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目的:在原核载体中表达、纯化金黄色葡萄球菌乳酸脱氢酶,免疫小鼠获得多克隆抗体,使用多克隆抗体分析与其它菌种的交叉反应性。方法:复苏p ET28a-ldh/Bl21重组菌,IPTG诱导重组融合蛋白表达、以抗HIS标签的单克隆抗体进行western-blot鉴定重组蛋白。使用纯化的重组蛋白以及相应的佐剂免疫小鼠,利用ELISA测定血清抗体效价,并使用小鼠抗血清进行免疫印迹法鉴定重组蛋白的反应原性及其与金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌的交叉反应性。结果:SDS-PAGE电泳在39 KDa处可见目的条带,免疫印迹法验证了重组LDH的表达,纯化后获得2.8 mg重组蛋白。纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性Ig G效价为1:50000,western-blot鉴定显示所制备的多克隆抗血清能分别识别金黄色葡萄球菌重组及天然乳酸脱氢酶,但不识别表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌中天然乳酸脱氢酶。结论:纯化的LDH具有良好的免疫活性,免疫小鼠获得高滴度的多克隆抗体。使用多克隆抗体western-blot显示与其它菌种LDH不存在交叉反应性,为后续使用该重组蛋白进行金黄色葡萄球菌感染的诊断研究奠定基础。
OBJECTIVE: To express and purify S.aureus lactate dehydrogenase in prokaryotic vector and to obtain polyclonal antibody in immunized mice. The polyclonal antibody was used to analyze the cross-reactivity with other strains. Methods: Recombinant p ET28a-ldh / Bl21 cells were resuscitated and induced by IPTG. The recombinant protein was identified by western-blot with anti-HIS monoclonal antibody. Mice were immunized with the purified recombinant protein and the corresponding adjuvant, the serum antibody titers were measured by ELISA, and the immunogenicity of the recombinant protein was tested by using mouse antiserum and its interaction with Staphylococcus aureus, Staphylococcus epidermidis , Streptococcus pneumoniae, Enterococcus faecalis, Escherichia coli cross-reactivity. Results: The target band was observed at 39 KDa by SDS-PAGE electrophoresis. The expression of recombinant LDH was verified by immunoblotting. After purification, 2.8 mg recombinant protein was obtained. Western blot analysis showed that the prepared polyclonal antiserum could recognize Staphylococcus aureus recombinant and natural lactic acid, respectively Dehydrogenase, but does not recognize Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Escherichia coli natural lactate dehydrogenase. Conclusion: The purified LDH has good immunocompetence and the immunized mice get high titers of polyclonal antibodies. Using polyclonal antibody western-blot showed no cross-reactivity with other strains of LDH, which laid the foundation for the subsequent diagnosis of Staphylococcus aureus infection using this recombinant protein.