副猪嗜血杆菌聚胺转运蛋白PotD重组亚单位疫苗的制备及其免疫保护效力的评价

发布时间：2018-05-12 16:46

本文选题：副猪嗜血杆菌 + PotD蛋白　；参考：《中国兽医科学》2017年08期

【摘要】：为研究副猪嗜血杆菌Pot D蛋白的免疫保护效力,本研究以副猪嗜血杆菌SC1401菌株的基因组DNA为模板,PCR扩增去除信号肽编码序列后的potD基因部分片段,将其克隆于pET-28a(+)载体中,构建重组表达质粒pET-potD,并转化至大肠杆菌BL21(DE3)中进行诱导表达,超声破碎后SDS-PAGE分析目的蛋白表达形式。纯化后的蛋白以每只50 g的剂量免疫2周龄的BALB/c小鼠,同时设置SC1401全菌灭活疫苗组、生理盐水对照组及佐剂对照组,一免后第14天进行一次加强免疫,二免后第14天断尾采血进行抗体效价测定,并以5×LD50的SC1401野毒株攻毒,评价其免疫保护效力。结果显示,目的蛋白的分子质量为44.3 ku,主要以包含体形式存在;ELISA测定的Pot D蛋白的血清抗体效价(最高稀释度为1∶20 000,P/N2.1),显著高于SC1401全菌甲醛灭活苗(最高稀释度1∶10 000)、生理盐水对照组及佐剂对照组的血清抗体效价(P0.05)。攻毒后显示Pot D蛋白组的保护力达80%,与SC1401全菌灭活疫苗组持平,远高于生理盐水对照组及佐剂对照组。结果表明,Pot D重组蛋白能够很好的激发小鼠体内的免疫应答,提供较好的免疫保护力,可以作为副猪嗜血杆菌病重组亚单位疫苗的候选抗原蛋白。
[Abstract]:In order to study the immuno-protective effect of Haemophilus parasuis Pot D protein, the genomic DNA of Haemophilus parasuis SC1401 strain was used as template to amplify part of the potD gene which was removed from the signal peptide coding sequence and cloned into pET-28a () vector. The recombinant expression plasmid pET-potDwas constructed and transformed into E. coli BL21DE3. The expression of the target protein was analyzed by SDS-PAGE after ultrasonic fragmentation. The purified protein was immunized with 50 g of each BALB/c mouse at the age of 2 weeks. At the same time, the whole SC1401 vaccine group, saline control group and adjuvant control group were immunized once on the 14th day after immunization. On the 14th day after the second immunization, the antibody titers were determined and the immune protective effect was evaluated with 5 脳 LD50 SC1401 wild strain. The results show that The molecular weight of the target protein was 44.3ku. the antibody titer of Pot D protein determined by Elisa in the form of inclusions was significantly higher than that of formaldehyde inactivated SC1401 vaccine (the highest dilution was 1:20 P / N 2.1g), and the highest dilution was 1:10 000g. The serum antibody titer of normal saline control group and adjuvant control group was P 0.05. The protective power of Pot D protein group was 80%, which was equal to that of SC1401 inactivated vaccine group, and was much higher than that of normal saline group and adjuvant control group. The results showed that Pot D recombinant protein could stimulate immune response in mice and provide better immune protection. It could be used as a candidate antigen protein for recombinant subunit vaccine of Haemophilus paraporus disease.
【作者单位】：四川农业大学动物医学院猪病研究中心;西南医科大学基础医学院病原生物学教研室;
【基金】：公益性行业(农业)科研专项(201303034)
【分类号】：S858.28

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