禽分枝杆菌副结核亚种重组蛋白r22-ag85B的表达及纯化 |
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引用本文: | 杨金国,王聃,刘慧芳,杜艳芬,司微,赵海玲,林靖凯,王春来,倪红波,赫明雷,彭玮,赵福广,刘思国.禽分枝杆菌副结核亚种重组蛋白r22-ag85B的表达及纯化[J].中国兽医寄生虫病,2009,17(2):59-62. |
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作者姓名: | 杨金国 王聃 刘慧芳 杜艳芬 司微 赵海玲 林靖凯 王春来 倪红波 赫明雷 彭玮 赵福广 刘思国 |
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作者单位: | 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;吉林农业大学生命科学学院,长春,130118 2. 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 3. 吉林农业大学生命科学学院,长春,130118 4. 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;延边大学,延吉,133400 |
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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-agS5B,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。
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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 |
EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN R22-AG85B OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS |
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Institution: | YANG Jin-guo, WANG Dan, LIU Hui-fang, DU Yan-fen, SI Wei, ZHAO Hai-ling, LIN Jing-kai, WANG Chun-lai, NI Hong-bo, HE Ming-lei, PENG Wei, ZHAO Fu-guang, LIU Si-guo(1. National Key Laboratory of Veterinary Biotechnology Division of Animal Bacteriosis , Harbin Veterinary Research Institute, CAAS, Harbin 150001 , China; 2. College of Life Science, J ilin Agricultural University, Changchun 130118, China 3 Yanbian Unirersity, Yanji 133400, China) |
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Abstract: | 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. |
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Keywords: | Mycobacterium avium subsp paratuberculosis 22KD gene ag85B gene splicing by overlapping extension (SOE) recombinant expression |
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