羊源多杀性巴氏杆菌sodA基因的克隆、表达及其生物信息学分析
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摘要
为了解羊源多杀性巴氏杆菌超氧化物歧化酶(SOD)的生物学功能,本试验对该菌sodA基因进行克隆及原核表达,并对克隆的sodA基因进行遗传进化树分析,对其表达的SOD蛋白进行生物信息学分析。参照GenBank中多杀性巴氏杆菌HN06株基因组中sodA基因序列信息设计引物进行PCR扩增,将产物与pET-28a(+)载体相连,构建pET-28a(+)-sodA重组质粒,将该质粒转化E.coli DH5α感受态细胞进行克隆,再转化E.coli BL21(DE3)感受态细胞进行表达,经IPTG诱导后对表达蛋白进行SDS-PAGE和Western blotting鉴定分析。结果显示,本试验成功扩增出大小为645bp的目的片段,并表达出大小约28ku的目的蛋白。遗传进化树分析表明,该基因与HN07(GenBank登录号:CP007040.1)和Pm70(GenBank登录号:AE004439.1)株亲缘关系较近,重组蛋白生物信息学分析显示,该融合蛋白为稳定的酸性亲水可溶性蛋白,分子式为C1085H1651N293O309S9,分子质量为24 032.36u,理论等电点为6.19,消光系数为45 170,不稳定系数为26.87(<40),在哺乳动物网织红细胞的半衰期预计为30h,疏水指数为82.15,总平均疏水性(GRAVY)为-0.282,二级结构以α-螺旋和无规则卷曲为主。以上研究结果为后续深入研究多杀性巴氏杆菌在羊体内的存活机制及研发预防巴氏杆菌病的疫苗提供了参考。
To investigate the biological functions of superoxide dismutase(SOD)of Pasteurella multocida,the sodAgene was cloned,expressed and analyzed by phylogenetic and bioinformatics,respectively.A pair of primers was designed based on the sodAgene sequence of Pasteurella multocida HN06 strain in GenBank.The PCR products were ligated with pET-28 a(+)vector to construct pET-28 a(+)-sodA recombinant plasmids.The recombinant plasmids were transformed into E.coli DH5αcompetent cells for cloning and then transformed them into E.coli BL21(DE3)competent cells for expression.The expressed proteins were induced by IPTG and identified by SDS-PAGE and Western blotting.The results showed that the target fragment with the length of645 bp was successfully amplified and the target protein which was 28 ku was successfully expressed.Phylogenetic analysis showed that the gene was closely related to the HN07(GenBank accession No.:CP007040.1)and Pm70(GenBank accession No.:AE004439.1)strains.Bioinformatics analysis results of the recombinant protein showed that it was a stable acidic hydrophilic soluble fusion protein.The molecular formula and molecular mass were C1085 H1651 N293 O309 S9 and24 032.36 u;The theoretical isoelectric point,extinction coefficient and instability coefficient were6.19,45 170 and 26.87(<40),repectively;The half-life of reticulocytes in mammals was estimated to be 30 h;The aliphatic index and total average hydrophobicity(GRAVY)were 82.15 and-0.282,respectively;And the secondary structure was mainly composed ofα-helix and random coil.The above results provided references for the further study of the survival mechanism of Pasteurella multocida and the development of a vaccine against pasteurellosis.
To investigate the biological functions of superoxide dismutase(SOD)of Pasteurella multocida,the sodAgene was cloned,expressed and analyzed by phylogenetic and bioinformatics,respectively.A pair of primers was designed based on the sodAgene sequence of Pasteurella multocida HN06 strain in GenBank.The PCR products were ligated with pET-28 a(+)vector to construct pET-28 a(+)-sodA recombinant plasmids.The recombinant plasmids were transformed into E.coli DH5αcompetent cells for cloning and then transformed them into E.coli BL21(DE3)competent cells for expression.The expressed proteins were induced by IPTG and identified by SDS-PAGE and Western blotting.The results showed that the target fragment with the length of645 bp was successfully amplified and the target protein which was 28 ku was successfully expressed.Phylogenetic analysis showed that the gene was closely related to the HN07(GenBank accession No.:CP007040.1)and Pm70(GenBank accession No.:AE004439.1)strains.Bioinformatics analysis results of the recombinant protein showed that it was a stable acidic hydrophilic soluble fusion protein.The molecular formula and molecular mass were C1085 H1651 N293 O309 S9 and24 032.36 u;The theoretical isoelectric point,extinction coefficient and instability coefficient were6.19,45 170 and 26.87(<40),repectively;The half-life of reticulocytes in mammals was estimated to be 30 h;The aliphatic index and total average hydrophobicity(GRAVY)were 82.15 and-0.282,respectively;And the secondary structure was mainly composed ofα-helix and random coil.The above results provided references for the further study of the survival mechanism of Pasteurella multocida and the development of a vaccine against pasteurellosis.
