The Affinity Of Beta 2-Glycoprotein I Binding To Hepatitis B Surface Antigen

Objective: Hepatitis B virus (HBV) infection is one of the most challenging global public health problems. Beta 2-Glycoprotein I (β2-GPI) is an enriched glycoprotein in plasma, which is a single-chain protein with 326 amino acids, containing four N-linked glycosylation sites.β2-GPI is one of the major autoantigen in the antiphospholipid antibody syndrome. A recent study showed thatβ2-GPI binding with HBsAg played a role in the process of HBV infection.So our research focused on the affinity betweenβ2-GPI and HBsAg and the influence of glycosylation ofβ2-GPI to the binding. This survey supplied the experimental evidences for HBV hepatotropic mechanism.Methods: We studied the affinity ofβ2-GPI binding to rHBsAg. rHBsAg was radiolabeled with Na 125I by using chloramine-t, and the affinity constant (Ka) was checked by radioimmunoassay (RIA). The optimization of temperature and time of the binding ofβ2-GPI and 125I-rHBsAg were observed. The doubling dilution of 125I– rHBsAg bound to gradient concentration ofβ2-GPI from the plasma. Then, the precipitates were separated by the method of competitive conjugation, and their radio-counting checked was drawn on the double logarithmic paper. Ka1 was calculated by means of the Adrion. Also, we had expressed rβ2-GPI in E.coli M15 (pQE30-hβ2-GPI) which was non- glycosylated. They were purified through nickel chromatography and renaturated. By the same above-mentioned way, the Ka2 of rβ2-GPI binding to 125I– rHBsAg was measured.Results: The specific activity of 125I-rHBsAg is (2.6-2.8)×106 Bq/μg. Following the binding curve, the best temperature and time is 37℃and 4 hours. The Ka1 of the group ofβ2-GPI binding to rHBsAg are (2.075±0.049)×10~8 L/mol,(2.25±0.099)×10~8 L/mol,(5.5±0.665)×10~8 L/mol,(1.355±0.064)×10~8 L/mol;The Ka2 of rβ2-GPI expressed by E.coil are(2.055±0.106)×108 L/mol,( 2.22±0.028)×10~8L/mol,(3.485±0.050)×10~8 L/mol,(4.245±0.134)×10~8 L/mol。According to the summary of β2-GPI from two separate sources binding to rHBsAg, there was no difference between Ka1 and Ka2 (P>0.05) by statistical analysis. Finally,the results of affinity constants of twoβ2-GPI groups were respectively (2.795±1.846)×10~8 L/ mol and (3.001±1.049)×10~8 L/ mol.Conclusion:The affinity ofβ2-GPI and HBsAg are a strong bind in plasma (2.795±1.846)×10~8 L/ mol) and the glycosylation ofβ2-GPI has no effect on this conjugation.