柯萨奇病毒A组16型实壳病毒颗粒与空壳病毒颗粒免疫原性的比较

Immunogenicity of full and empty particles of coxsackievirus A16

目的比较柯萨奇病毒A组16型（coxsackievirus A16,CVA16）实壳病毒颗粒（full particle,FP）和空壳病毒颗粒（empty particle,EP）的免疫原性。方法以CVA16病毒株CVA16-L731感染Vero细胞,采用10层细胞工厂培养工艺培养病毒,收获液经离心纯化后,用甲醛灭活,获得灭活纯化的CVA16-L731 FP和EP。采用BCA法检测CVA16-L731 FP和EP的蛋白浓度,4%~12%SDS-PAGE、Western blot及透射电镜对CVA16-L731 FP和EP进行鉴定。以氢氧化铝为佐剂,分别以EP、FP、EP/FP为抗原,于第0、14、28天经后肢肌肉注射免疫BALB/c雌性小鼠,于0、7、21、35、49、63、77、91 d对小鼠进行眼眶采血。采用微量中和试验法检测免疫小鼠血清中和抗体滴度,ELISA法检测免疫小鼠血清特异性Ig G抗体滴度,母传抗体保护试验检测CVA16-L731 FP和EP的垂直保护效果。结果纯化后的CVA16-L731 FP和EP的蛋白浓度分别达到420和150μg/ml;CVA16-L731 FP可见4条相对分子质量分别约为34 000（VP1）、26 000（VP2）、24 000（VP3）和8 000（VP4）的条带,CVA16-L731 EP可见3条相对分子质量分别约为36 000（VP0）、34 000（VP1）和24 000（VP3）的条带;CVA16-L731 FP和EP的纯度均可达95%;CVA16-L731 VP1兔多抗可与FP及EP的VP1、CVA16-L731 VP2兔多抗可与FP-VP2及EP-VP0发生特异性免疫反应;CVA16-L731FP和EP分别为内部不可被磷钨酸负染的FP致密圆形颗粒和内部可被磷钨酸负染的黑色EP圆型颗粒。小鼠免疫试验结果显示,第1次免疫后各抗原免疫组小鼠血清中和抗体阳性率均为100%;第2次免疫后血清中和抗体滴度和血清特异性Ig G水平均显著升高;第3次免疫后血清中和抗体滴度均无明显变化,但血清特异性Ig G水平均明显升高;第3次免疫后的63 d内,血清中和抗体滴度均无显著变化。各抗原免疫组乳鼠存活率均为100%。结论CVA16-L731 FP和EP均能诱导小鼠持续产生较高水平的血清中和抗体和特异性Ig G抗体,且母传抗体可保护2日龄乳鼠抵抗CVA16-S315的致死性攻击,为CVA16疫苗的研制提供了实验依据。

Objective To compare the immunogenicity of full particles（FPs）and empty particles（EPs）of coxsackievirus A16（CVA16）.Methods CVA16-L731 strain was infected to Vero cells and cultured in 10 layer cell factory,of which the harvest was inactivated with formaldehyde.The inactivated FPs and EPs were determined for protein concentration by BCA,and identified by 4% ~ 12% SDS-PAGE,Western blot and transmission electron microscopy.Female BALB / c mice were immunized i.m.with EPs,FPs,EPs / FPs using aluminum hydroxide respectively,of which the serum samples were collected on days 0,14 and 28 after immunization,and determined for neutralizing antibody titer by microneutralization assay,for specific Ig G titer by ELISA,and for vertical protective effect by maternal antibody protection test.Results The concentrations of purified CVA16-L731 FPs and EPs were 420 and 150 μg / ml respectively.The FPs showed four bands,with relative molecular masses of about 34 000（VP1）,about 26 000（VP2）,about 24 000（VP3）and about 8 000（VP4）,while EPs showed three bands with relative molecular masses of about 36 000（VP0）,about 34 000（VP1） and about24 000（VP3）,on SDS-PAGE profile,respectively.Both the FPs and EPs reached purities of 95%.The rabbit polyclonal antibody against CVA16-L731 VP1 showed specific reactions with the VP1 of FPs and EPs,while that against CVA16-L731 VP2 with FP-VP2 and EP-VP0.The FPs were dense and round,of which the inside was negatively stained with phosphotungstic acid;however,EPs were black and round,of which the inside was not negatively stained.The immuniza-tion test in mice showed that the neutralizing antibody positive conversion rates in sera of mice after first immunization with various antigens were 100%.Both neutralizing antibody titer and specific Ig G level in sera of mice after the second immu-nization increased significantly.The serum neutralizing titer after the third immunization showed no significant change,while the serum specific Ig G level increased significantly.The serum neutralizing antibody titer within 63 d after the third immunization showed no significant change.All the survival rates of suckling mice in various immunization groups were100%.Conclusion Both CVA16-L731 FPs and EPs induced high neutralizing antibody titer and specific Ig G level in sera of mice,and the maternal antibody protected 2-day-old suckling mice against the lethal challenge with CVA16-S315,which provided a laboratory basis for the development of CVA16 vaccine.