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[目的]检测布鲁氏菌BMEII0988基因的原核表达情况并对其免疫原性进行分析。[方法]以热灭活的布鲁氏菌16M标准株为模板,根据Gen Bank中公布的羊种布鲁氏菌16M的BMEII0988基因序列分别设计引物,用PCR方法扩增BMEII0988基因的编码序列,连接到T载体测序,将测序正确的基因序列克隆到原核表达载体p ET-32a上,转化入大肠杆菌DE3感受态细胞中诱导表达,将获得的目标蛋白用AKTAxpress智能多维纯化系统进行纯化,并用Western Blot分析其反应原性。[结果]BMEII0988基因长度为1 131 bp,编码377个氨基酸,SDS-PAGE表明BMEII 0988融合蛋白在大约66 k Da处出现条带,纯化后条带单一,BMEII 0988蛋白(NCBI标准序列号:WP_002966326.1)具有较好的反应原性。[结论]该研究为下一步建立相应蛋白标记的诊断方法和疫苗的研发奠定了基础。
[Objective] To detect the prokaryotic expression of Brucella BMEII0988 gene and analyze its immunogenicity. [Method] The heat-inactivated Brucella 16M standard strain was used as a template to design the primers according to the BMEII0988 gene sequence of Brucella 16M in GenBank. The coding sequence of BMEII0988 gene was amplified by PCR. Was ligated to T vector sequencing and the correct sequencing gene was cloned into prokaryotic expression vector p ET-32a and transformed into E. coli DE3 competent cells to induce expression. The obtained target protein was purified with AKTAxpress intelligent multi-dimensional purification system and used with Western Blot analysis of its reaction. [Result] The BMEII0988 gene was 1 131 bp in length and encoded a protein of 377 amino acids. SDS-PAGE showed that the band of BMEII 0988 fusion protein was about 66 kDa. The band of BMEII 0988 protein was single. The BMEII 0988 protein (NCBI standard serial number: WP_002966326 .1) has a good reaction. [Conclusion] This study lays the foundation for the next step to establish the diagnostic method of the corresponding protein markers and the development of the vaccine.