猪瘟病毒囊膜蛋白在杆状病毒表面的展示及展示蛋白的免疫原性试验

Baculovirus surface display of E0E1E2 envelope glycoprotein of CSFV and immunogenicity of the displayed proteins in mouse model

【目的】研制以杆状病毒表面展示系统为基础的猪瘟基因工程亚单位疫苗。【方法】利用杆状病毒表面展示(Baculovirus display)技术,构建展示猪瘟病毒(CSFV)囊膜蛋白的重组杆状病毒BacSC-E0E1E2,该重组杆状病毒带有His6标签,且胞质区域(CTD)和跨膜区域(TM)均来源于杆状病毒囊膜蛋白gp64的CTD和TM。对重组杆状病毒进行Western blot分析、激光共聚焦显微镜观察、免疫金电子显微镜检测、动物免疫试验、血清中和试验。【结果】Western blot分析结果表明,重组杆状病毒中表达了重组E0E1E2蛋白;激光共聚焦显微镜检测结果表明,重组杆状病毒感染昆虫细胞Sf9后在昆虫细胞膜上表达了重组E0E1E2蛋白;免疫金电子显微镜观察表明,重组蛋白展示在杆状病毒囊膜上;动物免疫试验表明,重组杆状病毒免疫小鼠血清OD450值达到1.82;血清中和试验表明,免疫小鼠的血清50%中和效价(PD50)达到512。【结论】成功构建了表面展示猪瘟囊膜蛋白的重组杆状病毒。

[Objective] The study developed classical swine fever genetic engineering subunit vaccine based on baculovirus surface display system. [Method] One recombinant baculoviruses BacSC-EOE1E2 expressing histidine tagged EOE1E2 with CTD and TM derived from baculovirus envelope protein gp64 were constructed. The recombinant baculoviruses were analyzed by Western blot,immunofluorescence confocal microscopy,immunogold electron microscopy, animal vaccine tests and serum neutralization test. [Result] After infection,EOE1E2 was expressed and anchored on the plasma membrane of Sf9 cells,as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that the BacSC-EOE1E2 displayed EOEIE2 glycoprotein on the viral surface. Animal vaccine tests showed that BacSC-EOE1E2 elicited significantly higher antibody titers （OD450 = 1.82）in the treated mouse models. Virus neutralization test showed that serum from the BacSC-EOEIE2 treated models had significant levels of virus neutralization activities （PD50= 512）. [Conclusion] The recombinant baculoviruses BacSC-EOE1E2 that surface display CSFV membrane protein were successfully built.