EV71临床毒株分离及其VP1单克隆抗体制备

目的对手足口病肠道病毒71型(EV71)流行临床株进行分型和鉴定,制备抗EV71外壳蛋白VP1的特异性单克隆抗体,为EV71生物学特征研究和疫苗株的筛选奠定基础。方法从患者咽拭子中分离出流行的EV71毒株,原核表达纯化该毒株EV71 VP1衣壳蛋白,以此蛋白为免疫原制备VP1的特异性小鼠单克隆抗体,采用免疫荧光法(ELISA)观察抗体能否特异性识别EV71感染的细胞。结果测序结果显示分离出的EV71病毒株为肠道病毒C4亚型。筛查出12株分泌针对VP1蛋白的抗体杂交瘤细胞株,将其中结合特异性和结合力最强的一株命名为11T12。复苏的11T12活化后接种于液体石蜡免疫的小鼠腹腔,收集腹水经过protein-G柱纯化后对抗体的效价进行ELISA评价,结果显示纯化抗体稀释10万倍后仍可以结合蛋白,证实成功制备了抗EV71 C4亚型外壳蛋白VP1单克隆抗体。抗体结合特征分析结果显示该抗体具有特异性识别能力。结论从感染的临床样本中分离到一株C4型EV71毒株,以该病毒VP1蛋白为免疫原获得一株相应的单克隆抗体11T12,该单克隆抗体的获得为病毒的检测、试剂盒的研发提供了核心材料。

Isolation of a clinical EV71 strain and a monoclonal antibody preparation towards its VP1

Objective The clinical strains of human enterovirus 71 (EV71) of hand-foot-mouth disease (HFMD) were classified and identified, to prepare specific monoclonal antibodies against EV71 shell protein VP1. It lays a foundation for the study of biological characteristics of EV71 and the screening of vaccine strains. Methods The prevalent strain of EV71 was isolated from the pharyngeal swab of the patient, and the capsid protein of the strain EV71 VP1 was purified by prokaryotic expression. The protein was used as the immunogen to prepare specific mouse monoclonal antibody of VP1, and immunofluorescence method (ELISA) was used to observe whether the antibody could specifically identify infected cells of EV71. Results Sequencing results showed that isolated EV71 virus strain was of enterovirus C4 subtype. Twelve antibody hybridoma cell lines secreting VP1 protein were screened out, and the one with the strongest binding specificity and binding force was named 11T12. Resuscitated 11T12 was activated and inoculated into the abdominal cavity of mice who were immunized with liquid paraffin. Collected ascites were purified by protein-G column, and the titer of the antibody was tested by ELISA evaluation. The results showed that the purified antibody could still bind to the protein after being diluted by 100 000 times, which confirmed the successful preparation of monoclonal antibody of anti-EV71 C4 subtype shell protein VP1. The analysis result of antibody binding characteristic proved that the antibody had specific recognition ability. Conclusion A C4 type EV71 strain was isolated from the infected clinical samples, and a corresponding monoclonal antibody 11T12 was obtained with the virus VP1 protein as the immunogen. The acquisition of monoclonal antibody provided the core material for virus detection and the development of the kit.