布氏杆菌表面特异性多表位抗原的串联表达与免疫磁珠的初步建立

目的为获得布氏杆菌表面多表位抗原的特异性抗体,用于布氏杆菌免疫磁珠的构建。方法 从布氏杆菌3个表面蛋白中分析到特异性抗原表位,人工合成其串联基因片段并插入原核表达载体,转化至E. coli BL21(DE3),获得重组质粒pET-32a(+)-CJMEA-A,IPTG诱导后表达出30 kDa的重组蛋白,亲和层析纯化后将其进行Western blot分析、间接ELISA鉴定和兔免疫试验,制备的抗血清经亲和层析纯化后,包被成免疫磁珠并用于布氏杆菌富集。结果 表达的重组蛋白能与牛抗布氏杆菌血清反应,其抗血清与布氏杆菌发生良好反应,纯化抗体包被的免疫磁珠能特异性吸附布氏杆菌。结论 获得的布氏杆菌特异性抗体和免疫磁珠具有良好的应用前景。

Design and expression of specific multi-epitope antigen of Brucella surface proteins and initial establishment of immunomagnetic beads method

In this study, we aim to obtain the specific antibody of multi-epitope antigen from Brucella surface proteins, and establish immunomagnetic beads method. Some unique antigenic epitopes were obtained from three surface proteins of Bru- cella, these epitopes were linked in series and its gene was synthesized. The recombinant plasmid was transformed into compe- tent E. coli BL21 （DE3） after the synthetic gene was inserted into pET-32a （＋） vector. The recombinant protein named BMEA-A was induced to express by IPTG, purified by affinity chromatography, and detected by Western blot, indirect EI.ISA and immunization of rabbits. After that, the anti-BMEA-A antibody was purified from the immune rabbit serum by affinity chromatography and was coated to magnetic beads. Then the immunomagnetic beads were preliminary applied in enriching Bru- celia. Results showed that the recombinant protein containing multi-epitope peptide was expressed in E. coli. It could react with the bovine anti-Brucella serum by Western blot and indirect ELISA and its antiserum had good reactogenicity to Brucella. The immunomagnetic beads tagged with the purified anti-BMEA-A antibody could selectively capture Brucella in enrichment culture and its sensitivity was higher than conventional bacteria isolation. The result of this research provides a highly sensitive method of enriching Brucella that will be valuable for clinical applications.