Epitope Presentation System Screening Multifunctional Monoclonal Antibodies Against Hepatitis B Virus And Virus-like Particles

Objective:HBc virus-like particles(HBc-VLPs)are formed by the self-assembly of Hepatitis B virus core proteins.HBc-VLPs can be used as epitope presentation system to carry foreign epitopes.Apart from as VLP vaccine and epitope vaccine candidates,some VLPs have successfully been used to study viral particle uptake,virion-medicated activation,and antigen presentation by dendritic cells(DCs).However, we notice that some commercial monoclonal antibodies against virus can not be used in the localization research of intracellular virus-like particles. It is difficult to get the commercial monoclonal antibodies specifically identifying the VLPs.Many antibodies must be purchased to find the suitable one,which is an annoying and expensive issue.So we expect to produce a HBc-VLP-specific antibody to meet the diverse need in many researchesMethods:①Construction and expression of Hepatitis B virus core antigen:The truncated HBc gene(aa 1-144)was amplified from the complete viral genome of Hepatitis B virus by PCR and was introduced into the expression vector pET28a.Escherichia coli BL21.(DE3)were transfected with the plasmids and the cells were induced by Isopropylβ-D-thiogalactoside;②Quick purification of recombinant HBc proteins: The cultured cells(typically 9 g wet weight)were harvested by centrifugation ang resuspended,then lysed by sonication.Inclusion bodies were separated by centrifugation Pellets were then resuspended in 10 ml denaturation buffer(50 mM Tris-HCl,pH8.0,and 0.1M urea), insoluble components were removed by centrifugation.Filtrated supernatant was determined the protein concentration and was diluted with refolding buffer at the concentration of 2 mg/ml.Refolded protein solution was dialysed agaist ddH2O(pH 6.0)in order to remove the survival urea.The refolded protein was quantified and its purity was determined by SDS-PAGE;③Transmission electron microscopic analysis VLP preparations;④Production of monoclonal antibody against HBc-VLPs:The purified HBc-VLPs(aa 1-144)were used as immunogen to immunize,female BALB/c mice.After immunization for several times,the splenocytes from the BALB/c mice with highest levels of antibodies were fused with SP2/0 myeloma cells.Nice hybridomas secreting the McAbs against HBc antigen were generated by ELISA and western blot analysis;⑤Phagocytosis of murine monocyte macrophage cell line RAW264.7;⑥Uptake of HBc-VLP vaccines was analyzed by confocal laser scanning microscopy;⑦Observation of the live HBV in HepG2.2.15 cells:The HepG2.2.15 cells were cultivated and seeded on cover slips.The cells were permeabilized,stained and observed as described previously.The HepG2 cells were used as negative control.Results:①Obtain a construct,pET-28a-HBc(aa1-144)and its molecular weight is 19kDa;②Western blotting assay using monoclonal antibody against HBc was performed to demonstrate that the 19 kDa protein possessed the immunogenicity of HBc antigen;③Electronic micrograph confirmed that the HBc proteins in the 0.1 M urea solution had formed particles;④By ELISA and western blot analysis,five of nine clones could secret the antibodies against the denatured HBc protein;⑤The purified HBc-VLP were used as immunogen to immunize female BALB/c mice,and screened three monoclonal antibodies specifically against HBc-VLPs taken up by APC.Their numbers are 3H2D7-F6, 1H7D7-D2 and 3B8E6-D3;⑥Screening only one monoclonal antibody could specifically bind HBc particles by HepG2.2.15,and its number is 3H2D7-F6.Conclusions:①The truncated HBc-VLP bone vector with special conformation was used as immunogen and the target hybidoma cell lines were screened by a series of tests on the epitope presentation system.The screened monoclonal antibody could be used to identify the HBc-VLP vector,chimeric HBc-VLP vaccine and intracellular HBV capsids.And we can save lots of money by using the screened multi-functional antibody.②the 19 kDa protein possessed the immunogenicity of HBc antigen.③The HBc proteins in the 0.1 M urea solution had formed particles.