摘要
对引起手足口病的肠道病毒A组4型进行生物化学特性及免疫原性的研究。利用大肠杆菌表达结构蛋白全长或部分片段的GST融合蛋白,纯化CV-A4病毒颗粒,制备兔抗CV-A4 VP1~VP4蛋白及抗CV-A4病毒颗粒抗血清。以免疫印迹法(Western blotting,WB),结合SDS-PAGE电泳法,对CsCl密度梯度纯化的CV-A4的空心颗粒(EP)与实心颗粒(FP)的蛋白组分、纯度、多聚蛋白酶剪切进行分析。以微量中和法测定以上6种抗原免疫的兔或小鼠血清中和抗体效价,评估免疫原性。WB和间接免疫荧光法证明了抗体特异性。EP由VP0、VP1和VP3构成,而FP的VP0被剪切,故由VP1-VP4构成。EP和FP比例分别为41%和59%。CsCl密度分别为1.26 g/cm^(3)和1.34 g/cm^(3),纯度分别为70.5%和96.3%。中和试验显示,兔抗全病毒抗体具有中和作用,而兔抗CV-A4VP1~VP4结构蛋白抗体不具有中和作用。免疫原性强弱为:FP>EP>VP1-VP4。本研究建立了CV-A4纯化病毒结构蛋白的分析方法,研究了重组病毒蛋白及2种病毒颗粒的免疫原性及免疫持久性。对包括CV-A4的手足口病多价疫苗的研发策略以及质量控制具重要意义。
To study the biochemical characteristics and immunogenicity of enterovirus coxsackievirus A4(CVA4)which causes hand,foot,and mouth disease.We Used a bacterial system to express the full-length or truncated GST-tagged structural proteins to prepare rabbit anti-CV-A4 VP1-VP4 antibodies,and purified CVA4 virions to prepare CV-A4 virus antiserum.Western Blotting(WB)combined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis were applied to analyze protein profiles,purity,polyprotein processing of the empty particles(EP)and full particles(FP)of CV-A4 purified by CsCl density gradient centrifugation.WB and indirect immunofluorescence demonstrated antibodies specificity.EP were composed of VP0,VP1 and VP3,whereas the precursor VP0 of FP was cleaved into VP2 and VP4,so it was composed of VP1-VP4.The ratio of EP and FP was 41%and 59%,the density was 1.26 g/cm^(3)and 1.34 g/cm^(3),and the purity was 70.5%and 96.3%,respectively,for EP and FP.Neutralization assays showed that the rabbit anti-whole virus antibody had neutralizing activity,whereas rabbit anti-CV-A4 VP1-VP4 antibodies did not have neutralizing activity.The immunogenicity of the different forms of antigens was FP>EP>VP1-VP4.An analytical method for the purified structural proteins of CV-A4 was established,and the immunogenicity and persistence of recombinant viral proteins and viral particles were studied.Our data are very important for the quality control of multivalent vaccines for hand,foot,and mouth disease due to CV-A4 infection.
作者
田宇璇
魏真妮
安欢欢
吴杰
申硕
TIAN Yuxuan;WEI Zhenni;AN Huanhuan;WU Jie;SHEN Shuo(Research Division of Viral Vaccines,Wuhan Institution of Biological Product Co.Ltd,Wuhan 430200,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2022年第3期581-590,共10页
Chinese Journal of Virology
基金
湖北省技术创新专项(重大专项)(项目号:2017ACA078)
题目:手足口病双价灭活疫苗的研制。
