滑液支原体醛缩酶的亚细胞定位及免疫原性
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摘要
【背景】滑液支原体(Mycoplasma synoviae,MS)感染能够引起鸡和火鸡的气囊炎、关节渗出性的滑液囊膜及腱鞘滑膜炎等。有研究表明,许多支原体中与代谢相关的酶类不仅分布在细胞质,也分布于细胞膜表面,通过结合宿主细胞的胞外基质蛋白,协助病原菌黏附入侵宿主细胞。已有报道牛支原体(Mycoplasma bovis)和鸡毒支原体(Mycoplasma gallisepticum)的醛缩酶(Fructose-bisphosphate aldolase,FBA)分布在膜蛋白和胞浆蛋白中,而MS的FBA蛋白还未见相关研究。【目的】对MSFBA蛋白进行生物信息学分析、原核表达、免疫原性分析及亚细胞定位检测,为进一步探索MS代谢相关酶类的生物学功能奠定基础。【方法】通过分析软件PSORTb、SignalP 4.1 Server和TMHMM Server在线预测MSFBA的亚细胞定位、信号肽及跨膜区,并用BLASTn和MEGA 5.0进行同源比对及进化树分析;通过Overlap PCR点突变扩增MS的fba全基因序列,连接表达载体pET-28a(+)并进行原核表达及纯化,获得纯化后的重组MSFBA(rMSFBA)蛋白;用MS阳性血清进行Westernblot鉴定rMSFBA的免疫原性;用纯化的rMSFBA蛋白制备兔多克隆抗体,与MS全菌蛋白、膜蛋白及胞浆蛋白进行Western blot分析,同时对MS全菌进行悬浮免疫荧光分析,检测MSFBA的膜定位情况。【结果】生物信息分析预测MSFBA分布在细胞质中,无信号肽,无跨膜区,在MS种内相似性高达99%,与其他种属FBA相似性在61%-78%之间,进化树显示其与牛鼻支原体(Mycoplasma bovirhinis)、仓鼠支原体(Mycoplasm acricetuli)等的FBA蛋白进化关系较近,与精氨酸支原体(Mycoplasma arginini)、人型支原体(Mycoplasma hominis)等的FBA蛋白进化关系较远;表达rMSFBA蛋白并纯化,经测定其相对分子质量大小约为33kD;rMSFBA能与MS阳性鸡血清特异性结合,证实其具有较好的免疫原性;Western blot显示抗rMSFBA的兔血清能与MS全菌蛋白和胞浆蛋白反应,而与膜蛋白不反应,说明MSFBA蛋白分布于胞浆中;悬浮免疫荧光实验证实MS的细胞膜上未见FBA蛋白分布。【结论】首次报道了滑液支原体的FBA蛋白是一个高度保守的免疫原性蛋白,主要分布在细胞质中,该结果为进一步研究MSFBA蛋白的生物学功能提供了分子基础。
[Background] Mycoplasma synoviae(MS) can infect chickens and turkeys to cause airsacculitis, joint exudative, synovial bursitis and tendon sheath synovitis. Previous studies have shown that many metabolism-related enzymes in mycoplasma are present not only in the cytoplasm but also on the cell membrane surface, which act as adhesion associated proteins and play roles on the pathogenicity.The fructose-bisphosphate aldolases(FBAs) have been identified on the membrane and cytoplasmic fractions of Mycoplasma bovis and Mycoplasma gallisepticum, however, the MS FBA has not been studied before. [Objective] To explore the biological functions of metabolism related enzymes in MS, the FBA protein was subjected to bioinformatics analysis, prokaryotic expression, as well as immunogenicity and subcellular localization determinations in this study. [Methods] Using softwares of PSORTb, SignalP 4.1 Server and TMHMM Server to predict the subcellular localization, signal peptides and transmembrane regions of MSFBA. Blastn and MEGA 5.0 were used to analyze the homology and construct the phylogenetic tree of MSFBA. The full length of MS fba gene was amplified by overlap PCR and inserted into the expression vector of pET-28a(+). The recombinant MSFBA(rMSFBA) protein was then expressed in Escherichia coli BL21(DE3) and purified. The immunogenicity of rMSFBA was determined by Western blot analysis using MS-positive chicken serum. The rabbit anti-rMSFBA sera were prepared, and used to perform Western blot against the whole-cell, membrane and cytoplasmic fractions of MS to determine the subcellular localization. Suspension immunofluorescence assay further confirmed the non-membrane surface localization of the MSFBA. [Results] The MSFBA is predicted to be a cytoplasmic protein with no signal peptide and no transmembrane region. It is highly conserved protein which shows up to 99%homology among different MS strains, and 61% to 78% homology with different species of mycoplasma.The phylogenetic tree shows that the MSFBA is evolutionally closed to the FBA from Mycoplasma bovirhinis and Mycoplasma cricetuli, but remote to that of Mycoplasma arginini and Mycoplasma hominis.The rMSFBA protein was successfully expressed and purified, and the relative molecular mass was approximately 33 kD. The rMSFBA presented a specific binding to MS-positive chicken serum, which proved that it had good immunogenicity. Western blot analysis showed the rabbit anti-rMSFBA sera can react with the whole-cell and cytoplasmic fractions of MS bacteria, but not the membrane fraction,indicating the MSFBA protein is distributed in the cytoplasma. Suspension immunofluorescence assay further confirmed the FBA was not displayed on the membrane surface of MS cells. [Conclusion] The MSFBA protein is a highly conserved, immunogenic and cytoplasmic localized protein. The results provide a molecular basis for further study of the biological function of MSFBA protein.
