抗猪流行性腹泻病毒N蛋白单克隆抗体制备及部分特性鉴定  

Production and identification of monoclonal antibodies against recombinant PEDV N protein

作　　者：裴敦国[1] 葛俊伟[1] 马广鹏[1] 姜艳萍[1] 李一经[1] 

机构地区：[1]东北农业大学动物医学院,哈尔滨150030

出　　处：《东北农业大学学报》2008年第7期84-89,共6页Journal of Northeast Agricultural University

摘　　要：以原核表达系统pGEX-6p-1-N/BL21经IPTG诱导表达的重组PEDV N-GST融合蛋白作为免疫抗原,免疫BALB/c小鼠,运用淋巴细胞杂交瘤技术,制备单克隆抗体,用原核表达系统T12-pPROHTa/BL21经IPTG诱导表达的PEDV N蛋白做筛选抗原,通过间接ELISA进行筛选,获得两株抗PEDV N蛋白杂交瘤细胞,命名为2E3、4C6。亚类鉴定为IgG1和IgG2a型,均为к链抗体,染色体数平均为(91±7)对。用制备的单抗与pGEX-6p-1-N/BL21、T12-pPROHTa/BL21诱导表达后的菌体裂解物做Western Blot试验,在不同载体表达的N蛋白处出现明显的特异性条带;通过间接ELISA做病原检测,这两株单抗不与TGEV、PRV和PrV发生交叉反应,而与PEDV显示良好的特异性反应。E.coli BL21 harboring recombinant pGEX-6P-1-N/BL21 was induced with IPTG to express the recombinant N-GST fusion protein. The N-GST protein was used as an immunogen. The BALB/c mice were immunized with purified recombinant N-GSTP（rN-GSTP）. T12-pPROHTa/BL21 was induced with IPTG to express recombinant N protein, which was used to detect the McAb. McAb was produced with the method of hybridoma technique, and indirect ELISA was used to screen two hybridoma cells against PEDV N protein which were named 2E3, 4C6. The isotypes of the McAbs were IgG1 and IgG2a, and the McAbs were correspond with K chain, the average number of chromosomes in tow hybridomas was （91±7） couples. To detect the specificity of McAbs, these McAbs were reacted with the lysate of pGEX-6P-1-N/BL21 and T12-pPROHTa/BL21 using Western Bolt after inducing expression. Specificity banding was appeared in the place of N protein expression. These McAbs could not react with TGEV, PRV and PrV, but they had specificity to PEDV when they were detected by indirect ELISA.

关 键 词：猪流行性腹泻病毒 单克隆抗体 WESTERNBLOT 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学] Q785[农业科学—兽医学]