基于rGroEL和rOmpC的牙鲆迟缓爱德华氏菌血清学诊断试纸条的制备

Development of Colloidal Gold Immunochromatography Assay Strip for rGroEL and rOmpC Based Serological Diagnosis of Edwardsiellosis in Flounder(Paralichthys olivaceus)

本实验旨在制备一种基于重组抗原rGroEL和rOmpC的迟缓爱德华氏菌(Edwardsiella tarda)血清学诊断试纸条,用于定性检测牙鲆(Paralichthys olivaceus)血清中抗迟缓爱德华氏菌的抗体水平。前期研究发现分子伴侣蛋白GroEL和外膜蛋白OmpC是迟缓爱德华氏菌的重要免疫保护性抗原。本研究重组表达获得高纯度的rGRoEL和rOmpC,以其免疫健康牙鲆制备抗血清,同时制备获得牙鲆抗迟缓爱德华氏菌全菌血清。ELISA结果显示rGroEL和rOmpC不存在抗原交叉,抗rGroEL和rOmpC血清均可与全菌发生阳性结合,且全菌抗血清也能识别rGroEL和rOmpC。将rOmpC、rGroEL及羊抗鼠IgG划线于NC膜分别作为检测线T1、检测线T2和质控线C,同时以制备的胶体金标记的鼠抗牙鲆IgM单克隆抗体2D8固定于结合垫,构建获得胶体金免疫层析试纸条。将试纸条分别用以检测不同稀释度的各抗血清,检测过程可在15min内完成,结果显示制备的灭活全菌、rGroEL和rOmpC牙鲆抗血清分别最高稀释200、600与400倍时,均可使检测线T1与T2显红色。同时,以试纸条检测牙鲆免疫全菌灭活疫苗后不同时间点的抗血清,结果显示3、4、5周检测结果为阳性,且检测线颜色随着免疫后时间延长而加深。实验结果表明,制备的基于重组抗原rGroEL和rOmpC迟缓爱德华氏菌血清学诊断试纸条,操作简单,结果快速可见,可以定性检测牙鲆血清中抗迟缓爱德华氏菌的抗体水平,可应用于全菌灭活疫苗、rGroEL和rOmpC亚单位疫苗免疫效果的评价,同时也具有牙鲆爱德华氏菌病的潜在诊断价值。

The aim of this workwas to develop a colloidal gold immunochromatography assay strip for the qualitative detection of the antibody against Edwardsiella tarda in flounder serum by using recombinant GroEL(rGroEL)and recombinant outer membrane protein C(rOmpC).In our previous study,molecular chaperone GroEL and OmpCof E.tarda were identified to be the important immune protective antigens.In the present work,high purerGroEL and rOmpC were prepared by inducing constructed recombinant E.coli and immunized flounder to get the antisera.Flounder antisera against E.tarda were also prepared.The results of ELISA assay showed that no antigenic cross-reactivity was observed between rGroEL and rOmpC,whereas antibodies induced by rGroEL and rOmpC could react with E.tarda,and the rGroEL and rOmpC could be also recognized by flounder anti-E.tardaantibodies.To develop the immunochromatography test strip,rOmpC,rGroEL and goat anti-mouse IgG were immobilized on the nitrocellulose membrane as test line 1(T1),test line 2(T2)and control line(C),meanwhile,colloidal gold labelled with monoclonal antibody against flounder IgM 2D8 was fixed on the bonding pad.The strips were then used to detect antisera with different dilutions,which appeared pink color lines at T1 and T2 positions within 15 min when the antisera against E.tarda,rGroEL and rOmpC with the highest dilutions of 200,600 and 400 folds,respectively.Meanwhile,the strips were applied to test the antisera that were collected at different weeks after immunization with inactivated E.tarda,which showed positive results at week 3,4 and 5,and the intensity of pink lines on test linesbecame deeper with the time after immunization.These results demonstrated that the test strips developed in this work have the advantages of rapid,easy operation and visible results for qualitative detection of the antibody against E.tarda,which would be applied in evaluating the immune effects of inactivated E.tardavaccine and subunit vaccines of rGroEL and rOmpC.In addition,it also has the potential value in diagnosis of edwardsiellosis in flounder.