| 梁秉绍 杨丽媛 董慧 李飞 黄艳梅 陈吟霜 谢永强 钟华敏 周珍文.金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性[J].,2016,16(33):6436-6439 |
| 金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性 |
| Preparation of Polyclonal Antibody Against Lactate Dehydrogenase ofand Research of Stahylococcus aureus Its Cross Reactivity |
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| DOI： |
| 中文关键词: 金黄色葡萄球菌 乳酸脱氢酶 多克隆抗体 交叉反应性 |
| 英文关键词: Lactate dehydrogenase Polyclonal antibody Cross reactivity |
| 基金项目:广州市医药科技重点项目（201102A212013）；广东省科技厅项目（2014A020212013） |
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| 中文摘要: |
| 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pET28a-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IgG 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。 |
| 英文摘要: |
| Objective:To express and purify lactate dehydrogenase (LDH) of Stahylococcus aureus in prokaryotic system, produce
polyclonal antibody from immune mice and use them to analyze the cross reactivity of Stahylococcus aureus LDH with other
species.Methods:Recombinant pET28a-ldh/Bl21 was resuscitated, isopropyl thiogalactoside (IPTG) was used to induce recombinant
LDH protein expression, and the recombinant LDH was identified by western-blot probed with HRP conjugated anti his-tag mouse monoclonal
antibody. Balb/c mice were immunized with purified recombinant LDH protein and adjuvant, then the polyclonal antibodies were
obtained. Antibody titer was measured by ELISA assay. The immunoreactivity of recombinant protein and its cross reactivity with Stahylococcus aureus,Stahylococcus epidermis,Stahylococcus pneumoniae, Enterococcus faecalis, Escherichia coli were identified by western-blot assay.Results:A recombinant protein band which is about 39 kDa was indentified by SDS-PAGE and western-blot, and 2.8 mg pure protein was obtained after purification. Specific immune response was induced by purified protein, the specific IgG antibody titer was 1:50000. western-blot assay showed that serum from immunized mice could recognize the recombinant and natural LDH form Stahylococcus aureus , but could not identify other natural LDH form Stahylococcus pneumoniae, Enterococcus faecalis, Escherichia coli.Conclusion:The purified protein showed good immunogenicity and immunoreactivity, and high titer polyclonal antibody
could be obtained, western-blot assay by serum from immune mice showed no cross reaction with LDH from other species. This
study provided useful information for further diagnosis research against Stahylococcus aureus infection. |
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