[Background] Mycoplasma synoviae(MS) can infect chickens and turkeys to cause airsacculitis, joint exudative, synovial bursitis and tendon sheath synovitis. Previous studies have shown that many metabolism-related enzymes in mycoplasma are present not only in the cytoplasm but also on the cell membrane surface, which act as adhesion associated proteins and play roles on the pathogenicity.The fructose-bisphosphate aldolases(FBAs) have been identified on the membrane and cytoplasmic fractions of Mycoplasma bovis and Mycoplasma gallisepticum, however, the MS FBA has not been studied before. [Objective] To explore the biological functions of metabolism related enzymes in MS, the FBA protein was subjected to bioinformatics analysis, prokaryotic expression, as well as immunogenicity and subcellular localization determinations in this study. [Methods] Using softwares of PSORTb, SignalP 4.1 Server and TMHMM Server to predict the subcellular localization, signal peptides and transmembrane regions of MSFBA. Blastn and MEGA 5.0 were used to analyze the homology and construct the phylogenetic tree of MSFBA. The full length of MS fba gene was amplified by overlap PCR and inserted into the expression vector of pET-28a(+). The recombinant MSFBA(rMSFBA) protein was then expressed in Escherichia coli BL21(DE3) and purified. The immunogenicity of rMSFBA was determined by Western blot analysis using MS-positive chicken serum. The rabbit anti-rMSFBA sera were prepared, and used to perform Western blot against the whole-cell, membrane and cytoplasmic fractions of MS to determine the subcellular localization. Suspension immunofluorescence assay further confirmed the non-membrane surface localization of the MSFBA. [Results] The MSFBA is predicted to be a cytoplasmic protein with no signal peptide and no transmembrane region. It is highly conserved protein which shows up to 99%homology among different MS strains, and 61% to 78% homology with different species of mycoplasma.The phylogenetic tree shows that the MSFBA is evolutionally closed to the FBA from Mycoplasma bovirhinis and Mycoplasma cricetuli, but remote to that of Mycoplasma arginini and Mycoplasma hominis.The rMSFBA protein was successfully expressed and purified, and the relative molecular mass was approximately 33 kD. The rMSFBA presented a specific binding to MS-positive chicken serum, which proved that it had good immunogenicity. Western blot analysis showed the rabbit anti-rMSFBA sera can react with the whole-cell and cytoplasmic fractions of MS bacteria, but not the membrane fraction,indicating the MSFBA protein is distributed in the cytoplasma. Suspension immunofluorescence assay further confirmed the FBA was not displayed on the membrane surface of MS cells. [Conclusion] The MSFBA protein is a highly conserved, immunogenic and cytoplasmic localized protein. The results provide a molecular basis for further study of the biological function of MSFBA protein.