引文
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[8]张振兴,聂鑫,杨小健,等.类鼻疽伯克霍尔德菌BPSS0180基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(11):3313-3319.ZHANG Z X,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformatics analysis of BPSS0180gene of Burkholderia pseudomallei[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3313-3319.(in Chinese)
[9]李国华,彭冬梅,李亚颖,等.马尔他布氏杆菌LpxK基因的克隆及原核表达[J].中国兽医科学,2015,45(9):959-962.LI G H,PENG D M,LI Y Y,et al.Cloning and prokaryotic expression of LpxK gene from Brucella melitensis[J].Chinese Veterinary Science,2015,45(9):959-962.(in Chinese)
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[11]李宝宝,聂鑫,杨小健,等.羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953.LI B B,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformation analysis of dhbC gene of Brucella melitensis[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1947-1953.(in Chinese)
[12]徐开莲,朱华培,赵天靖,等.羊种布鲁氏菌Omp10基因的克隆及原核表达[J].中国畜牧兽医,2014,41(12):122-125.XU K L,ZHU H P,ZHAO T J,et al.Cloning and prokaryotic expression of Omp10 gene of Brucella melitensis[J].China Animal Husbandry&Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[13]黄海峰,张振兴,杨小健,等.类鼻疽伯克霍尔德菌groEL基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2018,45(2):302-309.HUANG H F,ZHANG Z X,YANG X J,et al.Cloning,prokaryotic expression and protein bioinformatics analysis of groEL gene of Burkholderia pseudomallei[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):302-309.(in Chinese)
[14]华瑞其,赵新新,程安春.多杀性巴氏杆菌脂多糖的结构与功能研究进展[J].畜牧兽医学报,2016,47(10):1961-1968.HUA R Q,ZHAO X X,CHENG A C.Research progress in the lipopolysaccharide of Pasteurella multocida[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1961-1968.(in Chinese)
[15]高家登,扎西达瓦,曲久,等.多杀性巴氏杆菌病的研究[J].中国畜禽种业,2017,13(9):35-37.GAO J D,ZHA X D W,QU J,et al.Study on Pasteurella multocidasis[J].The Chinese Livestock and Poultry Breeding,2017,13(9):35-36.(in Chinese)
[16]HARPER M,BOYCE J D,ADLER B.The key surface components of Pasteurella multocida:Capsule and lipopolysaccharide[J].Current Topics in Microbiology and Immunology,2012,361:39-51.
[17]HARPER M,COX A D,ADLER B,et al.Pasteurella multocidalipopolysaccharide:The long and the short of it[J].Veterinary Microbiology,2011,153(1):109-115.
[18]SIEGERT P,SCHMIDT G,PAPATHEODOROU P,et al.Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade viathe Gα(q/11)-p63RhoGEF-RhoA axis[J].PLoS Pathogens,2013,9(5):e1003385.
[19]KATOCH S,SHARMA M,PATIL R,et al.In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA[J].Veterinary Research Communications,2014,38(3):183-191.
[20]BAGCHI A.Structural characterization of Fis-A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence factors[J].Gene,2015,554(2):249-253.
