多杀性巴氏杆菌OmpA基因的原核表达及生物信息学分析
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摘要
本试验旨在探究羊源多杀性巴氏杆菌OmpA基因的原核表达及其生物信息学特征。以羊源多杀性巴氏杆菌HN-01株基因组为模板,设计特异性引物扩增OmpA基因;构建pET-28a(+)-OmpA重组质粒后转化大肠杆菌BL21(DE3)感受态细胞,将鉴定正确的重组菌经IPTG诱导表达;通过SDS-PAGE及Western blotting分析表达蛋白的特征,并运用生物信息学工具对OmpA基因序列进行分析。结果显示,羊源多杀性巴氏杆菌OmpA基因大小约为1 044 bp,该基因序列与HN-06株的同源性达89.72%。通过诱导后发现,pET-28a(+)-OmpA重组菌最佳诱导条件为1 mmol/L IPTG 37℃诱导6 h,表达的重组蛋白大小约为40 ku,以包涵体的形式存在。Western blotting结果显示,约40 ku的重组蛋白携带His标签。经生物信息学分析,OmpA分子式为C_(1684)H_(2619)N_(457)O_(505)S_3,属碱性疏水蛋白,其多肽链的1-21位氨基酸为信号肽区域,并具有多种结构。综上所述,OmpA可能具有特殊结构,与众多外膜蛋白结构特点相似。本研究构建了多杀性巴氏杆菌OmpA基因原核表达系统,优化诱导条件后能稳定获得OmpA重组蛋白,为进一步探究巴氏杆菌的致病机理提供理论依据。
The aim of this study was to investigate the prokaryotic expression and bioinformatics character of Pasteurella multocida OmpA gene.Using the genome of Pasteurella multocida HN-01 strain as a template,specific primers were designed to amplify OmpA gene.The recombinant plasmid pET-28 a(+)-OmpA was constructed and transferred into E.coli BL21(DE3) competent cells,and the correct recombinant plasmid was identified and expressed by IPTG.The expressed proteins were analyzed by SDS-PAGE and Western blotting,and the OmpA gene sequence was analyzed by bioinformatics softwares.The results showed that the OmpA fragment of Pasteurella multocida was about 1 044 bp,and its coding region sequence was 89.72% homologous to HN-06 strain.After induction,the optimal induction condition of pET-28 a(+)-OmpA recombinant strain was 1 mmol/L IPTG at 37 ℃ for 6 h,and the expressed recombinant protein was about 40 ku in the form of inclusion bodies.Western blotting results showed that about 40 ku of recombinant protein carried His-tag.Bioinformatics analysis found that the OmpA had the formula C_(1684)H_(2619)N_(457)O_(505)S_3 and was a basic hydrophobin.The amino acids 1 to 21 of the polypeptide chain was signal peptide regions and had various structures.In summary,OmpA might had a special structure,consistent with the structural characteristics of many outer membrane proteins.In this study,the prokaryotic expression system of Pasteurella multocida OmpA gene was constructed,and the OmpA recombinant protein was stably obtained after optimized induction conditions,which provided a theoretical basis for further exploring the pathogenic mechanism of Pasteurella.
The aim of this study was to investigate the prokaryotic expression and bioinformatics character of Pasteurella multocida OmpA gene.Using the genome of Pasteurella multocida HN-01 strain as a template,specific primers were designed to amplify OmpA gene.The recombinant plasmid pET-28 a(+)-OmpA was constructed and transferred into E.coli BL21(DE3) competent cells,and the correct recombinant plasmid was identified and expressed by IPTG.The expressed proteins were analyzed by SDS-PAGE and Western blotting,and the OmpA gene sequence was analyzed by bioinformatics softwares.The results showed that the OmpA fragment of Pasteurella multocida was about 1 044 bp,and its coding region sequence was 89.72% homologous to HN-06 strain.After induction,the optimal induction condition of pET-28 a(+)-OmpA recombinant strain was 1 mmol/L IPTG at 37 ℃ for 6 h,and the expressed recombinant protein was about 40 ku in the form of inclusion bodies.Western blotting results showed that about 40 ku of recombinant protein carried His-tag.Bioinformatics analysis found that the OmpA had the formula C_(1684)H_(2619)N_(457)O_(505)S_3 and was a basic hydrophobin.The amino acids 1 to 21 of the polypeptide chain was signal peptide regions and had various structures.In summary,OmpA might had a special structure,consistent with the structural characteristics of many outer membrane proteins.In this study,the prokaryotic expression system of Pasteurella multocida OmpA gene was constructed,and the OmpA recombinant protein was stably obtained after optimized induction conditions,which provided a theoretical basis for further exploring the pathogenic mechanism of Pasteurella.
