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浙江大学学报(医学版)  2013, Vol. 42 Issue (2): 164-170    DOI: 10.3785/j.issn.1008-9292.2013.02.006
专题报道     
钩端螺旋体groEL基因原核表达及其产物免疫保护作用
李小余1,王银环1,严杰2,程东庆1
1.浙江中医药大学生命科学学院,浙江 杭州 310053;
2.浙江大学医学院病原生物学系,浙江 杭州 310058;
Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters
LI Xiao-Yu1, WANG Yin-Huan1, YAN Jie2, CHENG Dong-Qing1
1.Department of Microbiology and Immunology,College of Life Science,Zhejiang Chinese Medical University,Hangzhou 310053,China; 2.Department of Microbiology and Parasitology,Zhejiang University School of Medicine,Hangzhou 310058,China
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摘要:

目的:构建问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株groEL基因原核重组表达系统,了解重组表达的GroEL蛋白(rGroEL)对金地鼠的免疫保护作用。
方法:采用高保真PCR扩增问号钩体赖株groEL基因并测序,常规方法构建groEL基因原核表达系统。采用SDS-PAGE联合Bio-Rad凝胶图像分析系统检查rGroEL表达情况及其可溶性,Ni-NTA亲和层析法提纯rGroEL。观察rGroEL免疫金地鼠对问号钩体赖株感染的保护率。采用MAT法检测免疫动物血清与我国流行问号钩体血清群交叉凝集效价。
结果:所克隆的groEL基因核苷酸和氨基酸序列与GenBank中相应基因完全相同。所构建的groEL基因原核表达系统能表达可溶性rGroEL。100和200 μg rGroEL对金地鼠的免疫保护率分别为50.0%和75.0%。rGroEL免疫金地鼠血清可不同程度地凝集各问号钩体血清群。
结论:GroEL蛋白是问号钩体属特异性保护性抗原,可用于制备通用性钩体基因工程疫苗。

关键词: 问号钩端螺旋体GroEL蛋白质/遗传学重组遗传基因表达序列分析DNA免疫法被动groEL基因原核表达免疫保护    
Abstract:

Objective: To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai,and to determine the immunoprotective effect of recombinant GroEL protein(rGroEL) in LVG hamsters.
Methods: The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced.A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique.SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL.The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai.The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.
Results: The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank.The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL.The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0% and 75.0%,respectively.The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.
Conclusion: The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Key words: Leptospira interrogans    GroEL protein/genetics    Recombination,genetic    Gene expression    Sequence analysis,DNA    Immunization,passive    groEL gene    Prokaryotic expression    Immunoprotection
收稿日期: 2012-12-28 出版日期: 2013-03-25
:  R 377.5  
基金资助:

传染病国家科技重大专项资助项目(2008ZX10004-015).

通讯作者: 程东庆(1971-),女,副教授,主要从事微生物与免疫学方面的研究     E-mail: B2006069@163.com
作者简介: 李小余(1987-),女,硕士研究生,主要从事微生物与免疫学方面的研究.
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引用本文:

李小余, 王银环, 严杰, 程东庆. 钩端螺旋体groEL基因原核表达及其产物免疫保护作用[J]. 浙江大学学报(医学版), 2013, 42(2): 164-170.

LI Xiao-Yu, WANG Yin-Huan, YAN Jie, CHENG Dong-Qing. Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters. Journal of ZheJiang University(Medical Science), 2013, 42(2): 164-170.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2013.02.006        http://www.zjujournals.com/med/CN/Y2013/V42/I2/164


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