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  

Construction and immunogenicity of recombinant Listeria monocytogenes expressing Mycoplasma ovipneumoniae p74 gene

作　　者：卢海亭 乔军[1] 孟庆玲[1] 彭叶龙 陈诚[1] 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：《西北农林科技大学学报（自然科学版）》2017年第8期7-14,22,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31360596);国家国际科技合作专项(2014DFR31310)

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。【Objective】The recombinant Listeria Monocytogenes expressing Mycoplasma ovipneumoniae（MO）p74gene was constructed and its immunogenicity was analyzed to provide foundation for development of MO subunit vaccine.【Method】The rli60 gene homologous sequences a,b and p74 gene of MOwere amplified by PCR from genome of Listeria monocytogenes LM-SB5 strain and plasmid pET-32a（＋）-p74,respectively.The fusion fragment of ap74 b was constructed by splicing overlap extension PCR（SOEPCR）method and then inserted into cloning vector pMD19-T for sequencing.The ap74 bfragment was digested by double enzymes and then sub-cloned in shuttle vector pKSV7 to construct recombinant plasmid pKSV7-ap74 b.Then,the recombinant plasmid was introduced into competent LM-SB5 by electroporation.The recombinant bacteria strain（named as LM-Δrli60-ap74b）was obtained under double pressure screening of chloramphenicol and temperature selection.The expression of P74 protein was detected by SDSPAGE and Western blot and its immunogenicity was tested by immunization of mice.【Result】The homologous fragments a,b of rli60 gene flank and p74 gene were successfully amplified.Fusion fragment ap74 b was obtained using gene SOE-PCR method and then successfully sub-cloned into the shuttle plasmid pKSV7.In vitro stability assay showed that the target fragment ap74 b was correctly inserted into the desired site in-frame with rli60 and the recombinant strain had stable genetic characteristics.SDS-PAGE showed that recombinant protein was successfully expressed with a molecule weight of 17 ku.Western blot revealed that the recombinant LM-Δrli60-ap74 bcould specifically react with anti-MO positive serum.The immunization test showed that LM-Δrli60-ap74 bcan induce specific antibodies against MO in mice after immunization,which confirmed that recombinant LM-Δrli60-ap74 bhad immunogenicity.Recombinant bacteria can induce antibody titer of 1∶8to 1∶16.【Conclusion】The recombinant Listeria monocytogenes expressing MOp74 gene with good imm

关 键 词：绵羊肺炎支原体 单核细胞增生李斯特菌 p74基因 同源重组 

分 类 号：S858.26[农业科学—临床兽医学]