【摘 要】
：
目的：肺炎支原体(Mycoplasma pneumonia,Mp)作为人类呼吸道感染疾病的常见病原体,可引发小儿及青壮年的非典型性肺炎及其他急性呼吸道疾病,P1、P116和P30蛋白是密集分布于Mp菌体表明的膜蛋白,既可作为黏附细胞器使菌体黏附于宿主表面又能使菌体在宿主细胞表面滑动,将其抗原性强的蛋白片段P116N,P1C,P30分别进行表达,分析其免疫原性和抗原性.方法：采用生物信息学软件对肺炎
【机 构】
：
中国药科大学生命科学与技术学院,南京 210009;南京军区军事医学研究所,南京210002 南京
【出 处】
：
中国生物工程学会第六次全国会员代表大会暨第九届学术年会
论文部分内容阅读
目的：肺炎支原体(Mycoplasma pneumonia,Mp)作为人类呼吸道感染疾病的常见病原体,可引发小儿及青壮年的非典型性肺炎及其他急性呼吸道疾病,P1、P116和P30蛋白是密集分布于Mp菌体表明的膜蛋白,既可作为黏附细胞器使菌体黏附于宿主表面又能使菌体在宿主细胞表面滑动,将其抗原性强的蛋白片段P116N,P1C,P30分别进行表达,分析其免疫原性和抗原性.方法：采用生物信息学软件对肺炎支原体表面膜蛋白P1,P116及P30进行抗原表位分析,筛选其中抗原表位较富集的优势抗原片段作为目的蛋白；优化筛选的基因序列,使其能在合适的原核表达载体中进行高效表达.构建重组质粒pET28a(+)–P116N,pET28a(+)–P1C,pET28a(+)–P30,并分别转化到E.coli BL21(DE3)中进行IPTG诱导表达,采用亲和层析技术对含有GST标签的目的蛋白进行分离纯化,经过SDS-PAGE和Western blot的验证,获得了较高纯度的蛋白.分别使用三种膜蛋白抗原对采用三次免疫加强法对新西兰大耳白兔进行背部八点免疫,兔颈动脉取血制备兔抗血清.将制备的兔血清作为一抗,稀释比为1：200,而将酶标的羊抗兔IgG作为二抗,暗室胶片曝光,采用ELISA对其抗原性和免疫原性进行分析,并测定其效价.结果：分析E.coli 膜蛋白的全长序列,并将蛋白的抗原性、亲水性和表面可及性等参数作为分析指标,最终选取三个氨基酸片段作为研究对象,成功构建重组质粒pET28a(+)–P116N,pET28a(+)–P1C,pET28a(+)–P30,质粒转化入E.coli BL21(DE3),挑选转化子,经IPTG诱导后,SDS-PAGE电泳结果显示,重组蛋白为可溶性表达,采用谷胱甘肽溶液对目的蛋白进行梯度洗脱,条带明确较单一,与理论分子量相符.Western blot结果显示制备的多抗血清能特异性识别抗原,ELISA结果显示三种膜蛋白均能较好地与多抗血清进行特异性反应,效价可达1：107.结论：纯化获得的重组蛋白具有较好的免疫原性和抗原性,免疫效果良好,可作为肺炎支原体的候选疫苗,为肺炎支原体疫苗的进一步研究奠定了基础.
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