猪肺炎支原体部分血清体液免疫显性蛋白抗原的筛选鉴定
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摘要
旨在筛选猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)部分血清体液免疫显性蛋白抗原。利用生物信息学软件筛选到7个普遍存在于Mhp菌株中的蛋白Mhp104、Mhp299、Mhp367、Mhp462、Mhp465、Mhp477、Mhp488。将其对应的基因分别连接pGEX-6P-1载体后转化至大肠杆菌BL21(DE3),诱导表达目的蛋白。将诱导表达目的蛋白的7个重组菌裂解液和GST工程菌裂解液分别加入谷胱甘肽板进行抗原包被,以PBS+10%FBS+2.5%脱脂奶粉为封闭液,血清和酶标二抗分别按照1∶500,1∶40 000稀释,将抗原包被板分别与11份Mhp恢复期血清和7份Mhp阴性血清进行ELISA反应。结果表明,所有7个重组融合蛋白均能以可溶形式表达,且GST-Mhp299、GST-Mhp367、GST-Mhp488能被PreScission Protease酶切。最终共筛选出5个Mhp血清体液免疫显性蛋白抗原,分别为Mhp104、Mhp367、Mhp462、Mhp477、Mhp488,从而为Mhp基因工程亚单位疫苗的研发提供了抗原靶标。
The purpose of this research was to screen some Mycoplasma hyopneumoniae(Mhp) serological humoral immunodominant proteinic antigens.Seven universal existent Mhp proteins were analyzed by bioinformatics tools for their sub-localization.These proteins are Mhp104,Mhp299,Mhp367,Mhp462,Mhp465,Mhp477 and Mhp488.The corresponding genes of 7 proteins were ligated into pGEX-6 P-1,then transformed into E.coli BL21(DE3) and induced to express the recombinant proteins successfully.The lysates of 7 recombinant bacteria and engineering bacteria E.coli GST were added into the glutathione coated plates and reacted to 11 convalescent sera and 7 negative sera,respectively.Ten percent FBS and 2.5% skim milk in PBS was used as blocking buffer,and the serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were diluted by 500 times and 40 000 times for the ELISA assay,respectively.The result showed that all 7 recombinant fusion proteins can be expressed in soluble form,GST-Mhp299,GST-Mhp367,and GST-Mhp488 can be digested by PreScission Protease.Five serological humoral immunodominant protein antigens of Mhp were identified,namely Mhp104,Mhp367,Mhp462,Mhp477,and Mhp488.The result provided the antigen targets for the development of genetic engineering subunit vaccine against MPS.
The purpose of this research was to screen some Mycoplasma hyopneumoniae(Mhp) serological humoral immunodominant proteinic antigens.Seven universal existent Mhp proteins were analyzed by bioinformatics tools for their sub-localization.These proteins are Mhp104,Mhp299,Mhp367,Mhp462,Mhp465,Mhp477 and Mhp488.The corresponding genes of 7 proteins were ligated into pGEX-6 P-1,then transformed into E.coli BL21(DE3) and induced to express the recombinant proteins successfully.The lysates of 7 recombinant bacteria and engineering bacteria E.coli GST were added into the glutathione coated plates and reacted to 11 convalescent sera and 7 negative sera,respectively.Ten percent FBS and 2.5% skim milk in PBS was used as blocking buffer,and the serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were diluted by 500 times and 40 000 times for the ELISA assay,respectively.The result showed that all 7 recombinant fusion proteins can be expressed in soluble form,GST-Mhp299,GST-Mhp367,and GST-Mhp488 can be digested by PreScission Protease.Five serological humoral immunodominant protein antigens of Mhp were identified,namely Mhp104,Mhp367,Mhp462,Mhp477,and Mhp488.The result provided the antigen targets for the development of genetic engineering subunit vaccine against MPS.
引文
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[23] SIMIONATTO S,MARCHIORO S B,GALLI V,et al.Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins[J].Comp Immunol Microbiol Infect Dis,2012,35(2):209-216.
[24] MEENS J,BOLOTIN V,FRANK R,et al.Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array[J].Vet Microbiol,2010,142 (3/4):293-302.
[25] PENDARVIS K,PADULA M P,TACCHI J L,et al.Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232[J].BMC Genomics,2014,15:576.
