牛布鲁氏菌Wbkc基因原核表达及抗原性分析
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国家自然科学基金项目(31260608)、内蒙古自治区高等学校科学技术研究重点项目(NJZZ12117)和内蒙古自治区通辽市与内蒙古民族大学科技合作项目(sxzD2012131)


Expression and antigenicity analysis of Wbkc gene of Brucella abortus
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    摘要:

    克隆牛布鲁氏菌的甲酰基转移酶基因(Wbkc),在大肠杆菌中表达/纯化,并对甲酰基转移酶的抗原性进行分析。以牛布鲁氏菌的染色体DNA为模板,扩增Wbkc基因,双酶切后克隆至pET-28a上,在大肠杆菌BL21中诱导表达,His Trap FF层析柱纯化,Western blot鉴定甲酰基转移酶的抗原性。提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中。SDS-PAGE分析证明,表达产物为相对分子质量29 000的融合蛋白。Western blot结果表明,表达的蛋白具有特异的免疫反应原性。

    Abstract:

    In order to clone the formyltransferase gene (Wbkc) of Brucella abortus,express the gene in E.coli and detect the immunogenicity of the expressed protein,the Wbkc gene was amplified from the genomic DNA of Brucella abortus by PCR.The amplified fragments were digested with BamHⅠ and SalⅠ and then cloned into the pET28a vector.The constructed recombinant plasmid pET28a-Wbkc was transformed to E.coli BL-21 and induced to express the fusion protein.The protein was then purified by histidine-binding resin column chromatography and the immunogenicity was detected by Western blot.The recombinant plasmid was verified by PCR,double-enzyme cleavage and sequencing analysis.A specific protein band of 29 000 (relative molecular mass) was identified by SDS-PAGE and the expressed product showed good immunoreactivity by Western blot.

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李向阳,王学理,刘凯,霍晓伟,武迎红,张显华.牛布鲁氏菌Wbkc基因原核表达及抗原性分析[J].华中农业大学学报,2013,32(6):106-109

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  • 收稿日期:2013-04-22
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