利用单B细胞分选技术制备猪瘟病毒E2蛋白单克隆抗体及其在ELISA中的应用  

Development of Monoclonal Antibodies against Classical Swine Fever Virus E2 Protein by Single B Cell Sorting Technology and Its Application in ELISA

作　　者：马中元 郑君佐 梁志博 潘丽[2] 曾巧英[1] MA Zhongyuan;ZHENG Junzuo;LIANG Zhibo;PAN Li;ZENG Qiaoying(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《畜牧兽医学报》2024年第10期4579-4589,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家生猪技术创新中心战略重点研究项目(NCTIP-XD/C03)。

摘　　要：本研究旨在建立一种猪瘟病毒E2单抗竞争ELISA检测方法,用于评价猪瘟C株弱毒苗和E2亚单位疫苗的免疫效果。首先,构建猪瘟E2杆状病毒表达载体pFastBAC,通过悬浮培养SF9细胞高效表达E2蛋白;其次,用纯化的E2蛋白免疫BABL/c小鼠,通过流式分选IgM-PE-/E2-APC+型单个B细胞。然后,利用半巢式PCR分别扩增E2特异性IgG抗体的重链和轻链,测序后将抗体重链及轻链构建到pCDNA3.1载体,再将构建好的质粒共转染至HEK-293细胞,制备猪瘟E2单克隆抗体。结果表明,本研究通过单B细胞分选技术获得的两株单克隆抗体,即mAb3A9(IgG1,kappa)和mAb4F7(IgG2a,lambda),分别识别猪瘟E2蛋白B细胞线性表位25GLTTTWKEYSHDLQL^(39)和259GNTTVKVHASDERGP^(273)。此外,用上述两株单克隆抗体及E2蛋白建立的单抗竞争ELISA检测方法,在血清样本检测过程中,均展现出优异的诊断敏感性(97.49%,95.97%)及特异性(96.08%,94.38%),该研究为我国猪瘟的逐步净化提供了有利的技术支撑。The purpose of this study was to develop a classical swine fever virus(CSFV)E2 monoclonal antibody-based competition ELISA(Enzyme Linked Immunosorbent Assay)for evaluating the efficiency of the live attenuated C-strain and E2 subunit vaccines.Firstly,the E2 gene of CSFV was constructed into pFast BAC vector,and the E2 protein was efficiently expressed in SF9 cells;Secondly,6-8 weeks of BABL/c mice were immunized at intervals with purified E2 protein,and the single B cells were then sorted out at the gate of IgM-/E2+by flow cytometry.Then,the heavy and light chains of E2 antigen-specific IgG antibodies were amplified by seminested PCR,after sequencing,the heavy and light chains of the antibodies were constructed into pCDNA3.1vector,and then were co-transfected into HEK293 cells to prepare the monoclonal antibodies against CSFV E2.The results showed that the two monoclonal antibodies derived from single B cells,namely mAb3A9(IgG1,kappa)and mAb4F7(IgG2a,lambda),could efficiently reacted with the linear epitopes^(25) GLTTTWKEYSHDLQL^(39) and^(259) GNTTVKVHASDERGP^(273) of CSFV E2 protein,respectively.In addition,the competitive ELISAs developed using the mono-clonal antibodies and E2 protein mentioned above exhibited an excellent diagnostic sensitivity(97.49%,95.97%)and specificity(96.08%,94.38%)in the process of detecting serum sam-ples,which provides a favorable technical support for the gradual elimination of CSF in China.

关 键 词：猪瘟 E2 单B细胞 表位 ELISA 

分 类 号：S858.285.3[农业科学—临床兽医学]