钩端螺旋体主要属特异性蛋白抗原分布及其免疫性研究

孙百莉; 罗冬娇; 孙军德; 严杰

Abstract

孙百莉,罗冬娇,孙军德,严杰.钩端螺旋体主要属特异性蛋白抗原分布及其免疫性研究[J].Chinese journal of Epidemiology,2006,27(12):1073-1077

钩端螺旋体主要属特异性蛋白抗原分布及其免疫性研究

Study on the immnnogenicity of major leptospiral geuas-specific protein antigens and the distribution of antigens in different serogroups of Leptospira interrogans

Received:April 27, 2006

DOI：

KeyWord: 问号钩端螺旋体外膜蛋白属特异性抗原交叉凝集免疫性鉴定

English Key Word: Leptospira interrogans Outer envelope proteins Genus-specific antigen Cross agglutination Immune identification

FundProject:国家自然科学基金资助项目(39970678)；浙江省教育厅科研基金资助项目(20050716)

Author Name	Affiliation	E-mail
Sun Bai-li	College of Land and Enviromenty Shenyang Agriculture University, Shenyang 110161
Luo Dong-jiao	杭州师范学院基础医学部
Sun Jun-de	College of Land and Enviromenty Shenyang Agriculture University, Shenyang 110161
Yan Jie	浙江大学医学院	Med_bp@zju.edu.cn

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Abstract:

目的确定致病性问号钩端螺旋体(钩体)属特异性外膜蛋白的抗原性和交叉免疫反应性，为研制通用性钩体基因工程疫苗及检测试剂盒提供依据。方法采用Ni-NTA亲和层析法，提取了4种钩体外膜蛋白主要基因型重组表达产物rLipL21、rLipL32／1和rLipL32／2、rLipL41／1和rLipL41／2、rOmpL1／1和rOmpL1／2，并用SDS-PAGE检测表达及提纯效果。上述重组蛋白常规皮下免疫家兔获得抗血清，显微镜凝集试验(MAT)检测各抗血清交叉凝集效价。采用盐变法提取了我国15群问号钩体参考标准株及非致病的双曲钩体PatocⅠ株的外膜蛋白。以兔抗血清为一抗，采用Western blot检测上述4种外膜蛋白自然表达情况及其免疫反应性。结果rLipL21、rLipL32／1和rLipL32／2、rLipL41／1和r LipL41／2、rOmpL1／1和rOmpL1／2表达量分别约占细菌总蛋白的10％、40％和35％、15％和10％、30％和15％，各重组蛋白SDS-PAGE提纯后均仅见单一的蛋白条带。重组蛋白兔抗血清在同一基因不同基因型表达产物之间有广泛的交叉免疫反应性，与不同钩体血清群的MAT效价为1:2-1:128。各血清群钩体外膜中均可检出上述4种外膜蛋白，但LipL21仅存在于问号钩体中。结论LipL21、LipL32、LipL41、OmpL，1均为钩体属特异性表面抗原，可作为通用性钩体疫苗及检测试剂盒的候选抗原。

English Abstract:

Objective The determination of antigenicity and immunogenicity of Leptospira interrogans genus-specific outer envelope proteins(OEPs) will offer evidence for developing universal leptospiral genetic engineering vaccine and detection kit.Methods In this study, Ni-NTA affinity chromatography is used to purify the recombinant products rLipL21, rOmpLl/1, rOmpLl/2, rLipL32/1, rLipL32/2, rLipL41/1 and rLipL41/2 expressed by the major genotypes of four leptospiral OEPs of 15 serogroups.SDS-PAGE is applied to examine the expression and purity of the recombinant proteins.Rabbits are intracutaneously immunized with the recombinant proteins to obtain antisera.Microscope agglutination test ( MAT) is used to measure the cross inmmunoagglutination titers of antisera.The OMPs of the reference standard strains belonging to 15 serogroups of L.ittterrogarts in China and L.6i f Zexastrain Patoc I are prepared using salt-denature method.By each of the antisera as the first antibody, Western blot assay is established to detect the natural expressions and immunoreactivity of the four OEPs.Results The outputs of rLipL21, rLipL32/1, rLipL32/2, rLipL41/1, rLipL41/2, rOmpLl/1 and rOmpLl/2 are 10%, 40%, 35%, 15%，10%，30% and 15 %, respectively.Each the purified recombinant proteins shows a single ragment after SDSPAGE.Each the rabbit antisera displays extensive cross immunoreactivity between the products expressed by different genotypes of the same gene and the MAT titers ranging from 1:2-1:128.All the four OEPs can be detectable in the OEPs preparations. However, LipL21 is found to exist only in L.i n terrroga its.Conclusion The results of this study indicate that all the four OEPs are superficial genus-specific antigens of Leptospira which can be used as the candidate antigens of leptospiral universal vaccine and detection kit.

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