Kratz P A, Böttcher B, Nassal M
University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1915-20. doi: 10.1073/pnas.96.5.1915.
The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.
乙型肝炎病毒（HBV）的核衣壳，即乙肝核心抗原（HBcAg），是一种高度对称的结构，由单一核心蛋白的多个二聚体形成，该核心蛋白在其183个氨基酸序列中含有强效的T辅助表位。这两个因素使HBcAg成为一种异常强大的免疫原，并且是作为外源表位载体的有吸引力的候选物。衣壳上的免疫显性c/e1表位已被认为是向异源序列传递高免疫原性的优越位置。然而，由于其中心位置，任何c/e1插入都会破坏核心蛋白的一级序列；因此，只有肽段，或者更确切地说是小蛋白片段似乎与颗粒形成兼容。根据最近的结构数据，该表位位于由非常稳定的二聚体界面形成的突出表面刺突的尖端。因此我们推断，如果相应融合蛋白的各个部分能够独立折叠，可能会容忍更大的插入片段。以绿色荧光蛋白（GFP）作为模型插入片段，我们表明嵌合蛋白能有效形成荧光颗粒；因此，其所有结构上重要的部分都必须正确折叠。我们还证明GFP结构域暴露于表面，并且嵌合颗粒引发针对天然GFP的强效体液反应。因此，至少长达238个氨基酸的蛋白质可以天然地展示在HBV核心颗粒表面。这样的嵌合体不仅可能作为疫苗有用，而且还可能为通过电子显微镜对非组装蛋白进行高分辨率结构分析开辟道路。
