E2蛋白介导的猪瘟兔化弱毒疫苗株适应家兔的分子机制

C-strain, also known as hog cholera lapinized virus (HCLV) strain, is a highly efficacious and safe modified live vaccine that was developed through hundreds of passages of a highly virulent classical swine fever virus (CSFV) in rabbits in China. Our previous study has demonstrated that the C-strain E2 protein is indispensable to the adaptation of C-strain to the rabbit. However, the exact molecular mechanism of the E2-mediated adaptation of C-strain to the rabbit remains to be elusive. In this proposal, a panel of mutant viruses in the background of C-strain harboring the different domains or differential amino acids on the E2 protein of the highly virulent CSFV Shimen strain will be generated and evaluated in rabbits to map the critical domain(s) and site(s) on the E2 protein. Next, the roles of the E2 protein, the critical domain(s) and site(s) on the E2 protein in the adaptation will be determined through virus attachment and entry assays in rabbit-derived spleen lymphocytes, the target cells of C-strain. Finally, the host membrane proteins (HMP) interacting with the C-strain E2 protein will be screened and identified using Co-IP and GST pulldown assays and the functions of the above HMP in the adaptation will be determined by knocking out or over-expressing HMP and blocking assays using the soluble HMP or the anti-HMP antibodies in the rabbit lymphocytes. This project will clarify the E2-mediated adaptation mechanism of C-strain to the rabbit, which will provide a foundation for dissecting the attenuation mechanism of this excellent vaccine strain and generating high-yield vaccine strains.

猪瘟兔化弱毒疫苗株（C株）是我国学者将猪瘟病毒强毒株在兔体内连续传480余代后培育而成的一株非常安全和有效的疫苗。我们的前期研究证实，C株的E2蛋白是其适应家兔所必需的。然而，E2蛋白介导的C株适应家兔的确切机制尚不明确。本项目拟首先通过构建以C株为骨架、包含猪瘟病毒强毒石门株E2蛋白不同结构域和差异氨基酸的突变体病毒，确定C株适应家兔的E2蛋白关键结构域和位点；然后，通过不同突变体病毒在C株靶细胞上的吸附和侵入试验，明确E2蛋白及其关键结构域和位点在C株感染靶细胞中的作用；最后，通过Co-IP和GST pulldown等技术筛选和鉴定与C株E2蛋白相互作用的靶细胞膜蛋白，之后通过抗体封闭试验和可溶性蛋白阻断等试验，阐明与E2相互作用的细胞膜蛋白在C株吸附或侵入靶细胞中的作用。本项目可望揭示E2蛋白介导的C株适应家兔的分子机制，为揭示这株优异弱毒疫苗的致弱机制以及构建高产疫苗株奠定基础。

猪瘟兔化弱毒疫苗株（C株）是我国学者将猪瘟病毒强毒株在兔体内连续传480余代后培育而成的一株非常安全和有效的疫苗。E2蛋白介导的C株适应家兔的确切机制尚不明确。本项目主要发现如下：（1）通过构建以C株为骨架、包含猪瘟病毒强毒石门株E2蛋白不同结构域和差异氨基酸的突变体病毒，发现C株适应家兔的E2蛋白关键结构域I和关键位点P108和T109；（2）通过基于不同突变体病毒的囊膜蛋白构建的伪型病毒在C株靶细胞上的侵入试验发现，E2蛋白及其关键结构域和位点通过介导C株入侵靶细胞赋予了C株适应家兔的能力；（3）通过构建糖基化位点的突变体病毒，进行体内外试验发现，C株囊膜糖蛋白E2上特有的糖基化位点N986在病毒致弱和诱导免疫保护中的重要作用；（4）通过构建单克隆抗体识别位点的突变体病毒，进行体内外试验发现，C株可改造为具有DIVA特性的标记疫苗株；（5）经过Co-IP和GST-pulldown试验发现宿主膜分子ANXA1与E2的蛋白相互作用，并影响CSFV的复制。本项目揭示了E2蛋白介导的C株适应家兔的分子机制，为揭示这株优异弱毒疫苗的致弱机制以及构建高产疫苗株奠定基础。

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