引文
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[12] SOULAS C,BAUSSANT T,AUBRY J P,et al.Cutting edge:Outer membrane protein A (OmpA) binds to and activates human macrophages[J].Journal of Immunology,2000,165(5):2335-2340.
[13] MITTAL R,PRASADARAO N V.Outer membrane protein A expression in Escherichia coli K1 is required to prevent the maturation of myeloid dendritic cells and the induction of IL-10 and TGF-β[J].Journal of Immunology,2008,181(4):2672-2682.
[14] JEANNIN P,MAGISTRRLLI G,HERBAULT N,et al.Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs[J].European Journal of Immunology,2003,33(2):326-333.
[15] 李楚楚,刘畅,梁俊容,等.致病性小肠结肠炎耶尔森菌外膜蛋白A对SD大鼠免疫保护作用研究[J].中国人兽共患病学报,2018,34(6):495-500.LI C C,LIU C,LIANG J R,et al.Immunoprotection of outer membrane protein A from pathogenic Yersinia enterocolitica on rat[J].Chinses Journal of Zoonoses,2018,34(6):495-500.(in Chinese)
[16] 李燕.嗜麦芽窄食单胞菌外膜蛋白A的生物信息学分析及其粘膜免疫保护性的研究[D].合肥:安徽医科大学,2018.LI Y.Bioinformatics analysis and mucosal immune protection study of Stenotrophomonas maltophilia outer membrane proteine A[D].Hefei:Anhui Medical University,2018.(in Chinese)
[17] 安志远,黄骁舾,丁文一.鲍曼不动杆菌外膜蛋白A真核表达载体构建及其诱导HeLa细胞不完全自噬[J].免疫学杂志,2017,33(11):996-1002.AN Z Y,HUANG X X,DING W Y.Eukaryotic expression vector construction of outer membrane protein A from Acinetobacter baumannii and the imcomplete autophagy in HeLa cells induced by the protein[J].Immunology Journal,2017,33(11):996-1002.(in Chinese)
[18] LU Y S,LAI W C,PAKES S P,et al.A monoclonal antibodies against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis[J].Infection and Immunity,1991,59(1):172-180.
[19] 张萌萌,李宝宝,曹瑞勇,等.羊源类鼻疽伯克霍尔德菌BPSL1467基因克隆、表达及其蛋白生物信息学分析[J].中国畜牧兽医,2018,45(9):2401-2408.ZHANG M M,LI B B,CAO R Y,et al.Cloning,expression and protein bioinformatics analysis of BPSL1467 gene of Burkholderia pseudomallei[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2401-2408.(in Chinese)
[20] 李宝宝,张振兴,黄海峰,等.羊源多杀性巴氏杆菌ompW基因的克隆及生物信息学分析[J].中国畜牧兽医,2019,46(1):28-36.LI B B,ZHANG Z X,HUANG H F,et al.Cloning and bioinformatics analysis of ompW gene from Pasteurella multocida in goat[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):28-36.(in Chinese)
[21] HARPER M,BOYCE J D,ADLER B.The key surface components of Pasteurella multocida:Capsule and lipopolysaccharide[D].Berlin:Springer-Verlag Berlin Heidelberg,2012.
[22] BOYCE J D,ADLER B.The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2)[J].Infection and Immunity,2000,68(6):3463-3468.
[23] DEANGELIS P L,PADGETT-MCCUE A J.Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F[J].Journal of Biological Chemistry,2000,275(31):24124-24129.
[24] 韦东,吾鲁木汗·那孜尔别克,段世雄,等.禽多杀性巴氏杆菌C48-3株外膜蛋白H的致病作用[J].中国生物工程杂志,2014,34(6):31-39.WEI D,WULUMUHAN·NZEBK,DUAN S X,et al.Role of outer membrane protein H in the pathogenesis of avian Pasteurella multicida strain C48-3[J].China Biotechnology,2014,34(6):31-39.(in Chinese)
[25] 蒋玉亮,兰萍,李艳,等.一种基于OmpA的分泌型原核表达载体的构建及应用[J].成都医学院学报,2016,11(4):409-412.JIANG Y L,LAN P,LI Y,et al.Construction and application of a secreted prokaryotic expression vector based on OmpA signal peptide[J].Journal of Chengdu Medical College,2016,11(4):409-412.(in Chinese)
[26] MEUSKENS I,MICHALIK M,CHAUHAN N,et al.A new strain collection for improved expression of outer membrane proteins[J].Frontiers in Cellular and Infection Microbiology,2017,7:464.
