牛分枝杆菌mpb51-mpb70融合基因的原核表达与抗原活性初步鉴定
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摘要
目的通过获得牛分枝杆菌保护性抗原MPB51、MPB70的融合蛋白MPB51-MPB70,以提高单一蛋白的抗原性。方法采用重叠拼接PCR技术将牛分枝杆菌mpb51与mpb70基因融合,获得融合基因mpb51-mpb70,克隆至pMD19中,构建重组质粒pMD-51-70。酶切、纯化后连接至pET28a(+)中,构建重组表达质粒pET-51-70。通过SDS-PAGE和Western blotting分析、鉴定融合蛋白的表达及抗原性。以间接ELISA比较MPB51、MPB70、MPB51-MPB70的抗原性。结果表达、纯化了45ku的融合蛋白MPB51-MPB70,并与牛结核血清发生特异性反应,且比单一蛋白反应性强。结论成功地获得了具有良好抗原性的牛分枝杆菌融合蛋白MPB51-MPB70。
To improve antigenicity of the simple protein,Mycobacterium bovis MPB51 and MPB70 were fused using(Gly4Ser)3 to obtain a fusion protein MPB51-MPB70.The mpb51 and mpb70 genes were fused by splicing overlapping extension(SOE)polymerase chain reaction(PCR)to acquire the fusion gene mpb51-mpb70 which was then cloned into pMD19 vector to construct recombinant plasmid pMD-51-70.After enzyme digestion by BamH I and EcoR I,the purified fusion gene was subcloned into the expression vector pET28 a(+).The expression of mpb51-mpb70 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE)and the antigenic activity was determined by Western-blotting.Indirect-ELISA was used to compare the antigenicity among MPB51,MPB70 and MPB51-MPB70.A 1359 bp fusion gene was amplified and the recombinant plasmid pMD-51-70 was constructed successfully.The expressed fusion protein,approximately 45 ku,exhibited a reaction activity with bovine tuberculosis serum.Besides,the reaction activity was better than that of solo protein.These results indicated that the MPB51-MPB70 fusion protein was obtained successfully.
To improve antigenicity of the simple protein,Mycobacterium bovis MPB51 and MPB70 were fused using(Gly4Ser)3 to obtain a fusion protein MPB51-MPB70.The mpb51 and mpb70 genes were fused by splicing overlapping extension(SOE)polymerase chain reaction(PCR)to acquire the fusion gene mpb51-mpb70 which was then cloned into pMD19 vector to construct recombinant plasmid pMD-51-70.After enzyme digestion by BamH I and EcoR I,the purified fusion gene was subcloned into the expression vector pET28 a(+).The expression of mpb51-mpb70 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE)and the antigenic activity was determined by Western-blotting.Indirect-ELISA was used to compare the antigenicity among MPB51,MPB70 and MPB51-MPB70.A 1359 bp fusion gene was amplified and the recombinant plasmid pMD-51-70 was constructed successfully.The expressed fusion protein,approximately 45 ku,exhibited a reaction activity with bovine tuberculosis serum.Besides,the reaction activity was better than that of solo protein.These results indicated that the MPB51-MPB70 fusion protein was obtained successfully.
引文
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[16]姜秀云,王春凤,王春芳,等.牛分枝杆菌MPB70基因的克隆及其在E.coli中的表达[J].中国预防兽医学报,2005,27(6):486-489.
[17]Bethunaickan R,Baulard AR,Locht C,et al.Antibody response in pulmonary tuberculosis against recombinant 27kDa(MPT51,Rv3803c)protein of Mycobacterium tuberculosis[J]Scand J Infect Dis,2007,39(10):867-874.DOI:10.1080/00365540701402954
[18]Cho YS,Lee SE,Ko YJ,et al.Definition of purified enzymelinked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis[J].JImmunoass Immunoch,2009,30(3):291-304.DOI:10.1080/15321810903084483
[19]Wang LX,Nagata T,Tsujimura K,et al.Identification of HLA-DR4-restricted T-cell epitope on MPT51protein,a major secreted protein derived from Mycobacterium tuberculosis using MPT51overlapping peptides screening and DNA vaccination[J].Vaccine,2010,28(8):2026-2031.DOI:10.1016/j.vaccine.2009.10.063
[20]Hashimoto D,Nagata T,Uchijima M,et al.Intratracheal administration of third-generation lentivirus vector encoding MPT51 from Mycobacterium tuberculosis induces specific CD8+T-cell responses in the lung[J].Vaccine,2008,26(40):5095-5100.DOI:10.1016/j.vaccine.2008.03.101
[21]Uchijima M,Nagata T,Koide Y.Chemokine receptor-mediated delivery of mycobacterial MPT51protein efficiently induces antigen-specific T-cell responses[J].Vaccine,2008,26(40):5165-5169.DOI:10.1016/j.vaccine.2008.03.059
[22]Junqueira-Kipnis AP,de Oliveira FM,Trentini MM,et al.Prime-boost with Mycobacterium smegmatis recombinant vaccine improves protection in mice Infected with Mycobacteriumtuberculosis[J].PLoS ONE,2013,8(11):e78639-e78648.DOI:10.1371/journal.pone.0078639
[2]Ramalingam B,Baulard AR,Locht C,et al.Cloning,expression,and purification of the 27kDa(MPT51,Rv3803c)protein of Mycobacterium tuberculosis[J].Protein Expr Purif,2004,36(1):53-60.DOI:10.1016/j.pep.2004.01.016
