布鲁氏菌Ⅳ型分泌系统效应蛋白BspB的原核表达及布鲁氏菌免疫逃逸分析  

Prokaryotic expression of BspB protein of the type Ⅳ secretion system of Brucella and analysis of immune evasion by Brucella

作　　者：李默[1] 易德武 王远志[1] 陈创夫[2] 于潞[1] 张俊波[3] 郭飞[1] 

机构地区：[1]石河子大学医学院病原生物学与免疫学教研室,新疆石河子832002 [2]石河子大学动物科技学院,新疆石河子832003 [3]铜仁学院生物与农林工程学院,贵州铜仁554300

出　　处：《中国病原生物学杂志》2015年第9期778-781,815,共5页Journal of Pathogen Biology

基　　金：杰出青年科技人才培育计划项目(No.2013ZRKXJQ06);高层次人才科研启动资金专项(No.RCZX201228);博士科研启动基金项目(No.trxyDH1504)

摘　　要：目的克隆并表达布鲁氏菌Ⅳ型分泌系统效应蛋白BspB并推测其免疫逃逸机制。方法以羊种布鲁氏菌16M基因组为模板,利用PCR扩增布鲁氏菌分泌蛋白BspB(BAB1-0712)基因,连接pMD19-T载体后测序,将测序正确的基因片段经双酶切后克隆至pET-30a载体中,转化大肠埃希菌BL21(DE3),经IPTG诱导表达,通过SDS-PAGE和Western blot分析鉴定表达产物。结果成功克隆了BspB基因,片段大小为564bp;SDS-PAGE检测,BspB蛋白分子质量单位约为27.5ku;Western blot分析显示,BspB蛋白均可被布鲁氏菌阳性血清识别。该蛋白测序后与先天性免疫分子IL-1R、MyD88、TLR-2、TLR-4、SIGIRR比对,具有一定的同源性。结论重组蛋白BspB具有免疫反应原性,可作为候选诊断抗原;由于其序列与IL-1R、MyD88、TLR-2、TLR-4及SIGIRR有一定同源性,BspB可能在布鲁氏菌感染宿主过程中发挥免疫逃逸的作用。Objectives To clone and express the BspB （BAB1-0712） gene, a Brucella T4SS effector, and to predict its role in immune evasion by Brucella. Methods PCR was used to amplify the BspB gene from the 16M strain of Brucella melitensis and this gene was then cloned into the pMD18-T Simple Vector. The gene was sequenced to verify that the PCR product was correct, and then this product was cloned into the plasmid pET30a（-） and transformed into E. coli BL21 （DE3）. Expression of protein was induced by IPTG and this protein was analyzed and identified with SDS-PAGE and Western blotting. Results A 564-bp fragment of the BspB gene was successfully obtained and a protein with a mo lecular weight of 27.5 ku was obtained. The BspB protein reacted specifically with Brucella-positive serum. Moreover, the BspB protein had a certain degree of homology with the innate immune molecules IL-1R, MyD88, TLR-2, TLR-4, and SIGIRR. Conclusion The recombinant protein BspB triggered an immune response and this recombinant protein has potential for use as a diagnostic antigen. BspB protein may play an important role in evasion of host innate immunity by Brucella.

关 键 词：布鲁氏菌 效应蛋白 Ⅳ型分泌系统 原核表达 免疫逃逸 

分 类 号：R378.5[医药卫生—病原生物学]