绵羊肺炎支原体多抗原表位基因MO-meAg15重组单增李斯特菌的构建及其免疫原性研究

发布时间：2018-06-19 20:17

  本文选题：绵羊肺炎支原体 + 单增李斯特菌　； 参考：《西南农业学报》2017年09期

【摘要】：【目的】本文研发了MO重组活疫苗。【方法】利用PCR技术从LM-SB5株基因组中扩增出rli60基因上下游同源片段a、b,从pMD19-T-meAg15中扩增出MO meAg15目的片段,采用重叠延伸PCR(SOE-PCR)技术将扩增得到的3个基因片段融合成ameAg15b,将其克隆测序鉴定。将ameAg15b目的基因亚克隆到pKSV7穿梭载体,构建pKSV7-ameAg15b重组穿梭质粒,将该重组穿梭质粒电转化至LM-SB5感受态细胞,在氯霉素、高温双重压力筛选重组菌LM-Δrli60-ameAg15b。【结果】重组菌PCR鉴定表明,外源基因meAg15定点整合于LM基因组中。遗传稳定性试验表明,meAg15基因可以在LM基因组中稳定存在。SDS-PAGE结果表明,重组菌LM-Δrli60-ameAg15b可表达相对分子量为14.5 kDa的重组蛋白;Western blot分析说明,表达的重组蛋白能与MO阳性血清发生特异性免疫反应。【结论】小鼠免疫试验表明,重组菌LM-Δrli60-ameAg15b菌体可诱导机体产生抗MO特异性抗体,证实该重组菌具有一定的免疫原性。
[Abstract]:[methods] the upstream and downstream homologous fragment of rli60 gene was amplified from the genome of LM-SB5 strain, and the meAg15 fragment of MO was amplified from pMD19-T-meAg15. The three amplified gene fragments were fused to ameAg15b by overlapping extension PCR- SOE-PCR technique, and their cloning and sequencing were identified. The ameAg15b target gene was subcloned into pKSV7 shuttle vector, and the recombinant shuttle plasmid pKSV7-ameAg15b was constructed. The recombinant shuttle plasmid was electrotransformed into LM-SB5 competent cells. The recombinant strain LM- 螖 rliame60-Ag15b. was screened under chloramphenicol and high temperature double pressure. The foreign gene meAg15 was integrated into LM genome. The results of genetic stability test showed that the stably exists of the gene in LM genome. SDS-PAGE showed that the recombinant strain LM- 螖 rli60-ameAg15b could express the recombinant protein with relative molecular weight of 14.5 kDa. [conclusion] mice immunological test showed that the recombinant strain LM- 螖 rli60-ameAg15b could induce the production of anti-MO specific antibody, which proved that the recombinant strain had certain immunogenicity.
【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) 兵团中青年科技创新领军人才计划(2016BC001)
【分类号】：S852.4

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