[3]Cho YS,Jung SC,Kim JM,et al.Enzyme-Linked immunosorbent assay of bovine tuberculosis by crude Mycobacterial protein70[J].J Immunoass Immunoch,2007,28(4):409-418.DOI:10.1080/15321810701603781
[4]Aoshi T,Nagata T,Suzuki M,et al.Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51protein,a major secreted protein derived from Mycobacterium tuberculosis,by MPT51 Overlapping Peptide Screening[J].Infect Immun,2008,76(4):1565-1571.DOI:10.1128/IAI.01381-07
[5]Wiker HG.MPB70and MPB83-major antigens of Mycobacterium bovis[J].Scand J Immunol,2009,69(6):492-499.DOI:10.1111/j.1365-3083.2009.02256.x
[6]Waters WR,Buddle BM,Vordermeier HM,et al.Development and evaluationof an enzyme-linked immunosorbent assay for use in thedetection of bovine tuberculosis in cattle[J].Clin Vaccine Immunol,2011,18(11):1882-1888.DOI:10.1016/0022-1759(94)90144-9
[7]Casal C,Díez-Guerrier A,lvarez J,et al.Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing[J].Vet Microbiol,2014,170(3/4):342-351.DOI:10.1016/j.vetmic.2014.02.036
[8]Casal C,Infantes JA,Risalde MA,et al.Antibody detection tests improve the sensitivity of tuberculosis diagnosis in cattle[J].Res Vet Sci,2017,112:214-221.DOI:10.1016/j.rvsc.2017.05.012
[9]Souza IIF,MeloCarlos ESP,Ramos AN,et al.Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis[J].Springerplus,2012,1(1):77.DOI:10.1186/2193-1801-1-77
[10]Whelan C,Shuralev E,Kwok HF,et al.Use of a multiplex enzyme-linked immunosorbent assay to detect a subpopulation of Mycobacterium bovis-infected animals deemed negative or inconclusive by the single intradermal comparative tuberculin skin test[J].J Vet Diagn Invest,2011,23(3):499-503.DOI:10.1177/1040638711403410
[11]Liu,S,Guo S,Wang C,et al.A novel fusion protein-based indirect enzyme-linked immunosorbent assay for the detection of bovine tuberculosis[J].Tuberculosis,2007,87(3):212-217.DOI:10.1016/j.tube.2006.07.007
[12]de Sousa EM,da Costa AC,Trentini MM,et al.Immunogenicity of a fusion protein containing immunodominant epitopes of Ag85C,MPT51,and HspX from Mycobacterium tuberculosis in mice and active TB infection[J].PLoS One,2012,7(10):e47781-e47791.DOI:10.1371/journal.pone.0047781
[13]Parlane NA,Grage K,Mifune J,et al.Vaccines displaying mycobacterial proteins on Biopolyester beads Stimulate cellular Immunity and induce protection against tuberculosis[J].Clin vaccine Immunol,2012,19(1):37-44.DOI:10.1128/CVI.05505-11
[14]Lee JW.Parlane NA,Rehm BHA,et al.Engineering Mycobacteria for the production of self-Assembling biopolyesters displaying mycobacterial antigens for use as a tuberculosis vaccine[J].Appl Environ Microbiol,2017,83(5):e02289-16.DOI:10.1128/AEM.02289-16
[15]姜秀云,王春凤,王春芳,等.牛分枝杆菌MPB51基因的克隆及其在大肠杆菌中的表达[J].微生物学报,2005,45(2):298-300.
[16]姜秀云,王春凤,王春芳,等.牛分枝杆菌MPB70基因的克隆及其在E.coli中的表达[J].中国预防兽医学报,2005,27(6):486-489.
[17]Bethunaickan R,Baulard AR,Locht C,et al.Antibody response in pulmonary tuberculosis against recombinant 27kDa(MPT51,Rv3803c)protein of Mycobacterium tuberculosis[J]Scand J Infect Dis,2007,39(10):867-874.DOI:10.1080/00365540701402954
[18]Cho YS,Lee SE,Ko YJ,et al.Definition of purified enzymelinked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis[J].JImmunoass Immunoch,2009,30(3):291-304.DOI:10.1080/15321810903084483
[19]Wang LX,Nagata T,Tsujimura K,et al.Identification of HLA-DR4-restricted T-cell epitope on MPT51protein,a major secreted protein derived from Mycobacterium tuberculosis using MPT51overlapping peptides screening and DNA vaccination[J].Vaccine,2010,28(8):2026-2031.DOI:10.1016/j.vaccine.2009.10.063
[20]Hashimoto D,Nagata T,Uchijima M,et al.Intratracheal administration of third-generation lentivirus vector encoding MPT51 from Mycobacterium tuberculosis induces specific CD8+T-cell responses in the lung[J].Vaccine,2008,26(40):5095-5100.DOI:10.1016/j.vaccine.2008.03.101
[21]Uchijima M,Nagata T,Koide Y.Chemokine receptor-mediated delivery of mycobacterial MPT51protein efficiently induces antigen-specific T-cell responses[J].Vaccine,2008,26(40):5165-5169.DOI:10.1016/j.vaccine.2008.03.059
[22]Junqueira-Kipnis AP,de Oliveira FM,Trentini MM,et al.Prime-boost with Mycobacterium smegmatis recombinant vaccine improves protection in mice Infected with Mycobacteriumtuberculosis[J].PLoS ONE,2013,8(11):e78639-e78648.DOI:10.1371/journal.pone.0078639
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