嗜肺军团菌mip/flaA二联优势抗原表位基因融合表达载体的构建及表达
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摘要
目的选取嗜肺军团菌mip和flaA优势抗原表位基因,构建mip/flaA二联优势抗原表位基因融合表达载体,并在原核系统中表达,为后续制备嗜肺军团菌蛋白疫苗提供初步的实验基础。方法运用生物信息学方法对Mip和FlaA蛋白的二级结构和表面特性如理化性质、亲水性、可塑性、抗原指数以及胞外区等方面进行分析,选择其活性表位可能存在的区域为优势抗原表位区。通过PCR扩增和T4连接酶构建pET-mip、pET-flaA和pET-mip/flaA优势抗原表位基因融合表达载体,并诱导其在大肠杆菌中表达。结果 Mip和FlaA都存在多个潜在的抗原表位位点,选取其优势抗原表位区域进行克隆和表达获得成功,并成功表达了mip/flaA二联优势抗原表位融合蛋白。结论 DNA Star软件和Expasy在线蛋白分析系统能够成功预测嗜肺军团菌Mip和FlaA抗原的表位;选取其优势抗原表位成功构建了pET-mip/flaA二联原核表达载体,并高效表达。
Objective To generate and express fusion vector with mip/flaA advantages epitope genes of Legionella pneumophila by select mip and flaA advantages epitope genes for future research on Legionella pneumophila protein vaccine.Methods Following analysis of secondary structure and surface properties such as:physical and chemical properties,hydropathy,plasticity,antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods,the region which active epitope may exist was selected as advantages epitope region.Then,the recombinant plasmid pET-mip,pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification and T4 ligase connection,and induced the expression in E.coli.ResultsMany potential antigenic epitopes in Mip and FlaA were identified,and the selected advantages epitope regions were cloned and expressed successfully.Moreover,the mip/flaAtwo advantages associated epitope fusion proteins were also successfully expressed.Conclusion DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes for Legionella pneumophila Mip and FlaA.And prokaryotic expression vector pET-mip/flaA with advantages epitope genes has been successfully constructed and efficiently expressed.
Objective To generate and express fusion vector with mip/flaA advantages epitope genes of Legionella pneumophila by select mip and flaA advantages epitope genes for future research on Legionella pneumophila protein vaccine.Methods Following analysis of secondary structure and surface properties such as:physical and chemical properties,hydropathy,plasticity,antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods,the region which active epitope may exist was selected as advantages epitope region.Then,the recombinant plasmid pET-mip,pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification and T4 ligase connection,and induced the expression in E.coli.ResultsMany potential antigenic epitopes in Mip and FlaA were identified,and the selected advantages epitope regions were cloned and expressed successfully.Moreover,the mip/flaAtwo advantages associated epitope fusion proteins were also successfully expressed.Conclusion DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes for Legionella pneumophila Mip and FlaA.And prokaryotic expression vector pET-mip/flaA with advantages epitope genes has been successfully constructed and efficiently expressed.
引文
1 Newton HJ,Ang DK,van Driel IR,et al.Molecular pathogenesis of infections caused by Legionella pneumophila.Clin Microbiol Rev,2010;23(2):274-298.
2 Gomez-Valero L,Rusniok C,Rolando M,et al.Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires’disease.Genome Biol,2014;15(11):505.
3 Ditommaso S,Giacomuzzi M,Rivera SR,et al.Virulence of Legionella pneumophila strains isolated from hospital water system and healthcare-associated Legionnaires’disease in Northern Italy between 2004and 2009.BMC Infect Dis,2014;14 :483.doi:10.1186/1471-2334-14-483.
4 Jiang G,Charoenvit Y,Moreno A,et al.Induction of multiantigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys,in the absence of antigen interference,with heterologous DNA prime/poxvirus boost immunization.Malar J,2007;6:135.
5 Heppner DG Jr,Kester KE,Ockenhouse CF,et al.Towards an RTS,S-based,multi-stage,multi-antigen vaccine against falciparum malaria:progress at the Walter Reed Army Institute of Research.Vaccine,2005;23(17-18):2243-2250.
6 兰 希,赵 慧,丁剑冰等.寄生虫病疫苗研究新进展.中国病原生物学杂志,2014;9(11):3-5.
