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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (2): 164-170    DOI: 10.3785/j.issn.1008-9292.2013.02.006
    
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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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 wordsLeptospira interrogans      GroEL protein/genetics      Recombination,genetic      Gene expression      Sequence analysis,DNA      Immunization,passive      groEL gene      Prokaryotic expression      Immunoprotection     
Received: 28 December 2012      Published: 25 March 2013
CLC:  R 377.5  
Cite this article:

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.

URL:

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


钩端螺旋体groEL基因原核表达及其产物免疫保护作用

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 

[1]BHARTI A R,NALLY J E,RICALDI J N,et al.Leptospirosis: a zoonotic disease of global importance
[J].Lancet Infect Dis,2003,3(12):757-771.

[2]ZHANG C L,WANG H,YAN J.Leptospirosis prevalence in Chinese populations in the last two decades
[J].Microbes Infect,2012,14(4):317-323.

[3]YAN Jie,DAI Baoming,YU Enshu(严杰,戴保民,于恩庶).Leptospirology(钩端螺旋体病学)
[M].Third Edition,Beijing: The People′s Healthy Publication House,China,2006.(in Chinese)

[4]SUN Baili,GUO Zongqi,LUO Dongjiao(孙百莉,郭宗琪,罗冬娇,等),et al.Membrane location and naturally antibody responses of genus-specific antigen OmpLls of Leptospira interrogans 
[J].Acta Microbiol Sin(微生物学报),2007,47(2):329-334.(in Chinese)

[5]LUO Dongjiao,YAN Jie,MAO Yafei(罗冬娇,严杰,毛亚飞,等),et al.Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene
[J].Journal of Zhengjiang University: Medical Sciences(浙江大学学报:医学版),2005,34(1): 27-32.(in Chinese)

[6]YAN Jie(严杰).Progress of Leptospira pathogenic mechanisms and new vaccines and bacterial drug resistance
[J].Journal of Zhengjiang University: Medical Sciences(浙江大学学报:医学版), 2008,37(6):537-543.(in Chinese)

[7]SONRIER C,BRANGER C,MICHEL V,et al.Evidence of cross-protection with in Leptospira interrogans in an experimental model
[J].Vaccine,2000,19(1):86-94.

[8]HAAKE D A,CHAO G,ZUERNER R L,et al.The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection
[J].Infect Immun,2000,68(4):2276-2285.

[9]JULIO C,CLAUDIO P F,ELSIO A W J,et al.Targeted mutagenesis in pathogenic Leptospira species: Disruption of the LigB gene does not affect virulence in animal models of leptospirosis
[J].Infect Immun,2008,76(12):5826-5833.

[10]PICARDEAU M,BULACH D M,BOUCHIER C,et al.The OmpA-like protein Loa22 is essential for leptospiral virulence
[J].PLoS Pathog,2007,3(7):0894-0903.

[11]HAAKE D A,CHAMPION C I,MARFINCH C,et al.Molecular cloning and sequence analysis of the gene encoding OmpL1,a transmembrane outer membrane protein of pathogenic Leptospira spp
[J].J Bateriol,1993,175(13):4225-4234.

[12]LUO D J,XUE F,OJCIUS D M,et al.Protein typing of major outer membrane lipoproteins from Chinese pathogenic Leptospira spp.and characterization of their immunogenicity
[J].Vaccine,2010,28(1):243-255.

[13]DONG H Y,HU Y,XUE F, et al.Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein
[J].BMC Microbiol,2008,8:223.

[14]KHAN M,SHUKLA D,BANSAL A,et al.Immunogenicity and protective efficacy of GroEL(hsp60) of Streptococcus pneumoniae against lethal infection in mice
[J].FEMS Immunol Med Microbiol,2009,56(1):56-62.

[15]NOAH C E,MALIK M,BUBLITZ D A C,et al.GroEL and lipopolysaccharide from Francisella tularensis live vaccine strain synergistically activate human macrophages
[J].Infect Immun,2010,78(4):1797-1806.

[16]LIN J S,MARK A W.Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL
[J].J Biol Chem,2011,286(22):19459-19469.

[17]REN S X,FU G,JIANG X G,et al.Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing
[J].Nature,2003,422(6934):888-893.

[18]NATARAJASEENIVASAN K,ARTIUSHIN S C,VELINENI S,et al.Surface-associated Hsp60 chaperonin of Leptospira interrogans serovar Autumnalis N2 strain as an immunoreactive protein
[J].Eur J Clin Microbiol Infect Dis,2011,30(11):1383-1389.

[19]LO M,CORDWELL S J,BULACH D M,et al.Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature
[J].PLoS Negl Trop Dis,2009,3(12):e560.

[20]SAMBROOK J,FRITSCH EF,MANIATIS T.Molecular Cloning: A Laboratory Manual
[M].2nd edition,New York: Cold Sping Harbor Labooratory Press,1989,1.21-1.52,2.60-2.80,7.3-7.35,9.14-9.22.

[21]XU Lihui,YAN Jie,RUAN Ping(徐丽慧,严杰,阮萍,等),et al.Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans
[J].Journal of Zhengjiang University: Medical Sciences(浙江大学学报:医学版), 2005,34(1):9-14.(in Chinese)

[22]LEWIS S M,OSEI-BIMPONG A.Haemoglobinometry in general practice
[J].Clin Lab Haematol,2003,25(6):343-346.

[23]SCHREIER S,TRIAMPO W,DOUNGCHAWEE G,et al.Leptospirosis research: fast,easy and reliable enumeration of mobile leptospires
[J].Biol Res,2009,42(1):5-12.

[24]CULLEN P A,XU X,MATSUNAGA J,et al.Surfaceome of Leptospira spp
[J].Infect Immun,2005,73(8):4853-4863.

[25]PARK S H,AHN B Y,KIM M J.Expression and immunologic characterization of recombinant heat shock protein 58 of Leptospira species: a major target antigen of the humoral immune response
[J].DNA Cell Biol,1999,18(12):903-910.

[26]SUN Pingping,LIN Xv′ai,LI Liwei(孙萍萍,林旭瑷,李立伟,等),et al.Diversity of outer membrane protein expression of Leptospira interrogans during infection of human THP-1 monocytes
[J].Chinese Journal of Microbiology and Immunology(中华微生物学与免疫学杂志),2012,32(3):224-231.(in Chinese)

[27]XUE F,DONG H Y,WU J Y,et al.Transcriptional responses of Leptospira interrogans to host innate immunity: Significant changes in metabolism,oxygen tolerance,and outer membrane
[J].PLoS Negl Trop Dis,2010,4(10):e857.

[28]ATZINGEN M V,VIEIRA M L,OLIVEIRA R,et al.Evaluation of immunoprotective activity of six leptospiral proteins in the hamster model of leptospirosis
[J].Open Microbiol J,2012,6(1):79-87. 
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