[27] PENG B,WANG C,LI H,et al.Outer membrane proteins form specific patterns in antibiotic-resistant Edwardsiella tarda[J].Frontiers in Microbiology,2017,8:69.
[28] 白雪瑞,王权,陈永军,等.副溶血弧菌ompA基因缺失株的生物学特性及致病性分析[J].南京农业大学学报,2018,41(5):902-910.BAI X R,WANG Q,CHEN Y J,et al.Biological characteristics and pathogenicity of an ompA mutant of Vibrio parahaemolyticus[J].Journal of Nanjing Agricultural University,2018,41(5):902-910.(in Chinese)
[2] HARPER M,BOYCE J D,ADLER B.Pasteurella multocida pathogenesis:125 years after Pasteur[J].FEMS Microbiology Letters,2006,265(1):1-10.
[3] 孙贵.一例牛巴氏杆菌病的诊治[J].中国动物保健,2018,20(8):56-57.SUN G.Diagnosis and treatment of a case of bovine pasteurellosis[J].China Animal Health,2018,20(8):56-57.(in Chinese)
[4] 刘家平,蒋作吉.绵羊巴氏杆菌病的诊断与防治[J].新疆畜牧业,2018,33(2):58-59.LIU J P,JIANG Z J.Diagnosis and prevention of pasteurellosis in sheep[J].Xinjiang Xumuye,2018,33(2):58-59.(in Chinese)
[5] 王巍,乌云塔娜,格根塔娜,等.呼伦贝尔市首例羊源荚膜D群多杀性巴氏杆菌的分离与鉴定[J].中国动物传染病学报,2018,26(4):52-57.WANG W,WUYUN T N,GEGEN T N,et al.The first isolation and identification Pasteurella multocida serogroup-D from sheep in Hulunbuir[J].Chinese Journal of Animal Infectious Diseases,2018,26(4):52-57.(in Chinese)
[6] LIN J,HUANG S X,ZHANG Q J,et al.Outer membrane proteins:Key players for bacterial adaptation in host niches[J].Microbes and Infection,2002,4(3):325-331.
[7] 宋舟,陈伟,张立艳,等.大肠杆菌外膜蛋白A研究进展[J].中国畜牧兽医,2012,39(5):199-202.SONG Z,CHEN W,ZHANG L Y,at al.Research progress of outer membrane protein A of E.coli[J].China Animal Husbandry & Veterinary Medicine,2012,39(5):199-202.(in Chinese)
[8] JIN J S,KWON S O,MOON D C,et al.Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles[J].PLoS One,2011,6(2):e17027.
[9] OKAY S,?ETIN R,KARABULUT F,et al.Immune responses elicited by the recombinant Erp,HspR,LppX,MmaA4,and OmpA proteins from Mycobacterium tuberculosis in mice[J].Acta Microbiologica et Immunologica Hungarica,2019,66(2):219-234.
[10] SMITH S G,MAHON V,LAMBERT M A,et al.A molecular Swiss army knife:OmpA structure,function and expression[J].FEMS Microbiology Letters,2007,273(1):1-11.
[11] CHAUDHARY A,KAMISCHKE C,LEITE M,et al.β-barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses[J].Science Signaling,2018,11(558):7493.
[12] SOULAS C,BAUSSANT T,AUBRY J P,et al.Cutting edge:Outer membrane protein A (OmpA) binds to and activates human macrophages[J].Journal of Immunology,2000,165(5):2335-2340.
[13] MITTAL R,PRASADARAO N V.Outer membrane protein A expression in Escherichia coli K1 is required to prevent the maturation of myeloid dendritic cells and the induction of IL-10 and TGF-β[J].Journal of Immunology,2008,181(4):2672-2682.
[14] JEANNIN P,MAGISTRRLLI G,HERBAULT N,et al.Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs[J].European Journal of Immunology,2003,33(2):326-333.
