猪流行性腹泻病毒N蛋白特异性单克隆抗体的表达与鉴定

Expression and identification of porcine monoclonal antibodies against porcine epidemic diarrhea virus

摘要

旨在利用单个B细胞抗体技术研发猪流行性腹泻病毒（PEDV）N蛋白特异性单克隆抗体（mAb）。通过荧光激活细胞分选（FACS）从猪外周血单个核细胞（PBMCs）中分离出单个B细胞，使用套式PCR扩增猪抗体IgG重链/轻链可变区基因，并将其克隆到携带猪抗体恒定区基因的真核表达载体CH/CL-pcDNA3.4中。将获得的抗体表达质粒转染到HEK293F细胞中，表达猪源性mAb，并用间接ELISA、间接免疫荧光（IFA）试验和病毒中和试验进行验证。结果：成功分选出24个双阳性（N蛋白和IgG均阳性）单个B细胞，并成功配对8对重链/轻链序列，获得了8株N蛋白特异性mAb；经间接ELISA检测，表达的8株mAb中有7株对PEDV N蛋白均具有较好的亲和力，有3株（N-5、N-10和N-20）可通过IFA识别PEDV；Western blot分析表明，有2株mAb与变性的PEDV N蛋白反应；中和试验结果表明，8株mAb均无中和活性。综上，本研究成功建立了基于单个B细胞直接制备抗PEDV单抗的方法，为PEDV诊断试剂的研发提供了可靠的技术手段。

Abstract

To develop porcine epidemic diarrhea virus (PEDV) nucleocapsid (N) protein-specific monoclonal antibodies (mAbs) using the single B cell antibody technology, this study isolated single B cells from porcine peripheral blood mononuclear cells (PBMCs) by fluorescence-activated cell sorting (FACS). The variable region genes of porcine IgG heavy and light chains were amplified by nested PCR and were subsequently cloned into the eukaryotic expression vector CH/CL-pcDNA3.4 which carried porcine antibody constant region genes. The resulting antibody expression plasmids were transfected into HEK293F cells for expression of porcine-derived mAbs. The expressed mAbs were validated using indirect Enzyme-Linked Immunosorbent Assay (indirect ELISA), indirect immunofluorescence assay (IFA), and virus neutralization test. The results showed that 24 double-positive (N protein and IgG positive) single B cells were sorted, yielding 8 successfully paired heavy/light chain sequences and 8 N protein-specific mAbs. The indirect ELISA demonstrated that 7 out of the 8 mAbs exhibited high affinity with the PEDV N protein. Three mAbs (N-5, N-10, and N-20) recognized PEDV in IFA. The Western blot analysis indicated that 2 mAbs reacted with denatured PEDV N protein. However, none of the mAbs showed neutralizing activity in the neutralization assay. This study successfully established a method for preparing anti-PEDV mAb directly from single B cells, providing a reliable technical approach for development of PEDV diagnostic reagents.

关键词

猪流行性腹泻病毒 / 单个B细胞抗体技术 / 单克隆抗体

Key words

porcine epidemic diarrhea virus / single B cell antibody technique / monoclonal antibody

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基金资助

河南省自然科学基金项目(252300421418)

河南农业大学拔尖人才项目(30501277)