7 Sharon J,Rynkiewicz MJ,Lu Z,et al.Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.Immunology,2014;142(1):1-23.
8 Xu Y,Guan W,Xu JN,et al.Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.Vaccine,2011;29(23):4051-4057.
9 Burton DR,Poignard P,Stanfield RL,et al.Broadly neutralizing antibodies suggest new prospects to counter highly antigenically diverse viruses.Science,2012;337(6091):183-186.
10 Delfani S,Imani Fooladi AA,Mobarez AM,et al.In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus.Clin Exp Vaccine Res,2015;4(1):99-106.
11 Ceymann A,Horstmann M,Ehses P,et al.Solution structure of the Legionella pneumophila Mip-rapamycin complex.BMC Struct Biol,2008;8:17.doi:10.1186/1472-6807-8-17.
12 Khler R,Fanghnel J,Knig B,et al.Biochemical and functional analyses of the Mip protein:influence of the Nterminal half and of peptidylprolyl isomerase activity on the virulence of Legionella pneumophila.Infect Immun,2003;71(8):4389-4397.
13 Ricci ML,Torosantucci A,Scaturro M,et al.Induction of protective immunity by Legionella pneumophilaflagellum in an A/J mouse model.Vaccine,2005;23(40):4811-4820.
14 Pereira MS,Morgantetti GF,Massis LM,et al.Activation of NLRC4by flagellated bacteria triggers caspase-1-dependent and-independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.J Immunol,2011;187(12):6447-6455.
15 Xu JN,Yang ZW,Chen JP,et al.Protective immunity against Legionnaires’disease in an A/J mouse model using a DNA vaccine composed of an outer membrane protein(29kDa)and the PilE fusion protein.Diagn Microbiol Infect Dis,2012;73(1):9-15.
2 Gomez-Valero L,Rusniok C,Rolando M,et al.Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires’disease.Genome Biol,2014;15(11):505.
3 Ditommaso S,Giacomuzzi M,Rivera SR,et al.Virulence of Legionella pneumophila strains isolated from hospital water system and healthcare-associated Legionnaires’disease in Northern Italy between 2004and 2009.BMC Infect Dis,2014;14 :483.doi:10.1186/1471-2334-14-483.
4 Jiang G,Charoenvit Y,Moreno A,et al.Induction of multiantigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys,in the absence of antigen interference,with heterologous DNA prime/poxvirus boost immunization.Malar J,2007;6:135.
5 Heppner DG Jr,Kester KE,Ockenhouse CF,et al.Towards an RTS,S-based,multi-stage,multi-antigen vaccine against falciparum malaria:progress at the Walter Reed Army Institute of Research.Vaccine,2005;23(17-18):2243-2250.
6 兰 希,赵 慧,丁剑冰等.寄生虫病疫苗研究新进展.中国病原生物学杂志,2014;9(11):3-5.
7 Sharon J,Rynkiewicz MJ,Lu Z,et al.Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.Immunology,2014;142(1):1-23.
8 Xu Y,Guan W,Xu JN,et al.Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.Vaccine,2011;29(23):4051-4057.
9 Burton DR,Poignard P,Stanfield RL,et al.Broadly neutralizing antibodies suggest new prospects to counter highly antigenically diverse viruses.Science,2012;337(6091):183-186.
10 Delfani S,Imani Fooladi AA,Mobarez AM,et al.In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus.Clin Exp Vaccine Res,2015;4(1):99-106.
11 Ceymann A,Horstmann M,Ehses P,et al.Solution structure of the Legionella pneumophila Mip-rapamycin complex.BMC Struct Biol,2008;8:17.doi:10.1186/1472-6807-8-17.
12 Khler R,Fanghnel J,Knig B,et al.Biochemical and functional analyses of the Mip protein:influence of the Nterminal half and of peptidylprolyl isomerase activity on the virulence of Legionella pneumophila.Infect Immun,2003;71(8):4389-4397.
13 Ricci ML,Torosantucci A,Scaturro M,et al.Induction of protective immunity by Legionella pneumophilaflagellum in an A/J mouse model.Vaccine,2005;23(40):4811-4820.
14 Pereira MS,Morgantetti GF,Massis LM,et al.Activation of NLRC4by flagellated bacteria triggers caspase-1-dependent and-independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.J Immunol,2011;187(12):6447-6455.
15 Xu JN,Yang ZW,Chen JP,et al.Protective immunity against Legionnaires’disease in an A/J mouse model using a DNA vaccine composed of an outer membrane protein(29kDa)and the PilE fusion protein.Diagn Microbiol Infect Dis,2012;73(1):9-15.