[15] 李楚楚,刘畅,梁俊容,等.致病性小肠结肠炎耶尔森菌外膜蛋白A对SD大鼠免疫保护作用研究[J].中国人兽共患病学报,2018,34(6):495-500.LI C C,LIU C,LIANG J R,et al.Immunoprotection of outer membrane protein A from pathogenic Yersinia enterocolitica on rat[J].Chinses Journal of Zoonoses,2018,34(6):495-500.(in Chinese)
[16] 李燕.嗜麦芽窄食单胞菌外膜蛋白A的生物信息学分析及其粘膜免疫保护性的研究[D].合肥:安徽医科大学,2018.LI Y.Bioinformatics analysis and mucosal immune protection study of Stenotrophomonas maltophilia outer membrane proteine A[D].Hefei:Anhui Medical University,2018.(in Chinese)
[17] 安志远,黄骁舾,丁文一.鲍曼不动杆菌外膜蛋白A真核表达载体构建及其诱导HeLa细胞不完全自噬[J].免疫学杂志,2017,33(11):996-1002.AN Z Y,HUANG X X,DING W Y.Eukaryotic expression vector construction of outer membrane protein A from Acinetobacter baumannii and the imcomplete autophagy in HeLa cells induced by the protein[J].Immunology Journal,2017,33(11):996-1002.(in Chinese)
[18] LU Y S,LAI W C,PAKES S P,et al.A monoclonal antibodies against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis[J].Infection and Immunity,1991,59(1):172-180.
[19] 张萌萌,李宝宝,曹瑞勇,等.羊源类鼻疽伯克霍尔德菌BPSL1467基因克隆、表达及其蛋白生物信息学分析[J].中国畜牧兽医,2018,45(9):2401-2408.ZHANG M M,LI B B,CAO R Y,et al.Cloning,expression and protein bioinformatics analysis of BPSL1467 gene of Burkholderia pseudomallei[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2401-2408.(in Chinese)
[20] 李宝宝,张振兴,黄海峰,等.羊源多杀性巴氏杆菌ompW基因的克隆及生物信息学分析[J].中国畜牧兽医,2019,46(1):28-36.LI B B,ZHANG Z X,HUANG H F,et al.Cloning and bioinformatics analysis of ompW gene from Pasteurella multocida in goat[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):28-36.(in Chinese)
[21] HARPER M,BOYCE J D,ADLER B.The key surface components of Pasteurella multocida:Capsule and lipopolysaccharide[D].Berlin:Springer-Verlag Berlin Heidelberg,2012.
[22] BOYCE J D,ADLER B.The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2)[J].Infection and Immunity,2000,68(6):3463-3468.
[23] DEANGELIS P L,PADGETT-MCCUE A J.Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F[J].Journal of Biological Chemistry,2000,275(31):24124-24129.
[24] 韦东,吾鲁木汗·那孜尔别克,段世雄,等.禽多杀性巴氏杆菌C48-3株外膜蛋白H的致病作用[J].中国生物工程杂志,2014,34(6):31-39.WEI D,WULUMUHAN·NZEBK,DUAN S X,et al.Role of outer membrane protein H in the pathogenesis of avian Pasteurella multicida strain C48-3[J].China Biotechnology,2014,34(6):31-39.(in Chinese)
[25] 蒋玉亮,兰萍,李艳,等.一种基于OmpA的分泌型原核表达载体的构建及应用[J].成都医学院学报,2016,11(4):409-412.JIANG Y L,LAN P,LI Y,et al.Construction and application of a secreted prokaryotic expression vector based on OmpA signal peptide[J].Journal of Chengdu Medical College,2016,11(4):409-412.(in Chinese)
[26] MEUSKENS I,MICHALIK M,CHAUHAN N,et al.A new strain collection for improved expression of outer membrane proteins[J].Frontiers in Cellular and Infection Microbiology,2017,7:464.
[27] PENG B,WANG C,LI H,et al.Outer membrane proteins form specific patterns in antibiotic-resistant Edwardsiella tarda[J].Frontiers in Microbiology,2017,8:69.
[28] 白雪瑞,王权,陈永军,等.副溶血弧菌ompA基因缺失株的生物学特性及致病性分析[J].南京农业大学学报,2018,41(5):902-910.BAI X R,WANG Q,CHEN Y J,et al.Biological characteristics and pathogenicity of an ompA mutant of Vibrio parahaemolyticus[J].Journal of Nanjing Agricultural University,2018,41(5):902-910.(in Chinese)