PRRSV标记病毒样颗粒的制备及其鉴定

Preparation and identification of PRRSV virus-like particles with Myc and His tags

当今预防猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome,PRRS）的灭活苗和弱毒苗都存在不可避免的局限性,所以研制新型疫苗势在必行。病毒样颗粒（virus-like particles,VLPs）在形态结构上与天然病毒粒子无异,生物安全指数高,是理想的传统疫苗替代品。猪繁殖与呼吸综合征病毒（PRRSV）粒子主要由能够诱导产生中和抗体的GP5蛋白和具有强细胞免疫力的M蛋白组成基本框架结构。本研究以2型高致病性PRRSV的GP5蛋白和M蛋白为研究对象,通过Bac-to-Bac昆虫杆状病毒表达系统,构建标记型PRRSV VLPs,同时探索核衣壳蛋白N对VLPs形成的影响。在本试验中将含有Myc标签的GP5（Myc-GP5）和含有His标签的M（His-M）蛋白基因分别克隆至pFastBac dual载体的p10和pH双元启动子下;N蛋白基因克隆至pFastBac HTB载体上,转染sf9和Highfive细胞后,GP5、M和N蛋白均被成功表达。透射电镜观察结果表明,Myc-GP5和His-M能够自动组装成VLPs,并且与天然PRRSV形态一致,具有双层膜结构,直径大小在40-60 nm。采用抗M蛋白的家兔血清进行免疫电镜观察,结果显示,Myc和His双标记型VLPs与天然病毒一样都能够被金颗粒标记,而且共感染的N蛋白不影响标记型VLPs的组装。本研究首次采用昆虫杆状病毒表达系统成功制备了PRRSV的双标记型VLPs,具备与天然病毒相似的特性,为开发高效安全以及能够区分感染与免疫VLPs疫苗奠定了基础。

Up to now,vaccine inoculation is still the most effective measure for preventing porcine reproductive and respiratory syndrome（PRRS）.However,the inactivated vaccine and the attenuated vaccine have their own shortcomings.Therefore,it is necessary to develop new vaccines against PRRS.Virus-like particles（VLPs） is highly similar to natural virions,having good immunogenicity and containing no infectious viral nucleic acids,so they become the promising vaccine candidate.PRRSV GP5 protein encoded by ORF5 gene is the most important immunoprotective protein by eliciting neutralizing antibodies.M protein encoded by ORF6 can perform a major role in the induction of cell-mediated immunity.Co-expression of GP5 and M protein can form heterodimers,which are the basic elements for generating infectious virus particles.In this study,type 2 highly pathogenic PRRSV VLPs labelled with Myc and His tags were developed via Bac-to-Bac baculovirus system by co-expressing GP5 and M proteins.Furthermore,the influence of N protein on the formation of VLPs was explored.The genes encoding GP5 with Myc tag（Myc-GP5）and M with His tag（His-M）were cloned in frame into p Fast Bacdual vector under the control of p10 and pH promoter,respectively.ORF7 was directly inserted into multiple cloning site of p Fast Bac HTB with 6 x His tag. After transfection of insect cells of sf9 and highfive,Myc-GP5,His-M and His-N proteins were successfully expressed with the molecular weight of around 26,20 and 14 k Da,respectively.Transmission electron microscopy indicated that Myc-GP5 and His-M recombinant proteins could self-assemble into VLPs which are similar to PRRSV natural particles.The VLPs particles are round or oval,and have bilayer membrane structure with the diameter of40 nm to 60 nm.Immunoelectron microscopic observation with rabbit serum against PRRSV M protein showed that the prepared VLPs could be labeled with gold particles just like the natural virus,showing that the expression of N protein did not affect the assembly of labeled VLPs.In this study,PRRSV VLPs tagged with Myc and His were successfully produced by Bac-to-Bac baculovirus expression system for the first time.The VLPs with double tags were pretty similar to the characteristics of PRRS natural virions.Our study lays foundation for further developing novel VLPs vaccine differentiating the infected and immunized pigs.