口蹄疫病毒A-O型多表位二价融合抗原的构建及其在小鼠模型中的免疫学功能
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摘要
为了研制广谱高效的新型口蹄疫病毒(FMDV) A-O型重组多表位二价疫苗,以结核分支杆菌热休克蛋白70(mHSP70)的C端肽结合区作为多表位免疫原的载体蛋白,将A型和O型FMDV代表性毒株VP1上的B细胞表位以及非结构蛋白3A上的T细胞表位基因进行串联合成后与mHSP70的肽结合区编码基因相连接,构建重组质粒pAO-HSP,并转化至大肠杆菌BL21(DE3)中进行表达。重组蛋白经过纯化和鉴定后免疫BALB/c小鼠,检测相应的体液免疫应答和细胞免疫应答。结果显示:重组蛋白pAO-HSP在BL21(DE3)中能够以可溶性形式表达,纯化后经Western blot检测能够与感染了A型和O型FMDV猪的阳性血清发生特异性反应,并能够刺激小鼠产生抗A-O型FMDV特异性抗体。免疫后小鼠的脾淋巴细胞在A型、O型灭活FMDV刺激后,均出现显著的增殖现象,同时可以产生相关的Th1或Th2型细胞因子。本研究成功构建重组抗原pAO-HSP,并在小鼠模型中初步显示出了良好的免疫效果,为新型FMDV A-O型多表位二价疫苗的研发奠定基础。
In order to develop a novel type A-O bivalent multiepitope vaccine for foot-and-mouth disease(FMD),the C-terminal peptide binding region of heat shock protein 70 of Mycobacterium tuberculosis(mHSP70) was selected as the carrier protein of epitope-based immunogen.The genes of B cell epitopes on VP1 of type A and O FMDV and a helper T cell epitope on nonstructural protein 3A were synthesized in series and coupled with the encoding gene of the peptide binding region of mHSP70 to constructed recombinant plasmid p AO-HSP.The recombinant protein p AO-HSP was expressed in BL21(DE3) and purified by affinity chromatography and subcutaneously immunized BALB/c mice to detect corresponding humoral immune response and cellular immune response.The results show that the recombinant protein p AO-HSP can be expressed in soluble form in BL21(DE3).Western-blot analysis showed that the purified recombinant protein can react specifically with the positive serum from cattle infected with type A and O FMDV.After immunizing mice,the protein p AO-HSP can stimulate the production of specific antibodies against type A and O FMDV in mice.The spleen lymphocytes of immunized mice can proliferate significantly after stimulation of the inactivated type A or O FMDV and efficiently produce Th1 or Th2 type cytokines.In this study,the recombinant antigen p AO-HSP was successfully constructed and has shown a good immune effect in mouse model,which provide basis for the development of a new type A/O FMDV bivalent epitope-based vaccine.
In order to develop a novel type A-O bivalent multiepitope vaccine for foot-and-mouth disease(FMD),the C-terminal peptide binding region of heat shock protein 70 of Mycobacterium tuberculosis(mHSP70) was selected as the carrier protein of epitope-based immunogen.The genes of B cell epitopes on VP1 of type A and O FMDV and a helper T cell epitope on nonstructural protein 3A were synthesized in series and coupled with the encoding gene of the peptide binding region of mHSP70 to constructed recombinant plasmid p AO-HSP.The recombinant protein p AO-HSP was expressed in BL21(DE3) and purified by affinity chromatography and subcutaneously immunized BALB/c mice to detect corresponding humoral immune response and cellular immune response.The results show that the recombinant protein p AO-HSP can be expressed in soluble form in BL21(DE3).Western-blot analysis showed that the purified recombinant protein can react specifically with the positive serum from cattle infected with type A and O FMDV.After immunizing mice,the protein p AO-HSP can stimulate the production of specific antibodies against type A and O FMDV in mice.The spleen lymphocytes of immunized mice can proliferate significantly after stimulation of the inactivated type A or O FMDV and efficiently produce Th1 or Th2 type cytokines.In this study,the recombinant antigen p AO-HSP was successfully constructed and has shown a good immune effect in mouse model,which provide basis for the development of a new type A/O FMDV bivalent epitope-based vaccine.
引文
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[22] CAO Y,LU Z,LI D,et al.Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I∶C)[J].Veterinary Microbiology,2014,168:294-301.
[23] CAO Y,LI D,FU Y,et al.Rational design and efficacy of a multi-epitope recombinant protein vaccine against foot-and-mouth disease virus serotype A in pigs[J].Antiviral Research,2017,140:133-141.
[24] LEE H,PIAO D,LEE J,et al.Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate[J].Microbial Cell Factories,2017,54:16-33.
[25] S?RENSEN H P,MORTENSEN K K.Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[J].Microbial Cell Factories,2005,4:1-8.
[26] KAPUST R B,WAUGH D S.Escherichia coli maltosebinding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused[J].Protein Science,1999,8:1668-1674
[27] CHENG L,YU L,WU X,et al.Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector[J].PLoS One,2014,9(2):e88183.
[28] GE F,QIU Y,GAO X,et al.Fusion expression of major antigenic segment of JEV E protein-hsp70 and the identification of domain acting as adjuvant in hsp70[J].Veterinary Immunology and Immunopathology,2006,113:288-296.
[29] HEMANTA K M,SOHINI D,MADHAN M,SAGAR A,et al.Protective efficacy of a DNA vaccine construct encoding the VP2 geneof infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens[J].Vaccine,2015,33:1033-1039.
[30] KERRY M,JULIAN S,LUCY R,et al.Hsp70 enhances presentation of FMDV antigen to bovine CD4+T cells in vitro[J].Veterinary Research,2010,41:36-48.
[2] ACHARYA R,FRY E,STUART D,et al.The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution[J].Nature,1989,337:709–716.
