肺炎支原体CARDS毒素蛋白多表位拼接抗原的表达及鉴定

摘要

We expressed multi-epitope chimeric protein of CARDS toxin protein of Mycoplasma pneumonia (Mp) in prokaryotic cells,and purified and investigated its immunoreactivity.A recombinant multi-epitope chimeric gene including ten critical epitopes was connected by linker and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The antigenicity of expressed recombinant protein was identified with 6 × His monoclonal antibody and human positive serum by Western blot.The recombinant expression vector pET-CARDS was constructed and the about 30 kDa recombinant chimeric protein expressed in BL21(DE3) successfully.Western blot analysis showed that it can react respectively with 6 × His monoclonal antibodies and human positive serum.This study showed that the chimeric CARDS protein has an obvious immunoreactivity and a potential to be a new antigen for the diagnosis of Mp infection.%目的 探讨肺炎支原体CARDS毒素蛋白多表位拼接抗原的免疫反应活性.方法 对CARDS毒素蛋白的抗原表位进行分析,选取10个重要的表位进行拼接,反向翻译后合成并克隆,插入pET28a表达载体构建pET-CARDS表达质粒并转入受体菌.经IPTG诱导表达的多表位拼接CARDS蛋白使用6×His单克隆抗体和人阳性血清进行了免疫印迹的检测.结果 CARDS毒素蛋白的多表位拼接抗原表达载体构建成功,诱导后表达大小为30KDa的重组蛋白,Western blot测定其能与6×His单克隆抗体和人阳性血清发生反应.结论 本研究选取的CARDS毒素蛋白抗原表位具有较强的免疫活性,可为Mp感染的诊断提供新的候选抗原.