禽分枝杆菌副结核亚种重组蛋白r22-ag85B的表达及纯化  

EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN R22-AG85B OF MYCOBACTERIUM AVIUM SUBSP.PARATUBERCULOSIS

作　　者：杨金国[1,2] 王聃[1,3] 刘慧芳[1] 杜艳芬[1] 司微[1] 赵海玲[1] 林靖凯[1] 王春来[1] 倪红波[1] 赫明雷[1] 彭玮[1] 赵福广[2] 刘思国[1] 

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,哈尔滨150001 [2]吉林农业大学生命科学学院,长春130118 [3]延边大学,延吉133400

出　　处：《中国动物传染病学报》2009年第2期59-62,共4页Chinese Journal of Animal Infectious Diseases

摘　　要：为表达并纯化禽分枝杆菌副结核亚种主要抗原基因的串联重组蛋白r22-ag85B,期望研制出一种新型副结核病疫苗,以禽分枝杆菌副结核亚种参考株P18的基因组DNA为模板,扩增了22KD基因和ag85B基因。采用重叠延伸剪接PCR技术(SOE-PCR)获得了融合基因22-ag85B,将基因连接于表达载体pET32a(+),构建了重组质粒pET22-ag85B。将其转化到大肠杆菌感受态细胞BL21(DE3)中,以IPTG(终浓度1mmol/L)诱导后对表达产物进行Western blot检测。检测结果显示,成功表达并纯化了重组蛋白r22-ag85B,其分子质量约为65ku。Western blot检测表明此蛋白具有良好的免疫学活性。禽分枝杆菌副结核亚种22-ag85B重组蛋白的成功表达及纯化为副结核病疫苗的研究工作奠定了基础。The recombinant gene 22 - ag85B of Mycobacterium avium subsp, paratuberculosis was obtained by splicing by overlapping extension PCR （SOE-PCR） from Mycobacterium avium subsp, paratuberculosis P18 strain. The gene was inserted into the pET32a （ ＋ ） to construct the recombinant plasmid pET22-ag85B. Then the recombinant plasmid was transformed into Escherichia coli BL21 （DE3）. Protein expression was induced by IPTG with a final concentration of 1mmol/L. The expressed protein was purified by nickel-NTA chromatography and identified by Western blot analysis. The recombinant protein r22-ag85B had a MW of 65 ku and showed good immunological activity in Western blot analysis. Preliminary results have demonstrated that the recombinant protein r22-ag85B can be promising novel vaccine against paratuberculosis.

关 键 词：禽分枝杆菌副结核亚种 22KD基因 ag85B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]