【摘要】 将表达志贺样毒素II型变异体A亚单位重组蛋白的菌株ppSLT -IIeA在含氨苄青霉素的 2×YTA培养基中培养 ,至对数生长期时加入IPTG诱导 ,诱导的重组菌超声波裂解后 ,经聚丙烯酰胺凝胶电泳证明其已表达重组蛋白。用谷胱苷肽活化的Sepharose 4B亲和层析柱纯化重组蛋白 ,测定纯化蛋白的浓度 ,免疫BALB/c小鼠 ,细胞融合后经ELISA检测 ,得到一株阳性杂交瘤细胞 ,命名为A2 _D3 ,制备的单抗腹水ELISA效价为 2 4log2 ,免疫印迹结果表明此单抗仅与重组蛋白反应 ,而与载体蛋白不反应。利用此单抗检测 13株水肿病菌株 ,发现均产志贺样毒素II型变异体更多还原

【Abstract】 Recombinant E.coli ppSLT_IIeA which expressed fusion protion GST_SLT_IIeA was cultured in 2×YTA,,put IPTG in cuture to induce the fusion protein which would be purified by Glutation Sephrose 4B and detected by SDS_PAGE as antigen immunized BALB/c mice.GST exspressed in recombinant E.coli pGEX₆P₁ as negative antigen and SLT_IIeA as positive antigen absorbed in ELISA plate. SP2/0 myelomacells and spleen cells of the immunized BALB/C mice were fused by PEG₂000. The hybridoma culture supernatants were screened for anti_SLT_IIeA antibodies by indirect_ELISA.One monoclonal antibody A2_D3 was detected and only reacted with a 62kDa fusion protein band in Western_blotting. The Mab as probe has detected 13 strains of ED and recombinant E.coli TB1 by Western_blotting which shows 14 strains were all positive.更多还原