引文
[1]Lu F.Study on inactivated of Mycoplasma gallisepticum disease and Mycoplasma synovia disease combined vaccine[D].Lanzhou:Master’s Thesis of Gansu Agricultural University,2015(in Chinese)陆凤.鸡毒支原体、滑液支原体二联灭活苗的研究[D].兰州:甘肃农业大学硕士学位论文,2015
[2]Liu JS,Yin XT.Diagnosis and treatment of Mycoplasma synoviae disease[J].Shandong Journal of Animal Science and Veterinary Medicine,2009,30(12):91(in Chinese)刘建设,尹逊同.鸡滑液囊支原体病的诊疗[J].山东畜牧兽医,2009,30(12):91
[3]Bradbury JM,Howell LJ.The response of chickens to experimental infection‘in ovo’with Mycoplasma synoviae[J].Avian Pathology,1975,4(4):277-286
[4]He SB,Tan L,Bao SJ,et al.Prokaryotic expression and membrane localization of aldolase of Mycoplasma gallisepticum[J].Chinese Veterinary Science,2013,43(1):42-46(in Chinese)何随彬,谭磊,包世俊,等.鸡毒支原体醛缩酶的原核表达及膜定位鉴定[J].中国兽医科学,2013,43(1):42-46
[5]Wu YM,Ye YK.Mycoplasmology[M].2nd ed.Beijing:People’s Medical Publishing House,2008:69(in Chinese)吴移谋,叶元康.支原体学[M].2版.北京:人民卫生出版社,2008:69
[6]Chen HJ,Yu SQ,Shen XY,et al.The Mycoplasma gallisepticumα-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen[J].Microbial Pathogenesis,2011,51(4):285-290
[7]Bao SJ,Guo XQ,Yu SQ,et al.Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J].BMC Veterinary Research,2014,10:223
[8]Bao SJ,Chen DQ,Yu SQ,et al.Characterization of triosephosphate isomerase from Mycoplasma gallisepticum[J].FEMS Microbiology Letters,2015,362(17):fnv140
[9]Dumke R,Hausner M,Jacobs E.Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in mediating interactions with the human extracellular matrix[J].Microbiology,2011,157(8):2328-2338
[10]He SB,Qi JJ,Yu SQ,et al.Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum[J].Microbial Pathogenesis,2015,89:161-168
[11]Hegde S,Rosengarten R,Chopra-Dewasthaly R.Disruption of the pdhB pyruvate dehydrogenase gene affects colony morphology,in vitro growth and cell invasiveness of Mycoplasma agalactiae[J].PLoS One,2015,10(3):e0119706
[12]Bhattacharya S,Ploplis VA,Castellino FJ.Bacterial plasminogen receptors utilize host plasminogen system for effective invasion and dissemination[J].Journal of Biomedicine and Biotechnology,2012,2012:482096
[13]Wang SJ.Cloning and expression of FBA gene from Mycoplasma Hyopneumoniae,analysis of immunogenicity and activity[D].Taigu:Master’s Thesis of Shanxi Agricultural University,2014(in Chinese)王世杰.猪肺炎支原体FBA蛋白原核克隆表达、免疫原性及活性分析[D].太古:山西农业大学硕士学位论文,2014
[14]Wu ZF,Zhang W,Lu CP.Immunoproteomic assay of surface proteins of Streptococcus suis serotype 9[J].FEMS Immunology&Medical Microbiology,2008,53(1):52-59
[15]Mendon?a M,Moreira GMSG,Concei??o FR,et al.Fructose1,6-bisphosphate aldolase,a novel immunogenic surface protein on Listeria species[J].PLoS One,2016,11(8):e0160544
[16]Tunio SA,Oldfield NJ,Berry A,et al.The moonlighting protein fructose-1,6-bisphosphate aldolase of Neisseria meningitidis:surface localization and role in host cell adhesion[J].Molecular Microbiology,2010,76(3):605-615
[17]Gao X,Xing XY,Feng N,et al.Cloning,expression and subcellular localization of fba gene in Mycoplasma bovis Wuwei strain[J].Journal of Agricultural Biotechnology,2017,25(2):274-281(in Chinese)高翔,邢小勇,冯娜,等.牛支原体武威株fba基因的克隆、表达及亚细胞定位[J].农业生物技术学报,2017,25(2):274-281
[18]Sun ZY,Shen BB,Wu HZ,et al.The secreted fructose1,6-bisphosphate aldolase as a broad spectrum vaccine candidate against pathogenic bacteria in aquaculture[J].Fish&Shellfish Immunology,2015,46(2):638-647
[19]Chaves EGA,Weber SS,Báo SN,et al.Analysis of Paracoccidioides secreted proteins reveals fructose1,6-bisphosphate aldolase as a plasminogen-binding protein[J].BMC Microbiology,2015,15:53
[20]Wang J,Yu CX,Xiao D,et al.Patterns and diagnostic value of the antibody response to fructose-1,6-bisphosphate aldolase of Schistosoma japonicum[J].Journal of Pathogen Biology,2015,10(3):232-238(in Chinese)王玠,余传信,肖迪,等.日本血吸虫果糖二磷酸醛缩酶抗体反应模式及诊断价值的研究[J].中国病原生物学杂志,2015,10(3):232-238
