抗传染性法氏囊病病毒VP5蛋白单克隆抗体的制备与初步应用

Preparation and Primary Analysis of Monoclonal Antibodies against VP5 protein of chicken Infectious Bursal Disease Virus

原核表达的IBDVGx-VP5蛋白经纯化后免疫8周龄的BALB/c雌性小鼠,三次基础免疫后,融合前加强免疫,取脾细胞在PEG(MW1500)的作用下与SP2/0小鼠骨髓瘤细胞融合,经过三次亚克隆筛选,获得稳定分泌抗VP5蛋白的杂交瘤细胞,分别命名为4B4、6D12、3E8,以三株杂交瘤细胞制备的腹水ELISA效价分别为5×104、3.5×104、3×104,特异性实验表明三株单抗能与IBDVGt株反应。以单抗介导的间接免疫荧光检测表达Gt-VP5的VeroE6细胞,可以见到特异的荧光,能做为特异性的检测VP5蛋白的工具,为今后IBDVVP5蛋白的研究打下基础。

Infectious bursal disease virus （IBDV）, the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein （NS）. In this study, vvlBDV Gx-VP5 genes were cloned into plasmid pET30a（ ＋ ） and expressed in E. coli with IPTG inducing. BALB/c mice were immunized with the purified recombinant fusion protein. SP2/0 myeloma cells and spleen cells of BALB/c mice were fused by PEG（MW1500）, three hybridoma cell lines were examined by indirect ELISA and clone for three times by limited dilution, and were named as 4B4, 6D12, 3E8. The subtype of the monoclonal antibodies were IgG1 with a subtype identified ELISA kit, and light chains were kappa. The ascites titers of monoclonal antibodies were 5 × 10^4, 3.5 × 10^4, 3 × 10^4 by indirect ELISA, respectively. Indirect ELISA and Western blot results showed that the monoclonal antibodies only acted with VP5 protein, IF analysis indicated that three monoclonal antibodies acted with IBDV Gt.There were specific fluorescence in detected Vero E6 cells which transient expressed VP5 protein by IFA. Therefore, monoelonal antibodies specific to IBDV VP5 proteins are specific method for detected VP5 proteins, and base on establish stabilize expressed VP5 protein Vero cell lines to research IBDV VP5 protein function.