[3] LEE S Y,PARK M E,KIM R H,et al.Genetic and immunologic relationships between vaccine and field strains for vaccine selection of type A foot-and-mouth disease virus circulating in East Asia[J].Vaccine,2015,33:664–669.
[4] GRUBMAN M J,BAXT B.Foot-and-mouth disease[J].Clinical Microbiology Reviews,2004,17:465-493.
[5] BREHM K E,KUMAR N,THULKE H H,et al.High potency vaccines induce protection against heterologous challenge with foot-and-mouth disease virus[J].Vaccine,2008,26:1681-1687.
[6]刘在新.全球口蹄疫防控技术及病原特性研究概观[J].中国农业科学,2015,48,17:3547-3564.LIU Zai-xin.Progress and prospect of the technologies to control foot-and-mouth disease and its pathogen characteristics worldwide[J].Scientia Agricultura Sinica,2015,48,17:3547-3564.(in Chinese)
[7] RODRIGUEZ L L,GRUBMAN M J.Foot and mouth disease virus vaccines[J].Vaccine,2009,27:90-94.
[8] FAYNA D S,GISSELLE N M,CAROLINA S,et al.Footand-mouth disease vaccines[J].Veterinary Microbiology,2017,206:102-112.
[9]邵军军,常惠芸.口蹄疫多表位疫苗的研究进展[J].中国兽医科学,2008,38(11):1009-1012.SHAO Jun-jun,CHANG Hui-yun.Advances in multiple-epitopes vaccines against foot-and mouth disease[J].Chinese Veterinary Science,2008,38(11):1009-1012.(in Chinese)
[10] MATEU M G,CAMARERO J A,GIRALT E,et al.Direct evaluation of the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host[J].Virology,1995,206:298-306.
[11] BITTLE J L,HOUGHTEN R A,ALEXANDER H,et al.Protection against foot-and-mouth disease by immunization with chemically synthesized peptide predicted from the viral nucleotide sequence[J].Nature,1982,198:30-33.
[12] TANG H,LIU X S,FANG Y Z,et al.The epitopes of foot and mouth disease[J].Asian Journal of Animal and Veterinary Advances,2012,7:1261-1265.
[13] BOLWELL C,CLARKE B,PARRY N,et al.Epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies[J].Journal of General Virology,1989,70:59–68.
[14] BLANCO E,GARCI′A-BRIONES M M,SANZ-PARRA A,et al.Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus[J].Journal of Virology, 2001,35:3164-3174.
[15] BLANCO E,CUBILLOS C,MORENO N,et al.B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines[J].Clinical&Developmental Immunology,2013,2013:475960.
[16] JOSEA.B,SIRA D,SARA M,et al.A bivalent dendrimeric peptide bearing a T-cell epitope from foot-and-mouth disease virus protein 3A improves humoral response against classical swine fever virus[J].Virus Research,2017,238:8-12.
[17] WANG Y,KELLY C G,SINGH M,et al.Stimulation of Th1-polarizing cytokines,C-C chemokines,maturation of dendritic cells,and adjuvant function by the peptide binding fragment of heat shock protein 70[J].Journal of Immunology,2002,169(5):2422-2429.
[18] LI X,YANG X,LI L,LIU H,LIU J.A truncated C-terminal fragment of Mycobac-terium tuberculosis HSP70gene enhanced potency of HBV DNA vaccine[J].Vaccine,2006,24:3321-3331.
[19] FU F,TIAN H,LI X,et al.C-terminal heat shock protein 70 of Mycobacterium tuberculosis as a molecular adjuvant for DNA vaccination with the porcine circovirus type 2 ORF2(capsid)gene in mice[J].The Veterinary Journal,2013,195:244-247.
[20] BARRIOS C,LUSSOW A R,VAN J E,et al.Mycobacterial heat-shock proteins as carrier molecules.II:The use of the 70-kDa mycobacterial heat-shock protein as carrier for conjugated vaccines cancircumvent the need for adjuvants and Bacillus calmette Guérin priming[J].European Journal of Immunology,1992,22:1365-1372.
[21] KITCHING P,HAMMOND J,JEGGO M,et al.Global FMD control—is it an option?[J].Vaccine,2007,25:5660-5664.
[22] CAO Y,LU Z,LI D,et al.Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I∶C)[J].Veterinary Microbiology,2014,168:294-301.
[23] CAO Y,LI D,FU Y,et al.Rational design and efficacy of a multi-epitope recombinant protein vaccine against foot-and-mouth disease virus serotype A in pigs[J].Antiviral Research,2017,140:133-141.
[24] LEE H,PIAO D,LEE J,et al.Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate[J].Microbial Cell Factories,2017,54:16-33.
[25] S?RENSEN H P,MORTENSEN K K.Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[J].Microbial Cell Factories,2005,4:1-8.
[26] KAPUST R B,WAUGH D S.Escherichia coli maltosebinding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused[J].Protein Science,1999,8:1668-1674
[27] CHENG L,YU L,WU X,et al.Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector[J].PLoS One,2014,9(2):e88183.
[28] GE F,QIU Y,GAO X,et al.Fusion expression of major antigenic segment of JEV E protein-hsp70 and the identification of domain acting as adjuvant in hsp70[J].Veterinary Immunology and Immunopathology,2006,113:288-296.
[29] HEMANTA K M,SOHINI D,MADHAN M,SAGAR A,et al.Protective efficacy of a DNA vaccine construct encoding the VP2 geneof infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens[J].Vaccine,2015,33:1033-1039.
[30] KERRY M,JULIAN S,LUCY R,et al.Hsp70 enhances presentation of FMDV antigen to bovine CD4+T cells in vitro[J].Veterinary Research,2010,41:36-48.