| 布鲁氏菌分泌蛋白BspJ多克隆抗体的制备 |
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| 引用本文： | 马忠臣,胡尊楠,王震,郑炜,王勇,陈创夫.布鲁氏菌分泌蛋白BspJ多克隆抗体的制备[J].中国动物传染病学报,2020(3):61-67. |
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| 作者姓名： | 马忠臣 胡尊楠 王震 郑炜 王勇 陈创夫 |
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| 作者单位： | 石河子大学动物科技学院;西部地区高发病人兽共患传染性疾病防治协同创新中心 |
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| 基金项目： | 国家重大研发计划(2017YFD0500304)。 |
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| 摘 要： | 为获得一定纯度及浓度的布鲁氏菌分泌蛋白BspJ并制备多克隆抗体,本研究通过常规PCR方法扩增BspJ基因,连接至pMD19-T克隆载体并筛选阳性克隆;使用限制性内切酶切下目的片段后连接至pET28a(+)载体,双酶切、菌液PCR验证阳性克隆菌并送测序;表达载体pET28a-BspJ转入大肠杆菌DE3后经IPTG诱导表达,收集表达菌进行SDS-PAGE分析;使用His标签蛋白纯化柱纯化目的蛋白rBspJ;将rBspJ蛋白混入弗氏佐剂分4次免疫实验兔,收集兔血清,Western blot鉴定多克隆抗体,饱和硫酸铵法纯化多克隆抗体.结果显示:PCR方法扩增出大小534 bp的目的片段,测序结果正确,表明成功构建出pMD19-T-BspJ克隆载体及pET28a-BspJ表达载体;表达菌表达出大小约24 kDa的rBspJ,纯化后的rBspJ条带明显,基本无杂带,表明成功纯化出rBspJ;真核表达的rBspJ与制备的多克隆抗体反应,表明成功制备多克隆抗体.本研究结果为BspJ蛋白的亚细胞定位分析、功能研究及布鲁氏菌致病机制的研究积累实验数据和奠定基础.
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| 关 键 词： | 布鲁氏菌 分泌蛋白BspJ 原核表达 多克隆抗体 |
PREPARATION OF POLYCLONAL ANTIBODIES AGAINST BRUCELLA SECRETING PROTEIN BSPJ |
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| MA Zhong-chen,HU Zun-nan,WANG Zhen,ZHENG Wei,WANG Yong,CHEN Chuang-fu.PREPARATION OF POLYCLONAL ANTIBODIES AGAINST BRUCELLA SECRETING PROTEIN BSPJ[J].Chinese Journal of Animal Infectious Diseases,2020(3):61-67. |
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| Authors: | MA Zhong-chen HU Zun-nan WANG Zhen ZHENG Wei WANG Yong CHEN Chuang-fu |
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| Institution: | (College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for the Prevention and Control of Infectious Diseases in the Western Region,Shihezi 832000,China) |
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| Abstract: | In order to obtain pure and concentrated Brucella secreted protein BspJ for preparation of polyclonal antibodies,the target gene was amplified by conventional PCR,ligated into the pMD19-T cloning vector and screened for positive clones.Then,the target fragment was excised by restriction endonuclease and ligated into pET28a(+)vector followed by confirmation with double-enzyme digestion,bacterial solution and sequencing.Subsequently,the expression vector pET28a-BspJ was transferred into E.coli DE3 for expression with induction of IPTG.The rBspJ was purified using His-tag column and confirmed to be a protein with molecular mass of 24 kDa in SDS-PAGE.The rBspJ was emulsified with Freund’s adjuvant and used to immunize rabbits 4 times.Rabbit serum samples were collected and polyclonal antibodies were purified using saturated ammonium sulfate.The preparation of polyclonal antibodies reacted with rBspJ in Western blot.This study provided the reagent for future study on the subcellular localization and functions of BspJ protein as well as pathogenesis of Brucella infection. |
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| Keywords: | Brucella secreted protein BspJ prokaryotic expression polyclonal antibody |
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