[21]唐宇龙.猪链球菌2型Sortases、CcpA和SodA功能与致病力研究[D].杭州:浙江大学,2012.TANG Y L.Functional analysis of Sortases,CcpA and SodA of Streptococcus suis type 2in relation to pathogenicity[D].Hangzhou:Zhejiang University,2012.(in Chinese)
[22]赵战勤,乔鹏芸,刘倩玉,等.多杀性巴氏杆菌的分型及其灭活疫苗研究进展[J].中国预防兽医学报,2017,39(7):600-603.ZHAO Z Q,QIAO P Y,LIU Q Y,et al.Advances in the classification of inactivated Pasteurella multocida and its inactivated vaccines[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(7):600-603.(in Chinese)
[23]黄庶识,卢明倩,李冰,等.重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J].中国激光,2014,41(12):251-259.HUANG S S,LU M Q,LI B,et al.Real-time detection on the expression of soluble protein and inclusion body in the recombinant Escherichia coli with laser tweezers Raman spectroscopy[J].Chinese Journal of Lasers,2014,41(12):251-259.(in Chinese)
[24]石华建.生物螺旋结构的奥妙与应用[J].生物学通报,2008,42(9):28-29.SHI H J.The mystery and application of biological spiral structure[J].Bulletin of Biology,2008,42(9):28-29.(in Chinese)
[25]王镜岩,朱圣庚,徐长发.生物化学[M].北京:高等教育出版社,1980.WANG J Y,ZHU S G,XU C F.Biochemistry[M].Beijing:Higher Education Press,1980.(in Chinese)
[2]彭忠,梁婉,刘文静,等.多杀性巴氏杆菌HN06基因组分泌蛋白的预测与分析[J].动物医学进展,2015,36(12):6-10.PENG Z,LIANG W,LIU W J,et al.Prediction and analysis of secreted proteins encoding genes in genome of Pasteurella multocida HN06[J].Progress in Veterinary Medicine,2015,36(12):6-10.(in Chinese)
[3]曾东柱.多杀性巴氏杆菌HN06株新型免疫蛋白的鉴定及PGAM免疫功能研究[D].武汉:华中农业大学,2014.ZENG D Z.Identification of novel immunogenic proteins of Pasteurella multocida HN06and study on the immune function of PGAM[D].Wuhan:Huazhong Agricultural University,2014.(in Chinese)
[4]王松艳,蔡双虎,鲁义善,等.无乳链球菌sodA基因的克隆、序列分析及原核表达载体构建[J].广东海洋大学学报,2016,36(3):15-19.WANG S Y,CAI S H,LU Y S,et al.Cloning and bioinformatics analysis of the sodAgene fromStreptococcus agalactiae and constructing pET28a-sodA plasmid[J].Journal of Guangdong Ocean University,2016,36(3):15-19.(in Chinese)
[5]张健康,汪洋,易力,等.鼠伤寒沙门菌超氧化物歧化酶SodA基因的表达及其酶活性的测定[J].中国兽医科学,2017,47(8):975-980.ZHANG J K,WANG Y,YI L,et al.Expression of Salmonella TyphimuriumSodA gene and determination of activities of the recombinant sodA[J].Chinese Veterinary Science,2017,47(8):975-980.(in Chinese)
[6]迟阳.副猪嗜血杆菌的分离鉴定及其16SrRNA和sodA基因的序列分析[D].长春:吉林农业大学,2013.CHI Y.Isolation and identification of Haemophilus parasuis and its sequence analysis on 16SrRNA and sodAgene[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[7]GAUTIER A L,DUBOIS D,ESCANDE F,et al.Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene[J].Journal of Clinical Microbiology,2005,43(5):2307-2314.
