Liu Di, Li Bo, Bi Cheng, Qiao Hongping, Wu Xiaoying
Department of Biology and Center for Veterinary Medicine, Taiyuan Normal University, Jinzhong 030619, Shanxi, China.
Faculty of Science, The University of Sydney, New South Wales 2006, Australia.
Sheng Wu Gong Cheng Xue Bao. 2020 Jul 25;36(7):1440-1449. doi: 10.13345/j.cjb.190534.
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.
乙型肝炎病毒核心蛋白在体外可自组装成二十面体对称的病毒样颗粒（VLPs），并在其表面重复且密集地展示外源序列。VLPs还具有很强的免疫原性和生物活性。当这些纳米颗粒进入体内时，它们能迅速诱导机体对外源抗原产生特异性体液免疫和细胞免疫反应。在本研究中，我们设计了一种可在特定位点与抗原偶联的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，将肽段的第80位氨基酸由丙氨酸变为半胱氨酸，在HBc-VLPs的主要免疫显性区域插入一个特异性交联位点，并构建了原核表达载体pET28a(+)-hbc。表达纯化后，高纯度的HBc(A80C)单体蛋白在磷酸盐缓冲液中组装成HBc-VLPs纳米颗粒。粒径分析结果显示，纳米颗粒的平均粒径为29.8 nm。透射电子显微镜（TEM）显示，HBc-VLPs形成了粒径约为30 nm的球形颗粒，其形态与天然乙肝病毒颗粒相似。以流感病毒抗原M2e肽作为模型抗原，通过氨基巯基双功能交联剂磺基琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯（Sulfo-SMCC）将其连接到HBc-VLPs的半胱氨酸残基上，制备了M2e-HBc-VLPs模型疫苗。通过细胞荧光示踪验证了HBc-VLPs结构的完整性以及M2e的正确交联。动物免疫实验表明，该疫苗能有效刺激小鼠产生抗原特异性IgG抗体，证实了疫苗载体HBc-VLPs的有效性。本研究为HBc-VLPs作为疫苗载体的研究奠定了基础，有助于推动HBc-VLPs疫苗的研发及其在其他领域的应用。
