摘要
【目的】克隆和表达奶牛乳腺炎金黄色葡萄球菌纤连蛋白连接蛋白A(Fnbp A)的功能基因。【方法】采集奶牛急性乳腺炎乳样,分离金黄色葡萄球菌,提取其DNA作为模板,利用设计合成的特异性引物,对金黄色葡萄球菌进行FnbpA功能基因的PCR扩增。回收目的基因并连接到T载体,鉴定后进行测序,然后将FnbpA基因连接到pET-32a(+)质粒中,经鉴定后转化大肠杆菌BL21感受态细胞,IPTG诱导后采用SDS-PAGE分析蛋白质的表达情况。【结果】PCR产物经电泳成像,仅金黄色葡萄球菌在1.7 kb处出现特异性条带,测序发现其与GenBank公布的FnbpA序列的同源性为98%;蛋白诱导表达SDS-PAGE分析发现,在80 ku处出现特异性条带。【结论】试验成功地克隆到FnbpA基因,并在大肠杆菌中获得高效表达,蛋白量为33.4%,为进一步研究金黄色葡萄球菌粘附素基因工程疫苗奠定了基础。
[Objective] To clone and express the gene of fibronectin-binding protein （Fnbp A）, the Staphylococcus aureu were isolated from the milk samples collected from dairy cows with peracute clinic mastitis. [Method] The DNA of the Staphylococcus aureus was extracted with classical technique to be template. The Fnbp A gene was amplified by PCR and cloned into plasmid of pMD-18-T. The recombinant plasmid was transferred into competent E. coli. After the recombinants were screened and identified by res- trition analysis,the cloned gene was sequenced. The Fnbp A gene was cloned into pET-32a（＋）, with the inducement of IPTG,it is expressed at high levels in E. coli. BL21. [Result] Result showed that a specific protein band was found in SDS-PAGE with the molecular weight of approximately 80 ku, which accorded with the anticipated result. [Conclusion] The turnout of the protein was about 33.4% of the total protein of the bacterium analyzed with the BandScan 5.0. The successful expression of Fnbp A in E. coli BL21 constituted a solid foundation for Staphylococcal vaccine development.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2008年第1期54-58,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
教育部高等学校科技创新工程重大项目(706039)
山东省“三0”工程项目(2003-3009)
山东省农科院高技术自主创新基金项目(2006YCX027)
山东省农科院重大成果培育基金项目(2006YCG012)
