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  

Construction and Immunogenicity of Recombinant Listeria monocytogenes Expressing MO-meAg15 Gene of Multi-epitope in Mycoplasma ovipneumoniae

作　　者：卢海亭 乔军[1] 孟庆玲[1] 彭叶龙 田路路[1] 贡莎莎 才学鹏[3] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]阿克苏地区动物疫病控制诊断中心,新疆阿克苏843000 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《西南农业学报》2017年第9期2155-2160,共6页Southwest China Journal of Agricultural Sciences

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)

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。[ Objective] In this paper, the MO recombinant live vaccine was developed. [ Method] The rli60 gene homologous sequences a, band meAg15 gene of Mycoplasma ovipneumoniae （MO） from genome of LM-SB5 and pMD19-T-meAgl5 were amplified by PCR, respective-ly. The fusion gene of ameAgl5b was constructed by splicing overlap extention PCR （SOE-PCR） and then cloned and sequenced. ThenameAg15b was subcloned in shuttle vector pKSV7 to generate recombinant pKSV7-ameAgl5b. The recombinant plasmid was introduced intocompetent LM-SB5 by electroporation. The recombinant LM-ArIi60-ameAg15b was obtained under the double pressure screening of chloram-phenicol and temperature selection. [ Result] The target gene ameAg15b was proved to be correctly inserted into the genome of LM and therecombinant strain and had stable genetic characteristic. SDS-PAGE showed that recombinant protein was successfully expressed with a massof molecule of 14.5 kDa. Western blot revealed that the recombinant ameAglSb could specifically react with anti-MO serum. [ Conclusion]The immunization test showed that recombinant LM can induce specific antibodies against MO, confirming that recombinant LM has some im-munogenicity.

关 键 词：绵羊肺炎支原体 单增李斯特菌 meAg15基因 同源重组 

分 类 号：S852[农业科学—基础兽医学]