羊布氏菌外膜蛋白10基因的原核表达

Prokaryotic expression of outer membrane protein 10 gene of Brucella melitensis

目的原核表达并纯化羊布氏菌外膜蛋白10(Outer membrane protein,Omp10)。方法利用PCR技术扩增羊布氏菌16M株Omp10基因,插入原核表达载体pET-28a(+),构建重组原核表达质粒pET-28a-Omp10,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达的重组Omp10经HisTrapTMFF纯化后,Western blot分析表达产物的反应原性。结果重组原核表达质粒pET-28a-Omp10经双酶切及测序证明构建正确;表达的重组Omp10相对分子质量约15 000,能被羊布氏菌阳性血清所识别。结论已成功在大肠杆菌中表达了羊布氏菌Omp10,为羊布氏菌病血清学诊断方法的建立提供了材料。

Objective To express outer membrane protein 10 （Ompl0） gene of Brucella melitensis in prokaryotic cells and purify the expressed product. Methods The Ompl0 gene of B. melitensis was amplified by PCR and inserted into prokaryotic expression vector pET-28a （＋）. The constructed recombinant plasmid pET-28a-Omp10 was transformed to E. coli BL21 （DE3） and induced with IPTG. The expressed recombinant Ompl0 was purified by HisTrapTM FF chromatography and analyzed for reactogenicity by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a-Omp10 was constructed correctly. The expressed recombinant Ompl0, with a relative molecular mass of about 15 000, was recognized by B. melitensis positive serum. Conclusion The Ompl0 of B. melitensis was successfully expressed in E. coli, which provided a material for development of serological diagnostic method for B. melitensis infection