马金柱，崔玉东,，张晶，朱战波，朴范泽

1 黑龙江八一农垦大学生命科技学院，大庆 163319

2 黑龙江八一农垦大学动物科技学院，大庆 163319

金黄色葡萄球菌isdb基因的克隆表达及其小鼠免疫试验

马金柱1，崔玉东1,2，张晶1，朱战波2，朴范泽2

1 黑龙江八一农垦大学生命科技学院，大庆 163319

2 黑龙江八一农垦大学动物科技学院，大庆 163319

为了研究金黄色葡萄球菌表面Isdb蛋白的免疫原性，应用PCR方法扩增出金黄色葡萄球菌Wood46株的isdb基因并进行序列分析，再将isdb 基因插入到pET32-a(+) 载体上，构建了pET32-a(+)-isdb重组质粒，将重组质粒转化到宿主菌大肠杆菌BL21中并诱导表达和纯化Isdb蛋白。用纯化的Isdb蛋白免疫小鼠，检测小鼠血清中抗体水平；在二次免疫之后的第2周，用金黄色葡萄球菌Wood46、HLJ23-1株对小鼠攻毒，每组8只小鼠。研究结果发现：isdb基因在不同菌株中高度保守；Isdb蛋白在宿主菌中获得成功表达；在免疫后血清抗体效价与对照组相比，均显著升高 (P＜0.05)；攻毒结果表明Isdb蛋白对Wood46和HLJ23-1两种菌株攻毒保护率分别为62.5%和75%。以上结果表明Isdb蛋白具有较好的免疫原性和免疫保护作用。

金黄色葡萄球菌，isdb基因克隆，Isdb蛋白表达，免疫原性

Abstract:In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface Isdb, we amplified isdb gene from S. aureus Wood46 strain. The isdb gene was subsequently inserted into pET32a(+) vector and the recombinant plasmid was transformed into E. coli strain BL21. The recombinant Isdb was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level was measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus strains Wood46 and HLJ23-1. These results showed that isdb gene sequences were highly conserved, and the recombinant Isdb was successfully expressed. The antibody titer in the immunized groups was increased significantly (P<0.05) compared with the control, the protective rate of Isdb protein inducted by challenge with the two S. aureus stains Wood46 and HLJ23-1 was 62.5% and 75%, respectively. These results showed that the Isdb protein had high immunogenicity and immunoprotective capacity.

Keywords:Staphylococcus aureus, isdb gene cloning, Isdb expression, immunogenicity

金黄色葡萄球菌 Staphylococcus aureus是一种重要的致病菌，可以引起人和动物的多种疾病，尤其它是引起奶牛乳房炎的主要病原菌，给奶牛业造成了巨大的经济损失[1-3]。目前，抗生素和金黄色葡萄球菌全菌苗、类毒素等传统疫苗是临床上预防和治疗奶牛金黄色葡萄球菌性乳房炎的主要方法，但是抗生素的耐药性、药物残留和传统疫苗免疫原性低等问题，一直以来都是亟待解决的重要难题[4-6]。……