绵羊肺炎支原体p74基因重组单核细胞增生李斯特菌的构建及其免疫原性分析

发布时间：2018-09-17 11:46

【摘要】：【目的】构建绵羊肺炎支原体(MO)p74基因重组单核细胞增生李斯特菌(LM)并分析其免疫原性,为研发MO重组活疫苗奠定基础。【方法】采用PCR方法从LM-SB5株基因组中扩增出rli60基因的上、下游同源片段a和b,从质粒pET-32a(+)-p74中扩增出MOp74目的基因片段,利用重叠延伸PCR方法(SOE-PCR)将扩增的3个片段融合成ap74b基因片段,克隆入pMD19-T载体中进行测序鉴定。将ap74b片段从pMD19-T载体上切下亚克隆入pKSV7穿梭载体,构建pKSV7-ap74b重组穿梭质粒,电转化至LM-SB5株感受态细胞,用氯霉素和高温双重压力筛选得到重组菌LM-Δrli60-ap74b。并通过SDS-PAGE及Western blot分析重组菌P74蛋白的表达情况,通过小鼠免疫试验检测其免疫原性。【结果】成功扩增出了rli60基因的上下游同源片段a、b及p74目的片段,采用SOEPCR方法获得融合片段ap74b,并成功亚克隆于穿梭质粒pKSV7中。对重组菌PCR鉴定结果表明,外源基因MO p74定点整合于LM基因组中。体外稳定性检验表明,p74基因能在LM基因组中稳定存在。SDS-PAGE结果表明,重组菌LM-Δrli60-ap74b可表达蛋白分子质量约为17ku重组蛋白;Western blot分析表明,表达的重组蛋白能与MO阳性血清发生特异性免疫反应。小鼠免疫试验表明,重组菌LM-Δrli60-ap74b菌液可诱导机体产生抗MO特异性抗体,证实该重组菌具有一定的免疫原性,重组菌能诱导机体产生1∶8~1∶16的抗体效价。【结论】获得了具有免疫原性的绵羊肺炎支原体p74基因重组单核细胞增生李斯特菌。
[Abstract]:The expression of P74 protein was analyzed by SDS-PAGE and Western blot, and the immunogenicity of P74 protein was detected by mouse immunoassay. [results] the upstream and downstream homologous fragments of rli60 gene AGB and p74 were successfully amplified. The fusion fragment ap74b, was obtained by SOEPCR and subcloned into shuttle plasmid pKSV7. The results of PCR identification showed that the foreign gene MO p74 was integrated into the LM genome. The results of in vitro stability test showed that the p74 gene was stable in the LM genome. SDS-PAGE showed that the recombinant strain LM- 螖 rli60-ap74b could express the protein with molecular weight about the same as that of the 17ku recombinant protein. The expressed recombinant protein could react specifically with MO positive serum.
【作者单位】： 石河子大学动物科技学院;阿克苏地区动物疫病控制诊断中心;中国农业科学院兰州兽医研究所;
【基金】：国家自然科学基金项目(31360596) 国家国际科技合作专项(2014DFR31310)
【分类号】：S852.4
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