不同形式CA16病毒颗粒的分离鉴定与免疫原性初步分析

Isolation,identification and immunogenicity of various coxsackievirus A16 particles

目的:制备不同形式的CA16病毒颗粒,并对其免疫原性进行初步分析。方法:采用蔗糖密度梯度超速离心法和阴离子交换层析法,将CA16病毒进行分离纯化,收集相应组分,测定各组分抗原、蛋白含量,并进行SDS-PAGE、Western Blot、电镜鉴定。将分离到的不同病毒颗粒组分分别免疫小鼠,于0,14和28 d腹腔注射接种3次,末次免疫后14 d眼眶采血、分离血清。采用微量细胞病变抑制法检测血清中抗CA16病毒中和抗体效价。结果:CA16病毒液经超速离心和阴离子交换层析后,可分离得到2种形式的病毒颗粒:EP和FP。电镜下EP表现为空心颗粒、FP为实心颗粒。SDS-PAGE结果显示:EP可见VP0,VP1,VP3蛋白条带;FP可见VP1,VP2,VP4蛋白条带。EP,FP免疫小鼠后,均可诱导产生针对CA16病毒的中和抗体,但EP诱导产生的中和抗体效价远低于FP,两者间具有统计学差异。结论:体外培养的CA16病毒存在EP和FP 2种病毒颗粒形式,二者均具有免疫原性,但FP高于EP。

Objective： To prepare different forms of CA16 virus particles and to analyze their immunogenicity.Methods： The sucrose density gradient ultracentrifugation and anion exchange chromatography were used to isolate and purify CA16 virus. The corresponding components were collected and the contents of antigen and protein in each component were determined. SDS-PAGE,Western Blot,and electron microscopy were used for identification.Mice were immunized with different isolated virus particle fractions respectively and injected intraperitoneally 3 times at 0 d,14 d,and 28 d. Blood was collected on day 14 after the last immunization and serum was separated.The neutralizing antibody titers were detected by the cytopathic effect-based neutralization assay. Results： After the virus were subjected by ultracentrifugation and anion exchange chromatography,two forms of virus particles,EP and FP,could be isolated. Under electron microscope,EP showed hollow particles and FP was solid particles.SDS-PAGE results showed that VP0,VP1 and VP3 protein bands were observed in EP,and VP1,VP2 and VP4 protein bands were observed in FP. After the mice immunized with EP and FP,neutralizing antibodies against CA16 virus were induced,but the level of neutralizing antibodies induced by EP was much lower than that of FP.There was a statistical difference between them. Conclusion： In the cell culture,there are two types of virus particles EP and FP,both of which have immunogenicity. But the immunogenicity of FP is higher than that of EP.