脊髓灰质炎病毒Ⅲ型Sabin株C抗原和D抗原的分离及特性分析

目的分离脊髓灰质炎病毒Ⅲ型Sabin株(sPVⅢ)C抗原和D抗原,并分析其部分特性。方法采用CsCl密度梯度离心法分离sPVⅢ的C抗原和D抗原,电镜观察病毒形态;SDS-PAGE检测病毒结构蛋白组成;微量BCA法检测蛋白浓度,计算每种抗原所占比例;细胞病变法检测病毒滴度。同时检测上样浓度及超离次数对C抗原纯度的影响。结果sPVⅢC抗原和D抗原在透射电镜下均呈球形,C抗原病毒颗粒密度较小,外壳大多较为松散,D抗原病毒颗粒密度较大,结构更稳固;C抗原由VP0、VP1、VP3蛋白组成,D抗原由VP1、VP2、VP3和VP4蛋白组成;C抗原和D抗原分别约占总蛋白含量的10%和90%,部分C抗原在分离前及分离过程中易产生聚集;D抗原的病毒滴度高于C抗原。降低上样浓度及进行二次超离可提高C抗原纯度。结论采用CsCl密度梯度离心法成功分离了sPVⅢ的C抗原和D抗原,C抗原滴度远低于D抗原,且部分易产生聚集,较难达到较高纯度。

Isolation and characteristics of C and D antigens of poliovirus type Ⅲ Sabin strain

Objective To isolate C and D antigens of poliovirus type Ⅲ Sabin strain(sPV Ⅲ) and observe its partial characteristics. Methods The C and D antigens of sPV Ⅲ were separated by cesium chloride density gradient centrifugation and observed for morphology by electron microscopy. The composition of structural protein was determined by SDS-PAGE,while the protein concentration by BCA assay,and the proportion of each antigen was calculated. The virus titer was determined by cytovirus method. Meanwhile,the effects of sample concentration for loading and times of ultracentrifugation on the purity of C antigen were evaluated. Results Both C and D antigens of sPVⅢ were in spherical shapes under transmission electron microscope. The viral particles of C antigen were at a low density,of which the most shells were loose. However,the viral particles of D antigen were in a high density,of which the structure was more stable. The C antigen consisted of VP0,VP1 and VP3 proteins,while the D antigen consisted of VP1, VP2,VP3 and VP4. C and D antigens accounted for about 10% and 90% of total protein respectively,and partial C antigen was easy to be aggregated before and during separation. The virus titer of D antigen was higher than that of C antigen. The decrease of sample concentration for loading and ultracentrifugation again increased the purity of C antigen. Conclusion The C and D antigen of sPV Ⅲ were successfully separated by cesium chloride density gradient centrifugation. The titer of C antigen was remarkably lower than that of D antigen. Partial C antigen was easy to be aggregated,which was difficult to reach a high purity.