【摘要】 目的获得布鲁氏菌感染血清学诊断优势抗原BP26重组蛋白。方法应用PCR技术从布鲁氏菌减毒株(104M)中扩增出bp26目的基因片段,将其克隆于表达载体PET30a中,并转化入大肠杆菌BL21(DE3)中,IPTG诱导表达,用亲和层析纯化BP26重组蛋白,然后分别用Western-blot,间接ELISA检测其抗原性。结果DNA测序及酶切鉴定证实PET-30a/bp26原核表达载体构建成功,并在大肠杆菌中高效表达,免疫印迹和间接ELISA实验证明BP26重组蛋白可与布鲁氏菌阳性血清产生特异性结合。结论成功获得了布鲁氏菌中序列全长为696bp,编码232个氨基酸的BP26蛋白,通过血清学反应证实,该蛋白为人和动物布鲁氏菌病临床诊断试剂盒的研发奠定了基础。更多还原

【Abstract】 To obtain recombinant BP26 protein of brucella species,which was the predominant antigen for seroimmunological diagnosis of the brucella infection.The target fragment of bp26 gene was amplified by PCR from the template DNA of B.abortus 104M strain and was cloned into expression vector PET-30a.After transformed into competent E.coli BL21(DE3)cells and induced by IPTG,the recombinant protein BP26 was expressed and purified through affinity chromatography.As demonstrated by Western blot assay and indirect ELISA,this recombinant protein BP26 has satisfactory antigenicity.It was confirmed by DNA sequencing and restriction enzymes cleavage that the plasmid PET-30a/bp26 was correctly constructed and was highly expressed in E.coli.Furthermore the results of Western blot assay and indirect ELISA showed that this recombinant protein could be recognized by the positive anti-serum against Brucella.It is evident that this recombinant protein BP26 should be a potentially valuable antigen for the serological diagnosis and for the preparation of the clinical diagnostic kids for the detection of Brucella.更多还原