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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。
Objective To clone and express Brucella type Ⅳ secretion system effector protein BspB and speculate its immune escape mechanism. Methods Brucella spp 16M genome was used as a template to amplify the BspB (BAB1-0712) gene of Brucella by PCR, ligated with pMD19-T vector and sequenced. The correct gene fragment was double-digested Cloned into pET-30a vector, transformed into E. coli BL21 (DE3), induced by IPTG expression, and identified by SDS-PAGE and Western blot analysis. Results The BspB gene was successfully cloned and the fragment size was 564bp. The molecular mass unit of BspB protein was about 27.5ku by SDS-PAGE. Western blot analysis showed that the BspB protein was recognized by Brucella positive sera. The protein was sequenced and compared with the innate immune molecules IL-1R, MyD88, TLR-2, TLR-4 and SIGIRR, and had certain homology. Conclusion The recombinant protein BspB is immunogenic and can be used as a candidate diagnostic antigen. Because of its sequence homology with IL-1R, MyD88, TLR-2, TLR-4 and SIGIRR, BspB may be expressed in host of Brucella infection The process of immune escape play a role.