[2]Liu JS,Yin XT.Diagnosis and treatment of Mycoplasma synoviae disease[J].Shandong Journal of Animal Science and Veterinary Medicine,2009,30(12):91(in Chinese)刘建设,尹逊同.鸡滑液囊支原体病的诊疗[J].山东畜牧兽医,2009,30(12):91
[3]Bradbury JM,Howell LJ.The response of chickens to experimental infection‘in ovo’with Mycoplasma synoviae[J].Avian Pathology,1975,4(4):277-286
[4]He SB,Tan L,Bao SJ,et al.Prokaryotic expression and membrane localization of aldolase of Mycoplasma gallisepticum[J].Chinese Veterinary Science,2013,43(1):42-46(in Chinese)何随彬,谭磊,包世俊,等.鸡毒支原体醛缩酶的原核表达及膜定位鉴定[J].中国兽医科学,2013,43(1):42-46
[5]Wu YM,Ye YK.Mycoplasmology[M].2nd ed.Beijing:People’s Medical Publishing House,2008:69(in Chinese)吴移谋,叶元康.支原体学[M].2版.北京:人民卫生出版社,2008:69
[6]Chen HJ,Yu SQ,Shen XY,et al.The Mycoplasma gallisepticumα-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen[J].Microbial Pathogenesis,2011,51(4):285-290
[7]Bao SJ,Guo XQ,Yu SQ,et al.Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J].BMC Veterinary Research,2014,10:223
[8]Bao SJ,Chen DQ,Yu SQ,et al.Characterization of triosephosphate isomerase from Mycoplasma gallisepticum[J].FEMS Microbiology Letters,2015,362(17):fnv140
[9]Dumke R,Hausner M,Jacobs E.Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in mediating interactions with the human extracellular matrix[J].Microbiology,2011,157(8):2328-2338
[10]He SB,Qi JJ,Yu SQ,et al.Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum[J].Microbial Pathogenesis,2015,89:161-168
[11]Hegde S,Rosengarten R,Chopra-Dewasthaly R.Disruption of the pdhB pyruvate dehydrogenase gene affects colony morphology,in vitro growth and cell invasiveness of Mycoplasma agalactiae[J].PLoS One,2015,10(3):e0119706
[12]Bhattacharya S,Ploplis VA,Castellino FJ.Bacterial plasminogen receptors utilize host plasminogen system for effective invasion and dissemination[J].Journal of Biomedicine and Biotechnology,2012,2012:482096
[13]Wang SJ.Cloning and expression of FBA gene from Mycoplasma Hyopneumoniae,analysis of immunogenicity and activity[D].Taigu:Master’s Thesis of Shanxi Agricultural University,2014(in Chinese)王世杰.猪肺炎支原体FBA蛋白原核克隆表达、免疫原性及活性分析[D].太古:山西农业大学硕士学位论文,2014
[14]Wu ZF,Zhang W,Lu CP.Immunoproteomic assay of surface proteins of Streptococcus suis serotype 9[J].FEMS Immunology&Medical Microbiology,2008,53(1):52-59
[15]Mendon?a M,Moreira GMSG,Concei??o FR,et al.Fructose1,6-bisphosphate aldolase,a novel immunogenic surface protein on Listeria species[J].PLoS One,2016,11(8):e0160544
[16]Tunio SA,Oldfield NJ,Berry A,et al.The moonlighting protein fructose-1,6-bisphosphate aldolase of Neisseria meningitidis:surface localization and role in host cell adhesion[J].Molecular Microbiology,2010,76(3):605-615
[17]Gao X,Xing XY,Feng N,et al.Cloning,expression and subcellular localization of fba gene in Mycoplasma bovis Wuwei strain[J].Journal of Agricultural Biotechnology,2017,25(2):274-281(in Chinese)高翔,邢小勇,冯娜,等.牛支原体武威株fba基因的克隆、表达及亚细胞定位[J].农业生物技术学报,2017,25(2):274-281
[18]Sun ZY,Shen BB,Wu HZ,et al.The secreted fructose1,6-bisphosphate aldolase as a broad spectrum vaccine candidate against pathogenic bacteria in aquaculture[J].Fish&Shellfish Immunology,2015,46(2):638-647
[19]Chaves EGA,Weber SS,Báo SN,et al.Analysis of Paracoccidioides secreted proteins reveals fructose1,6-bisphosphate aldolase as a plasminogen-binding protein[J].BMC Microbiology,2015,15:53
[20]Wang J,Yu CX,Xiao D,et al.Patterns and diagnostic value of the antibody response to fructose-1,6-bisphosphate aldolase of Schistosoma japonicum[J].Journal of Pathogen Biology,2015,10(3):232-238(in Chinese)王玠,余传信,肖迪,等.日本血吸虫果糖二磷酸醛缩酶抗体反应模式及诊断价值的研究[J].中国病原生物学杂志,2015,10(3):232-238