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目的评价不同形式的脊髓灰质炎病毒(poliovirus,PV)颗粒的免疫原性。方法采用蔗糖密度梯度超速离心法分别将经56℃加热1.5 h处理和未经加热的PV进行分离纯化,收集相应组分,进行电镜观察及SDS-PAGE分析,并测定各组分的D抗原及蛋白含量。用不同PV颗粒组分分别免疫大鼠,均经腿部肌肉注射,共接种3次,每次间隔21 d,于免疫前和第1、2次免疫后21 d经眼眶采血,末次免疫后21 d经心脏穿刺采血,分离血清,微量细胞病变(CPE)抑制法检测血清中抗Ⅰ、Ⅱ、Ⅲ型PV中和抗体几何平均滴度(GMT)。结果未经加热处理的PV可分离获得EP(蔗糖浓度16%~19%)和FP(蔗糖浓度22%~24.5%)两种病毒颗粒,电镜观察分别为空心颗粒和实心颗粒,EP经SDS-PAGE分析可见VP0、VP1和VP3病毒蛋白条带,FP可见VP1、VP2、VP3和VP4条带;经56℃加热处理的PV可分离获得HP(蔗糖浓度15%~17%)一种病毒颗粒,电镜观察为空心颗粒,SDS-PAGE分析可见VP0、VP1、VP2、VP3病毒蛋白条带。EP、FP免疫大鼠后,均可诱导产生针对PV的中和抗体,但EP诱导产生的中和抗体GMT低于FP;HP不能诱导产生中和抗体。结论未经加热的PV可分离获得EP和FP两种病毒颗粒,均具有免疫原性;经加热处理的PV仅分离获得HP一种病毒颗粒,不具有免疫原性。
Objective To evaluate the immunogenicity of different forms of poliovirus (PV) particles. Methods The sucrose-density gradient ultracentrifugation method was used to separate and purify the PV after heating at 56 ℃ for 1.5 h and the corresponding components were collected and analyzed by electron microscopy and SDS-PAGE. The D antigen and Protein content. Rats were immunized with different components of PV granules. All of them were intramuscularly injected three times for 21 days at intervals of 21 d. Blood samples were taken from the orbital cavity 21 days before and after the first and second immunization. After the last immunization, d. Peripheral blood was collected by cardiac puncture, and the geometric mean titers (GMTs) of anti-type I, II, and III neutralizing antibodies in serum were detected by CPE inhibition. Results The two kinds of virus particles, EP (sucrose concentration 16% -19%) and FP (sucrose concentration 22% -24.5%), were isolated from the PV without heat treatment. Hollow particles and solid particles were observed under electron microscope. PAGE revealed VP0, VP1 and VP3 viral protein bands. The VP1, VP2, VP3 and VP4 bands were visible in FP. HP (heat-treated at 56 ℃) could be used to isolate HP with a concentration of 15% -17% Electron microscope observation of hollow particles, SDS-PAGE analysis showed VP0, VP1, VP2, VP3 virus protein bands. EP and FP immunized rats could induce neutralizing antibody against PV, but the neutralizing antibody GM induced by EP was lower than FP; HP could not induce neutralizing antibody. Conclusion The unheated PV can be isolated from both EP and FP virus particles, which are immunogenic. The heat-treated PV isolated only one HP virus particle and is not immunogenic.