重组EV71和CVA16型手足口病双价VLP疫苗构建及免疫保护效果评价

Expression of recombinant Enterovirus 71 and Coxsackievirus A group 16 bivalent virus-like particles in baculovirus expression system and evaluation of its immunogenicity

目的运用Bac-to-Bac杆状病毒表达系统表达EV71和CVA16的病毒样颗粒，并对纯化的重组EV71、CVA16双价病毒样颗粒疫苗进行免疫效果评价。方法构建重组杆状病毒Bacmid-P1-3CD，转染Sf9昆虫细胞，纯化EV71、CVA16的病毒样颗粒并检测其形态和生物特性；通过EV71、CVA16的病毒样颗粒免疫ICR雌鼠后，以EV71、CVA16强毒株腹腔攻击5日龄乳鼠，对重组EV71、CVA16型双价VLP免疫保护性进行评价。结果利用Bac-to-Bac杆状病毒表达系统成功构建并表达EV71和CVA16病毒样颗粒，颗粒大小约为23-30nm，存在Mr约39000VP1特异性蛋白表达。重组EV71、CVA16双价VLP疫苗免疫接种能够诱导小鼠机体产生高效价的特异性抗体（EV71中和抗体效价为1：960，CVA16中和抗体效价为1：624）并发挥优于单价VLP疫苗的有效免疫保护效果。结论重组EV71、CVA16型双价VLP疫苗免疫原性和保护性显著高于单价疫苗，为手足口病疫苗的研发提供了新的思路及实验基础。。

Enterovirus 71 （EV71） and Coxsackievirus A group 16 （CVA16） are viruses causing hand-foot- mouth disease. In this study, we aimed to expresse EV71 and CVA16 VLPs by Bac-to-Bac （r） baeulovirus expression system, and evaluate their immunogenicity. Firstly, EV71 and CVA16 were amplified by RT-PCR and cloned into pFastBacDual plasmids, then recombined with Bacmid DNA by transposition reaction was transfected into Sf9 cell. The expressed proteins were analyzed by electron microcopy examination, SDS-PAGE, and Western blotting, while the immunogenicity was measured by ELISA and micro-neutralization. These methods illustrated the VP1 protein of 39 KD was expressed, with antibody titers of 1：102 400 and 1：6 400, while the neutralization antibody titers were 1：960 and 1：624. In this study, EV71 and CVA16 VLP are successfully expressed by Bac-to- Bac（r） baculovirus expression system, and immunogenicity of EV71 and CVA16 bivalent VLP are preliminary evaluated, which are helpful for the researches of EV71 and CVA16 subunit vaccine.