Correlation of the CTD structural domain of hepatitis B virus core protein with the encapsulation effect of indocyanine green / 生物工程学报

Hepatitis B virus core protein (HBc) has become a hot spot in drug carrier protein research due to its natural particle self-assembly ability and ease of modification. The truncation of the C-terminal polyarginine domain (CTD, aa 151-183) of HBc does not affect the self-assembly of the particles. However, it does affect the internal and external charges of the particles, which may subsequently affect drug encapsulation. Thus, the truncated C-terminal polyarginine domain (CTD) of HBc and the inserted RGD peptide were selected to construct and express three HBc variants (RH) encapsulated with ICG (RH/ICG) with different C-terminal lengths to compare the stability and drug activity of their nanoformulations. RH160/ICG was found to have a great advantages in encapsulation efficiency and biological imaging. Compared with other HBc variants, RH160/ICG significantly improved encapsulation efficiency, up to 32.77%±1.23%. Cytotoxicity and hemolysis assays further demonstrated the good biocompatibility of RH160/ICG. Cell uptake and in vivo imaging experiments in mice showed that RH160/ICG could efficiently deliver ICG in tumor cells and tumor sites with good imaging effect. This research provides a new direction for further expanding the diagnosis and treatment application of ICG and development of HBc-based nanoparticle drug carrier platform.

Preparation and characterization of HBc virus like particles with site-directed coupling function / 生物工程学报

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.

Preparation of monoclonal antibodies against 3D protein of EV71 based on HBc particles as expression vector / 中国免疫学杂志

Objective:To prepare and preliminarily identify the monoclonal antibodies(mAbs) specifically against 3D protein of Enterovirus 71(EV71),using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier.Methods: Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region(MIR) area of Hepatitis B virus core protein(HBc),to construct the recombinant protein.BALB/c mice were immunized with the recombinant virus like particles(VLPs),to prepare the mAbs against 3D protein of EV71.Affinity chromatography technology was used to purify the mAb.The indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the mAb.Results: We displayed 3D(aa34-43),3D(aa61-76) and 3D(aa151-164) epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line(3E1) was selected by ELISA.The isotype of 3E1 was IgG2a.The results of immunofluorescence and immunohistochemistry staining assay showed that the mAb 3E1 could specifically recognize EV71.Conclusion: The prepared mAb 3E1 can specifically recognizes the EV71,which laid the foundation for the detection of virus and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare mAb quickly and efficiently.