the biological function of HMGN2, in the present study we investigated the anti-HBV effect of human HMGN2.Objective: 1. To isolate and purify antibacterial peptide HMGN2 from human monocyte THP-1 cells and determine its antimicrobial activity. 2. In order to develop new modality to treat hepatitis B infection, HepG2.2.15 cell line was used as an in vitro experimental model to study the anti-HBV activity of HMGN2. The level of HBsAg, HBeAg and HBV DNA in the supernatant of the HMGN2-treated HepG2.2.15 cells was assayed.Methods: Human monocyte cell line THP-1 cells were cultured in RPMI-1640 medium with 20% fetal bovine serum and collected by centrifugation. Acid-soluble components from THP-1 cells were prepared by homogenizing the cell pellet in 5% acetic acid. The supernatant was collected and dialyzed against water and lyophilized.The Acid-soluble components were subjected to RP-HPLC fractionation. The antimicrobial activities of the RP-HPLC fractions were determined by the agar radial diffusion assay and AU-PAGE and Tricine-SDS-PAGE were used to characterize the HMGN2 molecule.To investigate the in vitro anti-HBV effect of HMGN2, the HepG2.2.15 cells were treated with HMGN2 at a variety of concentrations. After inoculation for 4 days or 8 days, Supernatant of HMGN2-treated HepG2.2.15 cells were collected. The concentrations of HBsAg and HBeAg in the supernatant were measured by ELISA and the replication level of HBV DNA was detected with real-time quantitative PCR.Results: The agar radial diffusion assay indicated that the RP-HPLC fraction eluted at 21 min (fraction 21) had potent antimicrobial activity against E.coli ML-35p, and to some extent against E. Coli ATCC 25922 and clinical isolate 54080. However, this fraction was inactive against S. aureus ATCC25923 and E aeruginosa ATCC 27853 in this system. Analyzed by Tricine-SDS-PAGE, there was only one protein band in the fraction 21 which had an apparent molecular weight of approximately 20kDa. The biological characteristics of the protein in the fraction 21(RP-HPLC retention time, antimicrobial activities and apparent molecular weight) were identical to the previously reported characteristics of HMGN2.After incubation with HMGN2 at a dose of 2ug/ml for 4 days, the concentrations of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased by 30% and 40.8% respectively. HMGN2 also inhibited the replication of hepatitis B virus in HepG2.2.15 cells. After exposed to 2ug/ml of HMGN2 for 8 days, the level of extracellular HBV DNA in the supernatant of HepG2.2.15 cells decreased by 95.4%. The anti-HBV effect of HMGN2 was dose-dependent and the presence of serum weakened the anti-HBV activity. Although human neutrophilα-defensins showed anti-HBV activity, the potency of HMGN2 against HBV was more effective thanα-defensins.Conclusion: HMGN2 had potent antimicrobial activity against Gram-negative bacteria. Besides, in the presence of 2ug/ml of HMGN2, the level of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased significantly and the replication of hepatitis B virus in HepG2.2.15 cells is also inhibited. These results suggest that HMGN2 has direct anti-HBV effect in vitro.