VP60loop区的突变对RHDVVLP功能的影响

[目的] 从病毒样颗粒(VLP)的形成、血凝特性以及受体组织血型抗原(HBGA)结合特性方面,分析VP60 loop区在兔出血症病毒(RHDV)VLP功能中的作用。[方法] 针对RHDV VP60蛋白的loop区411~417位氨基酸对应的基因设计引物,通过SOE法将411~417位氨基酸等量替换成柔性氨基酸GSGGSGG,获得重组基因VP60-411-417,利用杆状病毒表达系统获得重组病毒rAc-VP60-411-417。将重组病毒接种Sf9细胞,通过IFA、SDS-PAGE和Western blot等方法证明嵌合蛋白P411-417的表达。通过负染透射电镜观察嵌合蛋白VLP的形成,利用血凝试验分析嵌合蛋白的血凝特性,利用合成多糖结合试验分析嵌合蛋白的受体结合特性。[结果] Western blot、IFA和负染透射电镜试验结果显示,嵌合蛋白P411-417得到有效表达,且能够形成完整的VLP;合成多糖结合试验结果显示,嵌合蛋白能够特异性结合HBGA,但其血凝特性消失。[结论] 411~417位氨基酸的突变显著影响RHDV 的血凝特性,不影响VLP的形成以及结合HBGA的能力,说明该区域是血凝特性的相关位点,但不是HBGA受体结合的关键位点。
[Objectives] Assembly of virus like particles(VLP),hemagglutination and binding activities to histo-blood group antigen(HBGA)of VLP were analyzed to explore effects of a VP60 loop on the structure and function of rabbit hemorrhagic disease virus(RHDV)VLP. [Methods] The primers were designed according to the sequences of VP60 and a loop domain(411-417aa). These quences of 411-417aa were replaced by flexible amino acids GSGGSGG. The recombinant virus rAc-VP60-411-417 was obtained by Bac-to-Bac Baculovirus Expression System. Chimera protein P411-417 were expressed and identified by IFA,SDS-PAGE and Western blot analysis. The formation of VLP was detected by the transmission electron microscope. To analyze the characteristics of chimera protein,hemagglutinating test and VLP binding to synthetic H type 2 blood group oligosaccharides assay were performed. [Results] Chimera protein P411-417 was expressed effectively in insect cells and could self-assemble into VLP,confirmed by IFA,Western blot and the transmission electron microscope. And P411-417 could also specifically bind to HBGA. However,P411-417 could not agglutinate human erythrocyte. [Conclusions] Therefore,411-417aa may be indispensable for the hemagglutination of RHDV,but not be crucial for binding to HBGA. It will lay the foundation for the research in the binding sites of RHDV to hemagglutinin and HBGA and be useful for future studies in the structure and function of RHDV