牛型布鲁氏菌铁离子转运蛋白基因的克隆及其表达蛋白的生物学特性

将通过PCR技术扩增的牛型布鲁氏菌铁离子转运蛋白(Brucellaferric uptake,BfuA)基因克隆至表达载体pCold TF中,构建重组表达质粒pCold TF-BfuA,转化大肠杆菌BL21(DE3)后,在IPTG诱导下表达出大小约为100ku的融合蛋白His-BfuA。Western-blot分析表明,His-BfuA能与布鲁氏菌阳性牛血清发生特异性反应,表明BfuA具有免疫原性。用His-BfuA作为包被抗原,对35份布鲁氏菌阳性牛血清进行ELISA检测,结果所有被检血清均为阳性反应,表明BfuA为潜在的可用于牛型布鲁氏菌诊断的靶蛋白。此外,对BfuA的亚定位结果表明,BfuA为膜蛋白;对His-BfuA的纤连蛋白结合活性及纤连蛋白溶酶原结合活性的试验结果表明,His-BfuA不具有上述2种活性。上述结果为牛型布鲁氏菌病诊断方法的建立及亚单位疫苗的研制奠定了基础。

Cloning ferric uptake protein gene from Brucella abortus A19 and biological characterization of the protein encoded by the gene

In this study,the immunogenicity,biological characterization and subcellular localization of iron-regulated transporter protein BfuA(Brucella ferric uptake) of Brucella abortus A19 was researched.The immunogenicity of bacterial-expressed BfuA protein(His-BfuA) and the subcellular localization were studied by Western-blot. The ELISA assay was developed to detect the collected clinical Brucella-positive serum. Western-blotting results showed that His-BfuA was an immunogenic protein.The established ELISA methods based on His-BfuA for the detection of 35 infected B.abortus sera are positive. Localization experiments showed that BfuA was a membrane protein.However,His-BfuA had no have the fibronectin and plasminogen-binding activity.BfuA was an immunogenic,membrane-associated protein and can be a potential diagnostic marker. The results of this study provide basis for the development of diagnosis and vaccines for B.abortus.