猪流行性腹泻病毒N蛋白单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against the nucleocapsid protein of porcine epidemic diarrhea virus

采用RT-PCR扩增猪流行性腹泻病毒（PEDV）N蛋白基因并克隆至原核表达载体pET-28a,转化到宿主表达菌BL21后经IPTG诱导表达和Ni柱纯化,SDS-PAGE和Western Blot鉴定重组N蛋白,其大小约为58 ku。将纯化重组N蛋白免疫BALB/c小鼠,通过杂交瘤细胞融合,PEDV间接ELISA方法筛选,制备获得4株能稳定分泌PEDV抗体的杂交瘤细胞株1C9、4C8、4F8和6A11。Western blot和间接免疫荧光试验证明其与PEDV均有特异性反应,单抗亚类鉴定均属于IgG1,轻链为κ型。杂交瘤细胞培养上清ELISA抗体效价为1∶1 600~6 400,腹水ELISA抗体效价达1∶1.024×10^5以上;4株细胞连续培养20代,其ELISA抗体效价基本一致。本研究为PEDV感染的诊断和致病机制研究提供了有用工具。

In this study, the nucleocapsid （N） protein gene of porcine epidemic diarrhea virus （PEDV） was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a. The recombinant N protein was induced to express in E. coli BL21 cells with IPTG, and confirmed by SDS-PAGE and western blot assays. Then the recombinant protein with the size of about 58ku was purified by Ni column. The monoclonal antibodies （MAbs） were prepared by fusing mouse myloma cells （SP2/0） with the spleen cells from BALB/c mice immu- nized with the purified recombinant N protein. Four hybridoma cell lines secreting MAbs were screened by indirect enzyme-linked immunosorbent assay （ELISA） and named as 1C9, 4C8, 4F8 and 6All, respectively. Western blot and indirect immunofluorescence assay （IFA） analyses showed that the four MAbs reacted specifically with the recombinant N protein and PEDV. The isotypes of four MAbs both belong to IgG1 subtype with K chain. Their ELISA titers in supernatant were 1 ： 1 600 - 1 ： 6 400, and titers in asicite were more than 1 ： 1. 024×10^5. It indicated that these MAbs may be useful for developing the diagnosis methods and investigating the pathogenicity of PEDV in the further study.