空肠弯曲菌PEB1重组蛋白的表达及单克隆抗体的制备

目的表达CJPEB1重组蛋白，制备并鉴定CJ PEB1蛋白的单克隆抗体。方法诱导培养工程菌E．co-liBL21（DE3）表达CJPEB1重组蛋白，以PEB1蛋白溶液与等体积的佐剂完全乳化作为免疫原常规免疫BALB／C小鼠，制备单克隆抗体。无菌取其脾细胞与小鼠骨髓瘤细胞SP2／0进行融合，建立杂交瘤细胞株。将建株的杂交瘤细胞注入小鼠腹腔诱生腹水制备抗体，应用间接ELISA法和双向琼脂扩散试验检测腹水中抗体的效价，应用Western-blot和玻片凝集试验检测抗体的特异性。结果得到高纯度的CJPEB1重组蛋白，蛋白质分子量大小与预计的理论值相符。获得两株稳定分泌单克隆抗体的杂交瘤细胞株。两株杂交瘤细胞诱生的腹水，经间接ELISA法检测，腹水中的抗体效价分别为8×10^4和5×10^4，经双向琼脂扩散试验检测效价均为1：16。通过Westem-blot鉴定，两株杂交瘤细胞分泌的抗体均与PEB1蛋白特异性地结合；其诱生的腹水与CJ菌液混合出现凝集现象，而与大肠杆菌、痢疾杆菌、伤寒杆菌等菌液混合均未出现凝集现象。结论成功建立了两株稳定分泌抗CJPEB1重组蛋白抗体的杂交瘤细胞株并制备了抗CJPEB1蛋白的单克隆抗体，为今后研究多种检测CJ的方法创造了条件。

Expression of recombinant PEB1 protein of campylobacter jejuni and preparation of monoclonal antibodies against it

Objective To express recombinant PEB1 protein of campylobacter jejuni （CJ）and prepare, identify monoclonal antibodies against it. Methods E. coli BL21 （ DE3 ） was raised and induced to express the recombinant CJ PEB1 protein. The BALB/C mice were routinely immunized by the immunogen PEB1 protein emulsified with identical volume adjuvant, then the monoclonal antibodies against PEB1 protein were prepared. The splenoeytes tissue were gathered and fused with SP2/0 cells, then the hybridoma cell lines were established. The hybridoma cells were used to induce ascites by intraperitoneal injection in BALB/C mice, then the antibody titer was detected with indirect ELISA and Double agar diffusion test ,and the antibody specificity was examined by Western-blot and Slide agglutination test. Results The recombinant PEB1 protein with high purity was obtained , and the relative molecular weight of which was identical with theory value. Two hybridoma cell lines which secreted monoclonal antibodies steadily were obtained, lsotypes of these two eeU strains showed that they belong to IgG1 and IgG3 respectively. The indirect ELISA showed that the antibodies titer of ascites induced by the two hybridoma cell lines were respectively 8 × 104 and 5 ×104, and the Double agar diffusion showed it was 1： 16. The monoclonal antibodies could specifically recogrtized PEB1 protein demonstrated by Western-blot. Slide agglutination test showed the ascites induced by two hybridoma cell lines appeared agglutinative mixing with CJ, but not with E toll, Bacillus dysenteriae, Bacillus typhosus and et al. Conclusion Two hybridoma cell lines stably secreting monoclonal antibodies against CJ PEB1 protein can be established , and the monoclonal antibodies against CJ PEB1 protein can be prepared successfully, which provids a basis for the detection of CJ.