EV71外壳蛋白表位肽多克隆抗血清的制备及鉴定  

Preparation and identification of the epitope peptide polyclonal antisera against capsid proteins of EV71

作　　者：李晨 张婷[1] 王志荣 许雪梅[1] LI Chen;ZHANG Ting;WANG Zhi-rong;XU Xue-mei(Department of Biophysics and Structural Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院生物物理及结构生物学系,北京100005

出　　处：《基础医学与临床》2022年第11期1661-1666,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(31970867);中国医学科学院医学与健康科技创新工程项目(2021-12M-1-043)。

摘　　要：目的制备肠道病毒71型(EV71)外壳蛋白表位肽多克隆抗血清。方法利用5种B细胞表位预测软件对EV71外壳蛋白进行分析,设计针对病毒蛋白(VP)的4种表位肽,即VP1(aa.204-223)、VP2(aa.133-163)、VP3(aa.139-148+aa.173-191)和VP4(aa.1-23),化学合成后与钥孔虫戚血蓝蛋白(KLH)偶联得到相应肽。然后,分别联合MF59/CpGC274佐剂皮下免疫BALB/c小鼠6次,间隔2周,采集血清,用ELISA和Western blot对其抗体活性进行分析。结果4种抗血清均可有效结合EV71病毒和其变性病毒,VP2抗血清与变性病毒的结合活性较高,其余3种与病毒及变性病毒的结合力相当;而且4种抗血清均可与病毒样颗粒(VLP)及变性VLP结合。VP1和VP2抗血清分别与EV71 VLP中的组成蛋白VP1和VP0(VP2和VP4前体蛋白)结合,其中VP2抗血清的结合活性最好,VP1抗血清次之;同时,VP1和VP2抗血清还可与EV71病毒特异性高效结合,且VP2抗血清可同时结合EV71病毒的空心颗粒的VP0及实心颗粒的VP2。VP3和VP4抗血清与VLP和病毒未显示明显的结合活性。结论成功制备了4种EV71表位肽多克隆抗血清,为ELISA和Western blot提供检测工具,并可用于EV71疫苗质控(QC)研究。Objective To prepare polyclonal antisera against enterovirus 71(EV71)capsid proteins.MethodsFive B cell epitope prediction softwares were used to analyze capsid proteins of EV71 and four peptides against viral protein were synthesized,VP1(aa.204-223),VP2(aa.133-163),VP3(aa.139-148+aa.173-191)and VP4(aa.1-23).They were conjugated with keyhole limpet hemocyanin(KLH).BALB/c mice were subcutaneously immunized with six doses of KLH-peptides adjuvanted with MF59/CpGC274 at two-week interval.Serum sample was collected and examined with ELISA and Western blot.Results The four antisera were bound to both EV71 virus and denatured virus effectively.VP2 antiserum showed activity with denatured virus and the other three had similar binding activity to EV71 virus and denatured virus.The four antiserum samples were bound to both EV71 virus-like particle(VLP)and denatured VLP effectively.VP1 and VP2 antiserum could bind to VP1,VP0 of EV71 VLPseparately.VP2 antiserum showed the highese binding activity and VP1 antiserum showed a inperior binding activity as compared to the formers.VP1 and VP2 antiserum were bound specifically and efficiently to EV71 virus.VP0 of empty particles and VP2 of intact EV71 virus particles could be simultaneously identified by VP2 antiserum.VP3 and VP4 antiserum failed to binding to no obvious binding to VLP and virus.Conclusions Four epitope peptide polyclonal antisera against EV71 are successfully prepared,which can provide ootential detection tools used in ELISA and Western blot and may be used for QC evaluation of EV71 vaccine.

关 键 词：肠道病毒71型 疫苗 外壳蛋白 表位肽 抗血清 

分 类 号：R373.2[医药卫生—病原生物学]