牛型结核分枝杆菌分泌性蛋白MPB70.MPB83基因串联重组质粒的构建及鉴定  

Construction and identification of a recombinant plasmid containing the mph 70 and mpb 83 genes coding for Mycobacterium bo vis secretory proteins

作　　者：叶俊 布日额 陈金龙[4,6] 吴金花[2,3,5] 锡林高娃 王金良 YE Jun;BU Ri-e;CHEN Jin-Long;WU Jin-hua;XI Lin gao-wa;WANG Jin-Liang(Animal Science College,Inner Mon golia University for Nationalities,Tongliao,China 028043;Inner Mongolia Autonomous Region Engineering Technology Research Center for Prevention and Control of Pathogenic Bacteria in Milk,Tongliao,China 028043;Life Science,Inner Mongolia Universily for Nationalities,Tongliao,China 028043;Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou,China 256600;Research Institute for Pathogens in Milk,Inner Mongolia University for Nationalities,Tongliao,China 028043;Shandong Ludu Bio sciences and Technology Co.,Ltd.,Binzhou,China 256600)

机构地区：[1]内蒙古民族大学动物科技学院,内蒙古通辽028043 [2]内蒙古自治区乳源性致病菌防控工程技术研究中心 [3]内蒙古民族大学生命科学学院 [4]山东省滨州畜牧兽医研究院 [5]内蒙古民族大学乳源性致病菌研究所 [6]山东绿都生物科技有限公司

出　　处：《中国病原生物学杂志》2019年第12期1429-1432,共4页Journal of Pathogen Biology

基　　金：内蒙古自治区科技创新引导项目(No.KCBJ2018026);内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题(No.MDK2018001,MDK2018004);内蒙古自治区"草原英才"人才工程项目(乳源性致病菌防控技术研究创新人才团队项目)(第7批)。

摘　　要：目的构建牛型结核分枝杆菌(MB)分泌性蛋白mpb70.mpb83基因串联重组表达质粒,为进--步利用工程菌表达、获得相应抗原蛋白奠定实验基础。方法参考NCBI上公布的MB全基因组序列(EU683972.1),在利用DNAstar7.0生物信息软件分析分泌性蛋白mpb70、mpb83抗原性基础上,选择mpb70、mpb83基因编码多肽主要抗原域,利用重叠延伸PCR技术构建pGEX-6p-mpb70+mpb83串联重组表达质粒。结果生物信息学预测MPB70、MPB83基因的抗原域分别在30-171位氨基酸和20-190位氨基酸。在mpb70、mpb83间加入16位柔性多肽序列,在线分析不影响mpb70和mpb83融合后的抗原性。pGEX-6p-1-mpb70+mpb83经双酶切获得966bp和4966bp两条片段,与预期一致;测序表明mpb70+mpb83融合基因无碱基突变和缺失,重组质粒构建正确。结论确定了MB早期标识性分泌性蛋白mpb70、mpb83基因的抗原域,获得了mpb70、mpb83串联基因,构建的重组原核表达载体柔性多肽的引人确保mpb70和mpb83的天然抗原构象且不影响融合蛋白的抗原性,为重组抗原的表达,单克隆抗体制备以及建立MB分泌性蛋白标识物的快速检测试剂盒奠定了基础。Objective To construct recombinant plasmids containing the mpb 70 and mpb 83 genes coding for Myco-bacterium bovis secretory proteins and to lay an experimental foundation for engineering the bacterium to further express and yield those proteins.Methods Based on the genome sequence of M.bovis published in the NCBI database(EU683972.1),the antigenicity of the secretory proteins mpb 70 and mpb 83 was analyzed using the bioinformatic soft-ware DNAstar 7.The main antigenic domains of the mpb 70 and mpb 83 genes encoding polypeptides were selected,and a pGEX-6p-1-MPB70+MPB83 tandem recombinant expression plasmid was constructed using overlap extension PCR.Results The antigenic domains of the MPB70 and MPB83 genes were predicted using bioinformatics,and the recombi-nant plasmid pGEX-6p-1 mpb70+mpb83 was successfully constructed using double digestion and sequencing.Conclu-sion This experiment has laid a solid foundation for further induction of antigen expression,preparation of those anti-gens,and preparation of monoclonal antibodies against them.

关 键 词：结核分枝杆菌 MPB70基因 MPB83基因 串联表达 

分 类 号：R383.33[医药卫生—医学寄生虫学]