摘要
为原核表达布鲁氏菌Bp26蛋白,利用布鲁氏菌M5-90疫苗株扩增得到Bp26基因片段,克隆构建pET28a-XXA-HRV 3C-Bp26原核表达载体,通过诱导表达和Ni-NTA亲和纯化得到XXA-HRV 3C-Bp26重组蛋白,再使用PreScission Protease酶切得到无标签的Bp26蛋白。通过SDS-PAGE和Western Blot试验结果,证明成功表达XXA-HRV 3C-Bp26重组蛋白,且蛋白是可溶性表达,并通过酶切获得了无任何标签的Bp26蛋白。以Bp26蛋白做为诊断抗原,初步建立布鲁氏菌间接ELISA检测方法,通过与商用布鲁氏菌ELISA检测试剂盒对256份羊血清样本进行检测,符合率为93.75%,证明建立的布鲁氏菌间接ELISA检测方法可以做为临床中的一种布鲁氏菌病检测方法。
In order to express the Bp26 protein of Brucella in prokaryotic expression,the Bp 26 gene fragment was amplified using the Brucella M5-90 vaccine strain.The pET28a-XXA-HRV 3C-Bp26 prokaryotic expression vector was cloned and constructed.The recombinant protein of XXA-HRV 3C-Bp26 was obtained through induction of expression and Ni-NTA affinity purification.Then,the unlabeled Bp26 protein was obtained by PreScission Protease digestion.The results of SDS-PAGE and Western Blot experiments confirmed the successful expression of the XXA-HRV 3C-Bp26 recombinant protein,which was soluble and obtained unlabeled Bp26 protein through enzymatic digestion.Using the Bp26 protein as the diagnostic antigen,a preliminary indirect ELISA detection method for Brucella was established.256 sheep serum samples were tested using a commercial Brucella ELISA detection kit,and the accuracy rate was 93.75%,proving that the established indirect ELISA detection method for Brucella can be used as a clinical detection method for Brucellosis.
作者
刘尚博
王振华
秦建华
郑可
LIU Shangbo;WANG Zhenhua;QIN Jianhua;ZHENG Ke(Hebei Agricultural University,Baoding Hebei 071000,China;Shijiazhuang Shengbo Biotechnoiogy Co.,Ltd,Shijiazhuang Hebei 050031,China;People's Hospital of Guangxi Zhuang Autonomous Region,Nanning Guangxi 530021,China)
出处
《现代畜牧科技》
2024年第10期27-31,共5页
Modern Animal Husbandry Science & Technology
基金
河北省现代农业产业技术体系第三期奶牛创新团队建设项目奶牛疫病防控与减抗(HBCT2023180201)。