[8]张振兴,聂鑫,杨小健,等.类鼻疽伯克霍尔德菌BPSS0180基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(11):3313-3319.ZHANG Z X,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformatics analysis of BPSS0180gene of Burkholderia pseudomallei[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3313-3319.(in Chinese)
[9]李国华,彭冬梅,李亚颖,等.马尔他布氏杆菌LpxK基因的克隆及原核表达[J].中国兽医科学,2015,45(9):959-962.LI G H,PENG D M,LI Y Y,et al.Cloning and prokaryotic expression of LpxK gene from Brucella melitensis[J].Chinese Veterinary Science,2015,45(9):959-962.(in Chinese)
[10]聂鑫,赵天靖,曹瑞勇,等.羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2016,43(7):1681-1687.NIE X,ZHAO T J,CAO R Y,et al.Cloning,prokaryotic expression and bioinformatics analysis of LpxB gene of Brucella melitensis[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1681-1687.(in Chinese)
[11]李宝宝,聂鑫,杨小健,等.羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953.LI B B,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformation analysis of dhbC gene of Brucella melitensis[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1947-1953.(in Chinese)
[12]徐开莲,朱华培,赵天靖,等.羊种布鲁氏菌Omp10基因的克隆及原核表达[J].中国畜牧兽医,2014,41(12):122-125.XU K L,ZHU H P,ZHAO T J,et al.Cloning and prokaryotic expression of Omp10 gene of Brucella melitensis[J].China Animal Husbandry&Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[13]黄海峰,张振兴,杨小健,等.类鼻疽伯克霍尔德菌groEL基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2018,45(2):302-309.HUANG H F,ZHANG Z X,YANG X J,et al.Cloning,prokaryotic expression and protein bioinformatics analysis of groEL gene of Burkholderia pseudomallei[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):302-309.(in Chinese)
[14]华瑞其,赵新新,程安春.多杀性巴氏杆菌脂多糖的结构与功能研究进展[J].畜牧兽医学报,2016,47(10):1961-1968.HUA R Q,ZHAO X X,CHENG A C.Research progress in the lipopolysaccharide of Pasteurella multocida[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1961-1968.(in Chinese)
[15]高家登,扎西达瓦,曲久,等.多杀性巴氏杆菌病的研究[J].中国畜禽种业,2017,13(9):35-37.GAO J D,ZHA X D W,QU J,et al.Study on Pasteurella multocidasis[J].The Chinese Livestock and Poultry Breeding,2017,13(9):35-36.(in Chinese)
[16]HARPER M,BOYCE J D,ADLER B.The key surface components of Pasteurella multocida:Capsule and lipopolysaccharide[J].Current Topics in Microbiology and Immunology,2012,361:39-51.
[17]HARPER M,COX A D,ADLER B,et al.Pasteurella multocidalipopolysaccharide:The long and the short of it[J].Veterinary Microbiology,2011,153(1):109-115.
[18]SIEGERT P,SCHMIDT G,PAPATHEODOROU P,et al.Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade viathe Gα(q/11)-p63RhoGEF-RhoA axis[J].PLoS Pathogens,2013,9(5):e1003385.
[19]KATOCH S,SHARMA M,PATIL R,et al.In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA[J].Veterinary Research Communications,2014,38(3):183-191.
[20]BAGCHI A.Structural characterization of Fis-A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence factors[J].Gene,2015,554(2):249-253.
[21]唐宇龙.猪链球菌2型Sortases、CcpA和SodA功能与致病力研究[D].杭州:浙江大学,2012.TANG Y L.Functional analysis of Sortases,CcpA and SodA of Streptococcus suis type 2in relation to pathogenicity[D].Hangzhou:Zhejiang University,2012.(in Chinese)
[22]赵战勤,乔鹏芸,刘倩玉,等.多杀性巴氏杆菌的分型及其灭活疫苗研究进展[J].中国预防兽医学报,2017,39(7):600-603.ZHAO Z Q,QIAO P Y,LIU Q Y,et al.Advances in the classification of inactivated Pasteurella multocida and its inactivated vaccines[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(7):600-603.(in Chinese)
[23]黄庶识,卢明倩,李冰,等.重组大肠杆菌表达可溶性蛋白和包涵体过程的拉曼光谱实时分析[J].中国激光,2014,41(12):251-259.HUANG S S,LU M Q,LI B,et al.Real-time detection on the expression of soluble protein and inclusion body in the recombinant Escherichia coli with laser tweezers Raman spectroscopy[J].Chinese Journal of Lasers,2014,41(12):251-259.(in Chinese)
[24]石华建.生物螺旋结构的奥妙与应用[J].生物学通报,2008,42(9):28-29.SHI H J.The mystery and application of biological spiral structure[J].Bulletin of Biology,2008,42(9):28-29.(in Chinese)
[25]王镜岩,朱圣庚,徐长发.生物化学[M].北京:高等教育出版社,1980.WANG J Y,ZHU S G,XU C F.Biochemistry[M].Beijing:Higher Education Press,1980.(in Chinese)