副结核分枝杆菌PanCD基因缺失株的构建及毒力评价  

Construction and virulence evaluation of PanCD gene-deleted strain of Mycobacterium avium subsp.paratuberculosis

作　　者：姜艳艳 鹿萍 崔宁 陈凡若 崔莹莹 党光辉 刘思国[1] JIANG Yanyan;LU Ping;CUI Ning;CHEN Fanruo;CUI Yingying;DANG Guanghui;LIU Siguo(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,哈尔滨150069

出　　处：《黑龙江畜牧兽医》2024年第7期36-42,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(32002256,32273005)。

摘　　要：为了开发出能有效防控副结核病的减毒疫苗,试验采用同源重组的方法构建MAP K-10ΔPanCD基因缺失菌株,以MAP K-10菌株基因组为模板,扩增PanCD基因的上下游同源臂,构建pHAE159-p0004s-PanCD重组质粒,转化入耻垢分枝杆菌(Mycobacterium smegmatis,Msg)感受态细胞中获得噬菌体,并感染MAP K-10菌株,然后挑取单克隆扩大培养,提取基因组进行PCR鉴定;绘制MAP K-10菌株与MAP K-10ΔPanCD基因缺失菌株的生长曲线;用MAP K-10ΔPanCD基因缺失菌株感染小鼠并评价其毒力,解剖取肝脏和脾脏,分析组织荷菌量和肝脏病理变化。结果表明:成功扩增出PanCD基因的上下游同源臂,经PCR扩增和酶切鉴定成功构建出pHAE159-p0004s-PanCD重组质粒,将重组质粒电转化入Msg感受态细胞中获得噬菌体,感染MAP K-10菌株后成功构建出MAP K-10ΔPanCD基因缺失菌株;与MAP K-10菌株相比,MAP K-10ΔPanCD基因缺失菌株的生长速率明显下降;攻菌2周时,MAP K-10ΔPanCD基因缺失菌株感染小鼠的肝脏和脾脏中荷菌量显著减少;小鼠肝脏病变明显减轻。说明PanCD基因是MAP的一个重要毒力因子,构建的MAP K-10ΔPanCD基因缺失菌株可作为MAP减毒活疫苗的候选菌株。In order to develop an effective attenuated vaccine against paratuberculosis,in this experiment,the MAP K-10ΔPanCD gene-deleted strain was constructed by homologous recombination.Using the genome of MAP K-10 reference strain as a template,the left and right homologous arms of PanCD were amplified,and the pHAE159-p0004s-PanCD recombinant plasmid was constructed.Subsequently,this plasmid was transformed into Mycobacterium smegmatis(Msg)competent cells to obtain a phage.The monoclonal expanded culture was selected to extract the genome for PCR identification after infecting MAP K-10 strain.The growth curves of MAP K-10 strain and MAP K-10ΔPanCD gene-deleted strain were measured.The virulence of MAP K-10ΔPanCD gene-deleted strain in mice was evaluated,and the liver and spleen were dissected;the number of bacteria in tissues and pathological changes of liver were analyzed.The results showed that the left and right homologous arms of PanCD gene were successfully amplified.The recombinant plasmid pHAE159-p0004s-PanCD was successfully constructed by PCR amplification and enzyme digestion identification.The recombinant plasmid was electroporated into Msg competent cells to obtain the phage which was then infected in MAP K-10 strain.After that,the MAP K-10ΔPanCD gene-deleted strain was successfully constructed.The growth rate of the MAP K-10ΔPanCD gene-deleted strain revealed a significant decrease,compared to the MAP K-10 strain.The number of bacteria in the liver and spleen of mice infected with MAP K-10ΔPanCD gene-deleted strain decreased significantly and the liver lesions of mice were significantly alleviated after 2 weeks of infection.The results suggested that PanCD gene served as a significant virulence determinant of MAP;the constructed MAP K-10ΔPanCD gene-deleted strain exhibited potentially as a candidate for the development of a MAP live-attenuated vaccine.

关 键 词：副结核分枝杆菌 PanCD基因 基因缺失株 毒力评价 减毒活疫苗 

分 类 号：S855.2[农业科学—临床兽医学]