布鲁氏菌BP26抗原制备及基于BP26的金标检测试纸的初步研究  

Expression and purification of Brucella BP26 antigen and preliminary study on colloidal gold test strip for Brucella

作　　者：孙田华 张祺 李朋伟 张正雷 王彩霞 张幸子 彭林峰 席仲兴 焦磊 王婷 SUN Tian-hua;ZHANG Qi;LI Peng-wei;ZHANG Zheng-lei;WANG Cai-xia;ZHANG Xing-zi;PENG Lin-feng;XI Zhong-xing;JIAO Lei;WANG Ting(The Third Research Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第三研究室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第5期16-20,共5页Progress In Microbiology and Immunology

基　　金：甘肃省重点研发计划项目(20YF3FA004)。

摘　　要：目的制备布鲁氏菌血清学优势抗原BP26的原核表达,对其进行鉴定,且进行基于BP26重组蛋白的布鲁氏菌抗体金标检测试纸的研究。方法检索人用疫苗株布鲁氏菌bp26基因序列进行基因合成,克隆至pET30a(+)载体,构建重组质粒pET30a(+)-bp26,转化大肠埃希菌BL21(DE3),IPTG诱导表达,亲和层析纯化BP26重组蛋白,纯化后重组蛋白进行蛋白免疫印迹鉴定。胶体金标记BP26,以BP26重组蛋白和BP26兔多抗血清分别包被检测线和质控线,制成布鲁氏菌抗体金标检测试纸。结果BP26重组蛋白在大肠埃希菌中成功表达,相对分子质量约为26000,与预期相符。亲和层析纯化后得到纯度较高的BP26重组蛋白。Western blot实验证明,该蛋白可与布鲁氏菌抗体阳性兔血清产生特异性结合。用布鲁氏菌抗体金标检测试纸检测兔阳性血清,检测线和质控线均显色明显。结论成功获得BP26重组蛋白,Western blot实验证明该蛋白具有良好的免疫反应性。研制的检测试纸成功检测出布鲁氏菌抗体阳性兔血清。该蛋白为布鲁氏菌病诊断试剂盒研制及疫苗研发奠定了基础。Objective Prokaryotic expression and identification of Brucella serological dominant antigen BP26.And the study of Brucella antibody gold label detection test paper based on recombinant BP26 protein.Methods The bp26 gene sequence of human Brucella vaccine strain was retrieved and synthesized,then cloned into pET30 a(+)vector to construct the recombinant plasmid pET30 a(+)-bp26,which was transformed into BL21(DE3).The recombinant BP26 protein was induced by IPTG,purified by affinity chromatography,and identified by Western blot.Colloidal gold was labeled with BP26.The detection line and control line were coated with BP26 and anti-BP26 rabbit antibody respectively.Results Recombinant BP26 protein was successfully expressed in E.coli with a molecular weight of about 26000 Da,which was consistent with the expected size.After purified by affinity chromatography,recombinant BP26 protein with high purity was obtained.Western blot result showed that the protein could specifically bind to Brucella rabbit positive serum and had good immune reactivity.Colloidal gold test was used to detect Brucella antibody positive rabbit serum,and the test result was positive.Conclusion The recombinant protein BP26 was successfully expressed in E.coli and had specific binding reaction with Brucella antibody positive serum.The colloidal gold test successfully detected Brucella antibody positive serum.The above work established a foundation for the development of brucellosis diagnostic kit and vaccine.

关 键 词：布鲁氏菌 BP26重组蛋白 蛋白免疫印迹 胶体金试纸条 

分 类 号：R378.5[医药卫生—病原生物学]