禽分枝杆菌副结核亚种map0862基因与map2154c基因的串联重组表达  

Fusion expression of map0862 gene with map2154c gene from Mycobacterium avium subsp. paratuberculosis

作　　者：迟磊[1] 刘思国[1] 王春来[1] 宫强[1] 赵昆[1] 刘建东[1] 王勇[1] 云孟克[1] 刘慧芳[1] 赵福广[2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室动物细菌病研究室,黑龙江哈尔滨150001 [2]吉林农业大学生命科学学院,吉林长春130118

出　　处：《中国兽医科学》2008年第10期856-859,共4页Chinese Veterinary Science

基　　金：中国农业科学院哈尔滨兽医研究所中央级公益性科研院所基本科研业务专项资金项目(2008-14)

摘　　要：以禽分枝杆菌副结核亚种参考株P18的基因组DNA为模板,扩增出了map0862基因和map2154c基因。采用重叠延伸剪接PCR技术(PCR-SOE)获得了融合基因map0862-2154c。将融合基因与原核表达载体pET32a(+)连接,构建了重组质粒pET0862-2154c。将重组质粒转化到大肠杆菌感受态细胞BL21(DE3)中,以IPTG(终浓度1 mmol/L)诱导后对表达产物进行SDS-PAGE分析和Western-blot检测。结果表明,表达的重组蛋白map0862-2154c的分子质量为76 ku,可用于建立诊断副结核病的ELISA。The map0862 and 2154c genes were amplified by PCR from Mycobacterium avium subsp. paratutuberculosis P18 strain. The fusion gene map0862-2154c was obtained by PCR SOE technique. Then the fusion gene was inserted into the pET32a（＋） to construct the recombinant plasmid pET0862-2154c. The recombinant plasmid was transformed into Escherichia coli BL21（DE3） to express the fusion protein which was induced with 1 mmol/L IPTG. The expressed fusion protein was insoluble in SDS PAGE analysis. It was purified by nickel-NTA chromatography. Western-blot detection showed that the purified pro tein was 76 ku in molecular weight and could be used in ELISA for immunodiagnosis of bovine paratuberculosis.

关 键 词：禽分枝杆菌副结核亚种 map0862基因 map2154c基因 重叠延伸剪接技术 重组表达 

分 类 号：S852.618[农业科学—基础兽医学]