牛结核分枝杆菌早期标识性分泌性蛋白MPB70、MPB83基因的串联表达及其抗原性鉴定  

Tandem expression of early secretory protein MPB70 and MPB83 genes of Mycobacterium bovis and identification of antigenicity

作　　者：布日额 陈金龙[2,6] 叶俊[1,4] 吴金花 锡林高娃 王金良 BU Ri-e;CHEN Jin-long;YE Jun;WU Jin-hua;XI Lin gao-wa;WANG Jin-liang(Inner Mongolia Autonomous Region Engineering Technology Research Center of Prevention and Control the Pathogenic Bacteria in Milk,Tongliao 028043,China;Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou 256600,China;Life Science,Inner Mongolia University for Nationalities,Tongliao 028043,China;Animal Science college,Inner Mongolia University for Nationalities,Tongliao 028043,China;Research Institute for Pathogenic in Milk of Inner Mongolia University for Nationalities,Tongliao 028043,China;Shandong Lvdu Bio-sciences and Technology Co.,LTD,Binzhou 256600,China)

机构地区：[1]内蒙古自治区乳源性致病菌防控工程技术研究中心,内蒙古通辽028043 [2]山东省滨州畜牧兽医研究院,山东滨州256600 [3]内蒙古民族大学生命科学学院,内蒙古通辽028043 [4]内蒙古民族大学动物科技学院,内蒙古通辽028043 [5]内蒙古民族大学乳源性致病菌研究所,内蒙古通辽028043 [6]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国预防兽医学报》2019年第6期622-626,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：内蒙古自治区科技创新引导项目(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(MDK2018001、MDK2018004);内蒙古自治区“草原英才”人才工程(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：为获得一种具有抗原性的牛结核分枝杆菌(MB)感染早期标识物MPB70、MPB83分泌性蛋白的串联抗原蛋白,本研究利用DNAStar生物信息软件对MB感染早期标识性分泌性蛋白MPB70、MPB83抗原结构区域进行预测后设计合成相应引物,并在两个片段之间引入16位柔性多肽,通过重叠延伸PCR技术获得MBP70与MBP83基因的抗原优势区核苷酸串联片段,将其连接至原核表达载体pGEX-6p-1中经诱导、表达,结果显示构建的重组表达载体获得高效表达,目的抗原蛋白的分子质量约58ku,纯化后重组蛋白的纯度>90%,经免疫BALB/c小鼠4次后,抗体效价达到1∶51200。本实验获得了具有明显抗原活性的MB感染早期分泌性蛋白串联抗原MPB70+MPB83,为后续进一步研发MB早期感染标识物快速检测试剂盒奠定了基础。To acquire a tandemly expressed protein of MPB70 and MPB83, the early infection markers of Mycobacterium bovis, the antigenic regions of MPB70 and MPB83 were predicted by DNAStar software. The tandem nucleotide fragments of MPB70 and MPB83 genes were amplified by overlapping extension PCR. In order to ensure the correct expression of the two genes, nucleotide sequence coding for sixteen flexible peptides were introduced between the two fragments. The fusion gene was cloned into prokaryotic expression vector pGEX-6p-1. After transforming of E.coli BL21 (DE3), the recombinant Escherichia coli expressed 58ku target protein after 1mmol/L IPTG induction. The fusion protein (purity>90%) was purified by affinity chromatography. Four BALB/c mice were subcutaneously immunized with the mixture of the recombinant protein and Freund's adjuvant four times at the interval of fourteen days. The titer of antibody against the recombinant protein reached up to 1∶51,200 after immunization, which indicated that the fusion protein had good antigenicity. This study laid a foundation for the development of rapid detection kit for early infection markers of bovine mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 MPB70 MPB83 串联表达 

分 类 号：S852.4[农业科学—基础兽医学]