目的初步探讨肺炎支原体CARDS毒素蛋白在检测肺炎支原体感染中的应用价值。方法 PCR扩增CARDS毒素基因并连接到PMD-19-T载体,经酶切、PCR及核苷酸测序分析后,将CARDS毒素基因片段克隆到pGEX-6p-1表达载体内并转入受体菌。IPTG诱导大肠埃希菌BL21重组株(pGEX-6p-1-CARDS)的表达。GST柱层析纯化重组CARDS毒素蛋白,以该重组蛋白和重组P1蛋白为抗原建立间接ELISA方法,检测85份感染肺炎支原体的血清标本,并与重组P1蛋白的ELISA结果进行比较。结果 CARDS毒素蛋白表达载体构建成功并表达大小为43kDa的重组蛋白,Western blot测定其能与兔抗Mp多价抗血清发生反应。ELISA结果显示,CARDS毒素蛋白的阳性率为70.59%(60/85),重组P1蛋白的阳性率为63.53%(54/85)。结论本研究成功构建了CARDS毒素蛋白表达载体并获得了稳定表达的重组菌株;在诊断肺炎支原体感染的血清学ELISA试验中,重组CARDS毒素蛋白的敏感性高于重组P1蛋白,为Mp感染的诊断提供了新的可用抗原。 Objective To investigate the value of CARDS toxin Mycoplasma pneumoniae in the detection of Mycoplasma pneumoniae infection. Methods CARDS toxin gene was amplified by PCR and ligated into PMD-19-T vector. After digestion, PCR and nucleotide sequencing, the CARDS toxin gene fragment was cloned into pGEX-6p-1 expression vector and transferred into recipient bacteria. IPTG induces the expression of recombinant Escherichia coli BL21 (pGEX-6p-1-CARDS). Recombinant CARDS toxin protein was purified by GST column chromatography. Serum samples of Mycoplasma pneumoniae were detected by indirect ELISA with the recombinant protein and recombinant P1 protein as antigen, and compared with ELISA results of recombinant P1 protein. Results The CARDS toxin protein expression vector was successfully constructed and expressed as a 43 kDa recombinant protein. Western blot analysis showed that it reacted with rabbit anti-Mp polyvalent antiserum. The results of ELISA showed that the positive rate of CARDS toxin protein was 70.59% (60/85), and the positive rate of recombinant P1 protein was 63.53% (54/85). CONCLUSIONS: The CARDS toxin protein expression vector was successfully constructed and a stable recombinant strain was obtained. In the serological ELISA for the diagnosis of Mycoplasma pneumoniae infection, the recombinant CARDS toxin protein is more sensitive than the recombinant P1 protein in diagnosis of Mp infection New available antigens are provided.