重组外膜脂蛋白LipL32与致病性钩端螺旋体抗体的免疫反应性及与不同血清群交叉反应性的Western blotting评价  

A Western-blot assay for the detection of antibodies against pathogenic Leptospira serogroups with recombinant outer membrane protein LipL32

作　　者：段鸿元[1] 刘志国[2] 邱少富[1] 何斌[1] 赵海[1] 宋利华[1] 朱虹[1] 端青[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [2]解放军总医院第一附属医院门诊部,北京100037

出　　处：《解放军医学杂志》2011年第3期241-243,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家“十一五”重大传染病防治专项课题(2008ZX10401)

摘　　要：目的研究钩端螺旋体外膜脂蛋白LipL32与致病性钩体抗体的免疫反应性以及与不同钩体血清群之间的交叉反应性,为钩体病的血清学检测提供抗原。方法依据黄疸出血群赖型56601株外膜脂蛋白LipL32基因序列设计引物,扩增出该基因片段DNA,将其克隆至表达载体pET-28a中。将构建好的重组质粒转化大肠埃希菌BL21并诱导表达,表达的重组蛋白纯化后采用Western blotting评价其与56601株及其他14个血清群致病性钩端螺旋体抗体的免疫反应性和交叉反应能力。结果扩增获得了56601株脂蛋白LipL32基因,成功构建了原核表达重组质粒pET28a-LipL32。重组蛋白rLipL32为包涵体表达,且与56601株及另外14个血清群的致病性钩端螺旋体的兔抗体发生了特异性免疫反应。结论原核表达重组外膜脂蛋白rLipL32在致病性钩体血清群之间有良好的交叉反应能力,可初步作为属范围内的钩端螺旋体抗体检测候选抗原。Objective To provide a possible antigen for rapid serodiagnosis of leptospirosis,the present study focused on the activity of immune-reaction and cross-reaction between outer membrane protein LipL32 and multi-serogroup anti-pathogenic Leptospira antibodies.Methods Based on the given sequence of LipL32 gene of Leptospira icterohaemorrhagiae strain 56601,the primer pair was designed and the DNA fragment was amplified by PCR.The amplified product was inserted into vector pET-28a-（c） to construct a recombinant plasmid.With E.coli BL21（DE3）,the recombinant protein was induced for expression.After purification,the Western-blot assay was applied as a method to evaluate immune-reaction between rLipL32 and pathogenic Leptospira antibodies of 15 serogroups including Leptospira icterohaemorrhagiae strain 56601.Results As the result,the recombinant protein rLipL32 coded by plasmid pET28a-LipL32 was expressed as an inclusion-body.The immune-reaction with Western blotting test truly occurred between rLipL32 and antibodies against 15 serogroups of Leptospira.Conclusions In conclusion,the expressed outer membrane protein rLipL32 do show cross-reaction among members of 15 serogroups of pathogenic Leptospira.It is possible to choose rLipL32 as the antigen for antibody detection in serodiagnosis of pathogenic Leptospira within genus.

关 键 词：钩端螺旋体 问号 黄疸出血型 抗体 交叉反应 

分 类 号：R539[医药卫生—内科学]