具备定点偶联功能的HBc-VLPs的制备与性质鉴定

Preparation and characterization of HBc virus like particles with site-directed coupling function

摘要:

乙型肝炎病毒核心蛋白HBc，可在体外自组装形成二十面体对称结构的病毒样微粒VLPs。VLPs可将外源序列重复且高密度地展示在表面，VLPs进入机体后能够快速诱导机体产生针对外源性抗原的特异性体液免疫及细胞免疫应答，具有极强的免疫原性与生物活性。因此，HBc-VLPs可以作为一种安全、有效的疫苗载体。文中设计了一种能够实现与抗原定点偶联的HBc-VLPs，并开发了一套高效制备HBc-VLPs的方法。通过定点突变技术，使翻译后的多肽序列第80位氨基酸由Ala变为Cys，在HBc-VLPs的主要免疫显性区域引入一个定点交联位点，构建了原核表达载体pET28a(+)-hbc，表达、纯化获得了高纯度的HBc(A80C) 单体蛋白；在PB缓冲体系中，HBc(A80C) 蛋白自组装形成HBc-VLPs纳米粒子。粒度仪的测定结果表明，HBc-VLPs纳米微粒的平均粒径为29.8 nm，透射电子显微镜观察到HBc-VLPs形成粒径约为30 nm的球形微粒，其形态与天然的HBV微粒相似。以流感病毒M2e抗原肽为模式抗原，通过Sulfo-SMCC氨基-巯基双功能交联剂，将M2e定点连接于HBc-VLPs通过突变引入的Cys残基处，制备了M2e-HBc-VLPs模式疫苗，通过细胞荧光示踪，验证了HBc-VLPs结构的完整性与M2e的正确交联。动物免疫实验表明该疫苗能够有效刺激小鼠产生抗原特异性的IgG抗体，验证了疫苗载体HBc-VLPs的有效性。研究结果为HBc-VLPs作为疫苗载体的研究奠定了基础，能够促进HBc-VLPs载体疫苗的研发以及HBc-VLPs在其他领域的应用。

Abstract:

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.