摘要
克隆羊外膜蛋白Omp25基因,在大肠杆菌中表达、纯化,并对Omp25蛋白的抗原性进行分析。以布鲁氏菌染色体DNA模板,扩增Omp25基因,双酶切后克隆至pET32a上,在大肠杆菌ER2566(DE3)中诱导表达,组氨酸结合树脂柱纯化,Western blotting鉴定Omp25蛋白的免疫原性。将Omp25克隆至载体pET32a,提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中。将重组质粒转化于大肠杆菌ER2566(DE3)中表达获得HIS融合蛋白,SDS-PAGE分析证明,表达产物为43 000的融合蛋白。Western blotting分析表明,所表达的蛋白具有免疫原性。结果表明,成功地表达并纯化了Omp25蛋白,而且纯化的蛋白具有一定的免疫原性。本试验为进一步研究Omp25蛋白的功能以及寻找布鲁氏菌的诊断性蛋白奠定了基础。
Clone and express the Omp25 protein of B. melitensis in E. coli, purify the expressed protein and detect its immunogenicity. A gene recoding outer membrane protein 25 000 （Omp25） was amplified from the genomic DNA of B. melitensis by PCR. The amplified fragments were digested with BamH I and Sal I,and then clone to the vector pET32a. The constructed recombinant plasmid pET32a-omp25 was transformed to E. coli ER2566 （DE3） and was induced to express the fusion protein. Then the protein was purified by histidine-binding resin column chromatogra- phy and the immunogenicity was detected by Western blotting. The PCR product of Omp25 gene was cloned to the vector pET32a and the recombinant vector was confirmed by colony PCR identification,recombinant vector digested identification and sequencing analysis. It was successfully expressed in E. coli ER2566 （DE3） as a fusion protein with histidine at the presence of IPTG,and a specific protein band of 43 000 was found when identified by SDS-PAGE. Western blotting showed good immunoreactivity of the expressed product. The conclusion is that the Omp25 was successfully cloned and expressed, and the purified fusion protein had immunogenicity. This study provide a solid foundation for the further study on the function of the protein and brucellosis diagnostic antigens.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第6期733-736,共4页
Chinese Journal of Veterinary Science
基金
国家“863”资助项目(2007AA02Z412)
