猪繁殖与呼吸综合征病毒GP3与GP5蛋白在杆状病毒表面的展示

Bsculovirus surface display of GP3 and GP5 proteins of porcine reproductive and respiratory syndrome virus

选择猪繁殖与呼吸综合征病毒(PRRSV) GP5和GP3蛋白作为免疫原,将杆状病毒的表面展示系统作为载体,成功构建了重组转座载体pBacSC-GP5与pBacSC-GP3,通过转化DH10Bac,筛选蓝白斑,转染昆虫Sf-9细胞,获得重组杆状病毒BacSC-GP5、BacSC-GP3。重组病毒感染Sf-9细胞后,经Western blot和激光共聚焦显微镜观察,目的蛋白在感染细胞中获得表达,并且成功展示在了感染细胞的质膜上。将重组病毒经蔗糖梯度超速离心纯化后,经Western blot和免疫透射电镜观察,目的蛋白在重组病毒上获得表达,展示在重组病毒的囊膜上。研究为开发PRRSV新型疫苗奠定基础。

The recombinant transfer vector pBacSC-GP5 and pBacSC-GP3 were successfully constructed,and then were transformed into competent E.coli DH10 Bac cells.After selecting colonies through blue/white selection,recombinant bacmids were isolated and transfected into Sf-9 cells,resulting in recombinant baculoviruses Bac SC-GP5 and Bac SC-GP3.After infection,the GP5 and GP3 proteins were expressed and anchored on the plasma membrane of Sf-9 cells,as revealed by Western blot and confocal microscopy.After the virus particles were purified by sucrose gradient ultracentrifugation,the purified virions were detected by immunogold electron microscopy.The result showed that the recombinant GP5 or GP3 protein was displayed successfully on the viral surface.