猪传染性胃肠炎病毒NSP5蛋白单克隆抗体的制备及抗原表位鉴定

Antigenic epitope identification of NSP5 protein in TGEV

为制备猪传染性胃肠炎病毒(TGEV)NSP5蛋白的单抗隆抗体(MAb)及鉴定其抗原表位,本研究以原核表达并纯化的重组NSP5蛋白(r NSP5-GST)免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0细胞进行融合,通过间接ELISA方法筛出一株稳定分泌抗TGEV NSP5蛋白的MAb杂交瘤细胞株(5C3)。MAb亚类鉴定结果显示其抗体亚类为Ig G1/κ链。杂交瘤细胞培养上清液和腹水的效价分别为1∶6 400和1∶105。Western blot鉴定表明该MAb能够识别原核及真核表达的NSP5重组蛋白。利用肽扫描法鉴定显示,该MAb识别的抗原表位为286EFTPTEVIR QMYGVN300。本研究制备的MAb及其抗原表位的鉴定,为TGEV NSP5蛋白结构和功能的研究奠定了基础。

To prepare the monoclonal antibodie （MAb） against non-structure protein 5 （NSP5） of porcine transmissible gastroenteritis virus （TGEV） and identify the epitope of NSP5, SP2/0 myeloma cells were fused with spleen cells from 6 week-old BALB/c mice immunized with purified recombinant NSP5 of TGEV expressed in E.coli. One hybidoma cell lines stably secreting MAb against NSP5 of TGEV was identified by indirect ELISA. The MAb belonged to IgG1 subtype with K chain. The titers in cell culture medium of the hybidomas and ascites were 1：6 400 and 1：105, respectively. Western blot assay showed that the MAb was able to recognize the recombinant NSP5. Furthermore, epitope on NSP5 was identified by pepscan technique and the linear epitope of 286EFTPTEVIRQMYGVN300 was identified by western blot and ELISA with the MAb. These results demonstrated that preparation of the MAb and identification of the epitope would facilitate the further study on the structure and function of TGEV NSP5.