摘要
【目的】制备金黄色葡萄球菌纤连蛋白结合蛋白A(FnbpA)基因工程亚单位疫苗,并以小白鼠为试验模型,检测其免疫保护效果。【方法】诱导表达FnbpA,经组氨酸标签亲合纯化后用Bradford法测定纯化的目的蛋白的含量,并检测其黏附活性及对动物的安全性。制备FnbpA亚单位疫苗,经无菌检验及安全检验后,进行免疫保护试验,用ELISA方法检测被免疫小鼠血清中的抗体效价,通过腹腔攻毒试验检测疫苗的免疫保护力。【结果】纯化的目的蛋白在80 ku处出现单一条带,蛋白质量浓度为0.245 mg/mL。细菌黏附抑制试验发现,经FnbpA蛋白预处理过的MDBK细胞黏附的金黄色葡萄球菌数较对照极显著减少;动物安全性试验显示,小鼠注射FnbpA亚单位疫苗后均健活,表明制备的FnbpA亚单位疫苗安全性良好。所有免疫组小鼠于首免后7 d即可在血清中检测到特异性抗体,抗体效价逐渐上升,在21 d达到最高值,之后逐渐降低。【结论】纯化的FnbpA蛋白达到了电泳级纯度,且依然保持良好的黏附活性;制备的FnbpA亚单位疫苗作用机体产生的抗体水平符合抗体消长规律,且表现出良好的免疫保护力。
【Objective】 Fibronectin A of Staphylococcus aureus,which was expressed in E.coli BL21（DE3） with recombinant pET32a＋-Fnbp plasmid,was purified with Gel filtration chromatography（GFC）,and then the engineered subunit vaccine was developed.The immunity effectiveness of this vaccine was evaluated on mouse models.【Method】 The purified fusion protein was analyzed in SDS-PAGE,and subjected to the evaluation of its adhering function on MDBK cell.Protein concentration was determined by the method of Bradford.Antibody titers were evaluated on ELISA,and then challenged to gain the immunity protect index.【Result】 There was an expected protein band with molecular mass of 80 ku in SDS-PAGE,and the concentration was 0.245 mg/mL.Specific antibodies were acquired in blood-serum from mice after vaccined and the antibody titers kept rising until it arrived at the max on 21 d,then went down.【Conclusion】 The purified fusion protein has good fineness and adhering activity,the antibody titers initiated by the protein vaccine go with regulation and the immunoprotection is satisfactory.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2011年第5期39-43,50,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
山东省中青年科学家基金项目(BS2009NY002)
山东省农业重大应用技术创新课题(2009)
现代农业产业技术体系(奶牛疾病)
