羊布氏菌vjbR基因的克隆及原核表达

目的克隆并原核表达羊布氏菌强毒株16M vjbR基因。方法 PCR扩增羊布氏杆菌16M株vjbR基因,亚克隆至原核表达载体pET-28a中,构建重组原核表达质粒pET-28a-vjbR,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经His Trap FF纯化后,进行Western blot分析。结果扩增的vjbR基因大小为708 bp,与预期一致;重组原核表达质粒pET-28a-vjbR经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为29 000,诱导4 h表达量可达6.84 mg/ml;纯化的重组蛋白纯度为65.7%,能被羊布氏杆菌阳性血清所识别。结论成功在大肠杆菌中表达了羊布氏菌VJBR蛋白,为其功能的研究、羊布病诊断试剂盒的研制及亚单位疫苗的研发奠定了基础。

Cloning and prokaryotic expression of vjbR gene of Brucella melitensis

Objective To clone the vjbR gene of virulent Brucella melitensis 16M strain and express in prokaryotic cells.Methods The vjbR gene of 16M strain was amplified by PCR and subcloned into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-vjbR was transformed to competent E.coli BL21(DE3) and induced with IPTG.The expressed recombinant protein was purified by His Trap FF column chromatography and analyzed by Western blot.Results The length of amplified vjbR gene was 708 bp,which was consistent with that expected.Restriction analysis and sequencing proved that recombinant plasmid pET-28a-vjbR was construced correctly.The expression level of recombinant protein,with a relative molecular mass of about 29 000,reached the maximum of 6.84 mg / ml 4 h after induction.The purified recombinant protein reached a purity of 65.7% and was recognized by B.melitensis-positive sera.Conclusion The VJBR protein of B.melitensis was successfully expressed,which laid a foundation of study on function of the protein,preparation of diagnostic kit for B.melitensis infection and development of subunit vaccine.