猪传染性胃肠炎病毒S基因主要抗原位点A和D的原核表达及间接ELISA检测方法的建立

Expression of major antigenic sites A and D in S gene of transmissible gastroenteritis virus of swine(TGEV) in Escherichia coli and development of indirect ELISA for detection of the antibody against TGEV

为建立一种检测猪传染性胃肠炎病毒血清抗体的间接ELISA,将猪传染性胃肠炎病毒S基因主要抗原位点A和D克隆到pET-28a(+)载体中,在大肠杆菌Rosetta(DE3)pLysS中进行表达。表达产物的分子质量为38ku,表达形式为包涵体。通过Western-blot证实,表达产物与猪传染性胃肠炎病毒多克隆抗体能够特异性结合。将重组蛋白经过8mol/L的尿素溶解后,作为间接ELISA的包被抗原,通过优化条件初步建立了检测猪传染性胃肠炎病毒血清抗体的间接ELISA。该方法与纯化的猪传染性胃肠炎病毒包被的间接ELISA相比,符合率达到91.53%。本研究结果对大规模地推广应用猪传染性胃肠炎间接ELISA诊断方法具有重要意义。

To establish an indirect ELISA technique for the detection of the serum antibody to porcine transmissible gastroenteritis virus of swine(TGEV),the major antigenic sites A and D in spike gene were amplified from TGEV and cloned into the pET-28a(+)vector.The recombinant AD protein(TGEV AD protein)could be expressed in Escherichia coli Rosetta(DE3)pLysS by IPTG induction,mainly presenting with inclusion bodies.The recombinant TGEV AD protein was 38 ku in molecular weight and could well detect with the polyclonal antibody against TGEV by Western-blot test.The TGEV AD protein diluted in8mol/L urea was used as an indirect ELISA coating antigen.We established an indirect ELISA to detect TGEV-specific antibodies in serum.Compared with coating with purified TGEV,the coincidence rate was91.53%.In conclusion,this study result had an important significant to detect porcine transmissible gastroenteritis in the large-scale application.