以乙肝病毒核心蛋白为载体制备肠道病毒71型非结构蛋白3D单克隆抗体

Preparation of monoclonal antibodies against 3D protein of EV71 based on HBc particles as expression vector

目的:预测肠道病毒71型(EV71)非结构蛋白3D的表位,以HBc蛋白为载体展示多肽,制备并鉴定抗EV71-3D的特异性单克隆抗体(m Ab)。方法:应用生物信息学方法分析预测出EV71 3D蛋白亲水性和免疫原性指数较高的多肽片段,并运用HBc颗粒型蛋白载体展示肽段,构建多肽融合蛋白,免疫BALB/c雌鼠,通过杂交瘤技术和亲和层析技术制备和纯化抗EV71-3D蛋白的特异性m Ab,用间接ELISA、ELISPOT、IFA和IHC对m Ab的性质进行初步鉴定。结果:构建表达分别嵌合3D蛋白34~43位氨基酸残基、61~76位氨基酸残基、151~164位氨基酸残基的HBc重组蛋白,免疫并经过多轮克隆化筛选,获得抗EV71-3D单克隆抗体3E1,其亚类为Ig G2a;免疫荧光试验、ELISPOT法和免疫组织化学染色结果显示其可与EV71特异性结合。结论:成功制备可特异性识别EV71的单克隆抗体3E1,为病毒的检测及进一步研究3D蛋白的功能奠定了基础,同时还验证了生物信息学技术与HBc颗粒型载体展示多肽技术相结合可快速高效地制备单克隆抗体。

Objective To prepare and preliminarily identify the monoclonal antibodies（mAbs） specifically against 3D protein of Enterovirus 71（ EV71 ）,using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier. Methods： Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region（MIR） area of Hepatitis B virus core protein（HBc）,to construct the recombinant protein. BALB/c mice were immunized with the recombinant virus like particles（ VLPs）,to prepare the mAbs against 3D protein of EV71. Affinity chromatography technology was used to purify the mAb. The indirect ELISA, ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the mAb. Results ： We displayed 3D（ aa34-43 ） , 3D（ aa61-76 ） and 3D（ aa151 -164） epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line（3E1） was selected by ELISA. The isotype of 3 E1 was IgG2a. The results of immunofluorescence and immunohistochemistry staining assay showed that the mAb 3 E1 could specifically recognize EV71. Conclusion： The prepared mAb 3E1 can specifically recognizes the EV71, which laid the foundation for the detection of vims and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare mAb quickly and efficiently.