绵羊肺炎支原体多表位融合基因MO-meAg1的构建、表达及重组蛋白免疫原性分析

摘要

为获得具有良好免疫原性的绵羊肺炎支原体(MO)重组蛋白,利用ABCpred等软件预测分析MOEF-P、PDHA、P97相似蛋白和EF-Ts蛋白的优势线性抗原表位,构建多表位融合基因MO-meAg1,合成后将其亚克隆至表达载体pET-32a(+),构建原核表达载体pET-32a(+)-MO-meAg1.将pET-32a(+)-MO-meAg1转化至感受态细胞E.coli BL21(DE3),IPTG诱导表达后检测重组蛋白反应原性和免疫原性.SDS-PAGE结果显示,MO-meAg1重组蛋白分子质量约34.5 ku;Western blot分析结果显示,该重组蛋白可被MO阳性血清特异性识别,具有良好的反应原性.小鼠免疫试验结果表明,重组蛋白MO-meAg1具有较强的免疫原性,诱导机体产生的抗血清其间接血凝效价可达1∶128以上.获得的多表位融合蛋白MO-meAg1为绵羊支原体肺炎的免疫学诊断及新型疫苗研究奠定基础.%To develop highly specific and sensitive antigen of Mycoplasma ovipneumoniae (MO),the dominant linear antigen epitopes of EF-P,PDHA,P97-1ike protein and EF-Ts using ABCpred softwares were analyzed,and then multi-epitope fusion gene MO-meAg1 was constructed.It was synthesized and subcloned to pET-32a (+) to generate pET-32a(+)-MO-meAg1.Then the pET-32a(+)-MO-meAg1 was transformed into competent cells of E.coli BL21 (DE3) for expression after inducing by IPTG.The reactogenicity and immunogenicity of recombinant were analyzed,respectively.The re sults of SDS-PAGE showed that expressed MO-meAg1 was about 35.4 ku.The analysis of Western blot showed that recombinant MO-meAg1 can be specifically recognized by anti-MO positive serum,displaying strong reactionogenicity.The results of immunization test in mice showed that recombinant MO-meAg1 had high immunogenicity,which can induce the specific antibody with indirect hemagglutination titer of 1 ∶ 128.The results showed that MO-meAg1 is a promising antigen,which provided a potentially valuable antigen for the development of the immunological diagnostic method and vaccine against MO infection.