O型FMDV适应酸性胁迫的分子机制研究及耐酸空衣壳样颗粒的分子组装

We have assembled empty capsid-like particles of serotype Asia I in silkworm larvae using silkworm-baculovirus expression system. Based on above research, we have developed the type O and Asia 1 bivalent vaccine (empty capsid vaccine compound) which composed of type O inactivated antigens and type Asia 1 empty capsid-like particles. It was licensed for use in eight provinces and autonomous regions of China in 2008. But we did not assembled empty capsid-like particles of serotype O in silkworm larvae or pupae. The empty capsid-like particles of type O are more difficult to obtain by genetic engineering techniques as the viral capsid of type O is more acid-sensitive than other types of FMDV. To overcome this problem, some amino acids in capsid protein will be replaced to improve acid resistance of empty capsid particles in this research. But the molecular determinants in viral capsid that mediate seotype O FMDV dissociation into pentameric subunits at acidic pH have not been elucidated. So we don't know which amino acids should be replaced. The infectious FMDV mutants with increased acid resistance were isolated by biological cloning after acid treatment in our proviously research. Based on these mutants, we will address the study to find the molecular determinants of viral resistance to acid-induced disassembly.The complete capsid coding regions of mutants and the parental virus will be sequenced and compared to find the nucleotide and amino acids replacements. The reversed genetics technique will be used to identify whether these amino acid replacements are sufficient to confer resistance to acid induced inactivation and find the molecular determinants finally. Based on above research, we will use the silkworm-baculovirus expression system to assemble the empty capsid-like particles of serotype O with an increased resistance to acid induced dissociation into pentameric subunits and evaluated the immunogenicity of these empty capsid-like particles. The results will not only provide a deeper insight into acid-dependent uncoating mechanism, but also provide an approach to develop the empty capsid-like particles vaccine for the acid-sensitive virus.

本课题组利用家蚕杆状病毒表达系统组装成功AsiaI型FMDV空衣壳样颗粒，以此研制的复合型疫苗已获农业部临时生产文号。由于O型FMDV衣壳在酸性环境中更易解离，我们未能用此系统组装出空衣壳样颗粒。为解决该问题，我们拟对衣壳蛋白进行耐酸改造以提高其对酸性环境的耐受力。但目前尚未在O型FMDV衣壳蛋白上鉴定出可上调衣壳耐酸能力的关键位点。本项目在课题组已获得耐酸突变株的基础上开展这一关键分子位点的鉴定工作：依据突变株与母本病毒衣壳蛋白上的氨基酸差异，构建不同的感染性克隆分析相关位点的氨基酸替代现象与上调突变株耐酸能力的关系，以期从中鉴定出关键分子位点。在此基础上开展空衣壳样颗粒的分子构建工作，并分析相关位点改造对空衣壳样颗粒的耐酸能力、组装率及免疫原性的影响。研究结果有助于阐明FMDV适应酸性胁迫的分子机制，丰富对酸依赖性脱壳分子机制的认识，并为酸敏感性病毒空衣壳疫苗的研制提供新的方法和思路。

口蹄疫（Foot-and-mouth disease, FMD）由口蹄疫病毒(Foot-and-mouth disease virus, FMDV)引起的一种动物烈性传染病，该病一旦发生对畜牧业造成毁灭性打击。目前，接种疫苗仍然是防制 FMD最重要的手段，但由于现今使用的灭活苗存在一定的安全隐患，因此研究安全、高效、价廉的口蹄疫基因工程疫苗是当前研究热点。本课题组利用家蚕杆状病毒表达系统组装成功 AsiaI 型 FMDV 空衣壳样颗粒, 以此研制的复合型疫苗已获农业部临时生产文号。由于 O 型 FMDV 衣壳在酸性环境中更易解离，我们未能用此系统组装出空衣壳样颗粒。为解决该问题，我们拟对衣壳蛋白进行耐酸改造以提高其对酸性环境的耐受力。本项目取得以下研究结果：.1. 通过酸性环境诱变方式筛选获得6株O型FMDV耐酸突变株，将诱变病毒与亲本病毒衣壳蛋白编码区序列比对后发现存在于VP1上的K41E、Q25R和N85A及VP3上K154Q 的4个共有氨基酸突变。依据筛选出的耐酸氨基酸位点对母本病毒衣壳蛋白基因进行了定点突变后，构建了含上述诱变氨基酸的重组杆状病毒。将重组杆状病毒感染细胞后，经间接免疫荧光、抗原捕获双抗体夹心ELISA、western blot等方法检测到外源基因表达。通过免疫金标电镜技术，观察到了直径约27－30nm的口蹄疫空衣壳样颗粒，表明O型FMDV空衣壳在杆状病毒表达系统中组装成功。并经世界FMD参考实验室检测，确定其空衣壳含量达到160.29 µg/ml。上述研究工作证实基于筛选诱变氨基酸位点对衣壳基因的改造工作可以使我们在昆虫细胞酸性环境下获得大量的耐酸空衣壳，能够解决O型FMDV在昆虫细胞酸性环境下难以组装的难题。将获得的空衣壳抗原免疫小鼠可诱导小鼠产生高水平的特异性抗体及IFN-γ及IL-4细胞因子。免疫豚鼠后，利用同源强毒攻击，可获得高水平保护率。证实利用耐酸改造工作制备的空衣壳抗原具有与亲本病毒灭活抗原相似的免疫原性。.2、基于可在酸性环境下组装的AsiaI型FMDV空衣壳骨架，构建出7组含不同O型和Asia 1型组合基因的嵌合杆状病毒。Western blotting检测结果表明上述嵌合体均可在昆虫细胞中得到表达；双抗体夹心ELISA 检测结果表明表达的嵌合体蛋白具有抗原性。组装的空衣壳能够使小鼠产生同传统疫苗组抗体水平相当的抗体水平。免