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

Development of a Haemophilus parasuis polyamine transport protein PotD subunit vaccine and evaluation of its immune efficacy

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/N>2.1),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(P<0.05)。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。

To evaluate the immunoprotective efficacy of Haemophilus parasuis polyamvne transport protein potD,the gene without signal peptide coding sequence was amplified from SC1401 genome DNA and subsquently subcloned and expressed in E.coli pET28a（＋） and BL21（DE3）,respectively.The expression form of recombined Pot D was confirmed by ultrasonication,followed by SDS-PAGE.The mice in vaccination group was immunized with 50 g purified product emulsified with Freund＇s complete and incomplete adjuvant（FCA and FIA,respectively）every two weeks,which was followed by antibody titer determination（ELISA） and mice challenge experiment.The results demonstrated that the recombined protein was nearly44.3 ku,mainly in a form of inclusion bodies.The antibody titer in recombined protein group was significantly higher compared with the formalin-killed vaccine group（P〈0.05）under the dilution of 1 ∶ 20 000（with the P/N2.1）,and both the vaccine groups of recombined Pot D and formalin-killed H.parasuis had a protective rate of 80%.Taken together,our studies demonstrate that the recombined Pot D can actively induce immune responses in mice and to protect them in challenge experiment,which can be served as a candidate subunit vaccine in protection of virulent H.parasuis.