[2] 陈财,华利忠,甘源,等.猪支原体肺炎净化研究进展[J].中国动物保健,2014,16(11):16-19.
[3] SIBILA M,BERNAL R,TORRENTS D,et al.Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion,and pig lung lesions at slaughter[J].Vet Microbiol,2008,127(1/2):165-170.
[4] MEYNS T,DEWULF J,DE KRUIF A,et al.Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations[J].Vaccine,2006,24(49/50):7081-7086.
[5] VILLARREAL I,MEYNS T,DEWULF J,et al.The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions[J].Vet J,2011,188(1):48-52.
[6] VILLARREAL I,MAES D,VRANCKX K,et al.Effect of vaccination of pigs against experimental infection with high and low virulence Mycoplasma hyopneumoniae strains[J].Vaccine,2011,29(9):1731-1735.
[7] COOK B S,BEDDOW J G,MANSO-SILVáN L,et al.Selective medium for culture of Mycoplasma hyopneumoniae[J].Vet Microbiol,2016,195:158-164.
[8] 周瑶琴,游凤,钟杰,等.一种快速鉴定猪肺炎支原体免疫显性蛋白抗原的ELISA方法的建立[J].生物工程学报,2018,34(1):44-53.
[9] BENDTSEN J D,NIELSEN H,WIDDICK D,et al.Prediction of twin-arginine signal peptides[J].BMC Bioinformatics,2005( 6):167.
[10] DILK S K,ROSE R W,HARTMANN E,et al.Prokaryotic utilization of the twin-arginine translocation pathway:a genomic survey[J].J Bacteriol,2003,185(4):1478-1483.
[11] PETERSEN T N,BRUNAK S,VON H G,et al.SignalP 4.0:discriminating signal peptides from transmembrane regions[J].Nat Methods,2011,8(10):785-786.
[12] HILLER K,GROTE A,SCHEER M,et al.PrediSi:prediction of signal peptides and their cleavage positions[J].Nucleic Acids Res,2004,32(Web Server issue):W375-W379.
[13] BAGOS P G,TSIRIGOS K D,LIAKOPOULOS T D,et al.Prediction of lipoprotein signal peptides in Gram-positive bacteria with a hidden markov model[J].J Proteome Res,2008,7 (12):5082-5093.
[14] K?LL L,KROGH A,SONNHAMMER E L.A combined transmembrane topology and signal peptide prediction method[J].J Mol Biol,2004,338(5):1027-1036.
[15] KAHSAY R Y,GAO G,LIAO L.An improved hidden markov model for transmembrane protein detection and topology prediction and its applications to complete genomes[J].Bioinformatics,2005,21(9):1853-1858.
[16] LITOU Z I,BAGOS P G,TSIRIGOS K D,et al.Prediction of cell wall sorting signals in Gram-positive bacteria with a hidden markov model:application to complete genomes[J].J Bioinform Comput Biol,2008,6(2):387-401.
[17] YU N Y,WAGNER J R,LAIRD M R,et al.PSORTb 3.0:improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes[J].Bioinformatics,2010,26(13):1608-1615.
[18] YU C S,LIN C J,HWANG J K.Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions[J].Protein Sci,2004,13(5):1402-1406.
[19] YU C S,CHEN Y C,LU C H,et al.Prediction of protein subcellular localization[J].Proteins,2006,64(3):643-651.
[20] SHEN H B,CHOU K C.Gpos-mPLoc:a top-down approach to improve the quality of predicting subcellular localization of Gram-positive bacterial proteins[J].Protein Pept Lett,2009,16(12):1478-1484.
[21] REOLON L A,MARTELLO C L,SCHRANK I S,et al.Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach[J].PLoS One,2014,9(11):e112596.
[22] MARCHIORO S B,SIMIONATTO S,GALLI V,et al.Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae [J].Vet Microbiol,2012,155(1):44-52.
[23] SIMIONATTO S,MARCHIORO S B,GALLI V,et al.Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins[J].Comp Immunol Microbiol Infect Dis,2012,35(2):209-216.
[24] MEENS J,BOLOTIN V,FRANK R,et al.Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array[J].Vet Microbiol,2010,142 (3/4):293-302.
[25] PENDARVIS K,PADULA M P,TACCHI J L,et al.Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232[J].BMC Genomics,2014,15:576.
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