羊源多杀性巴氏杆菌ompW基因的克隆及生物信息学分析
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摘要
试验旨在克隆羊源多杀性巴氏杆菌ompW基因,并对其序列进行生物信息学分析。根据GenBank中多杀性巴氏杆菌HN07株ompW基因序列(登录号:CP007040.1),使用DNAMAN 5.0软件设计1对引物,选取高保真酶PrimeSTARMax DNA Polymerase进行PCR反应获取目的基因片段,并对ompW基因的核苷酸序列及预测的氨基酸序列进行生物信息学分析。结果表明,PCR扩增产物约为615bp,编码204个氨基酸。核苷酸同源性比对分析显示,羊源多杀性巴氏杆菌ompW基因与猪源、牛源、禽源多杀性巴氏杆菌同源性较高,而与兔源同源性较低。系统进化树结果发现,羊源多杀性巴氏杆菌ompW基因与猪源多杀性巴氏杆菌ompW基因亲缘关系最近。经生物信息学分析发现,ompW蛋白分子式为C1007H1567N257O283S3,分子质量为21.90ku,理论等电点(pI)为9.16,属碱性蛋白质,疏水指数为96.57,总平均疏水性(GRAVY)为0.173(>0),属于疏水类蛋白;前21位氨基酸为信号肽,第5-27位氨基酸区域存在1个跨膜区,存在N-糖基化位点及磷酸化位点,不存在O-糖基化位点,具有多个B细胞、CTL细胞及Th细胞抗原表位;二级结构的α-螺旋、延伸链、β-转角和无规则卷曲分别占17.65%、35.29%、3.92%和43.14%;三级结构是呈β-桶状的单聚体,隶属于外膜蛋白家族成员之一。本研究结果为进一步阐明羊源多杀性巴氏杆菌侵染宿主过程中自身的抗宿主免疫胁迫机制及疫苗的开发与研制提供了理论依据。
This study was aimed to clone ompW gene of Pasteurella multocidain goat and carry out bioinformatics analysis.According to the ompWgene sequence of Pasteurella multocidastrain HN07(accession No.:CP007040.1)in GenBank,one pair of primers were designed using DNAMAN 5.0software,and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase was se-lected for PCR reaction to obtained the target gene fragment,the nucleotide and amino acid sequences of ompW gene were analyzed by bioinformatics softwares.The results showed that the length of PCR product was about 615 bp,which encoded 204 amino acids.The nucleotide homology analysis result showed that ompW gene of Pasteurella multocidain goat was highly homologous with porcine,bovine and avian-derived Pasteurella multocida,but not highly homologous to Pasteurella multocidain rabbit.The phylogenetic tree result showed that the relationship was the closest between ompW gene of Pasteurella multocida in goat and porcine.The bioinformatics analysis results showed that the molecular formula of ompW protein was C1007H1567N257O283S3,the molecular weight was 21.90 ku,the theoretical isoelectric point(pI)was 9.16,which was a basic protein with a hydrophobic index of 96.57 and a total average hydrophobicity(GRAVY)of 0.173(>0),it belonged to a hydrophobic protein.The first 21 amino acids were signal peptides,the 5th to 27 th amino acid regions had a transmembrane region,there were N-glycosylation and phosphorylation sites,no O-glycosylation site,and there were multiple B cell,CTL cell and Th cell epitopes.The alpha helix,extended chain,beta turn and random coil of the secondary structure accounts for 17.65%,35.29%,3.92% and 43.14%,respectively.The tertiary structure was a beta barrel monomer belonging to one of the members of the outer membrane protein family.These results provided a theoretical basis for further elucidating the mechanism of anti-host immune stress and the development of vaccines in the process of infecting host of Pasteurella multocida.
This study was aimed to clone ompW gene of Pasteurella multocidain goat and carry out bioinformatics analysis.According to the ompWgene sequence of Pasteurella multocidastrain HN07(accession No.:CP007040.1)in GenBank,one pair of primers were designed using DNAMAN 5.0software,and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase was se-lected for PCR reaction to obtained the target gene fragment,the nucleotide and amino acid sequences of ompW gene were analyzed by bioinformatics softwares.The results showed that the length of PCR product was about 615 bp,which encoded 204 amino acids.The nucleotide homology analysis result showed that ompW gene of Pasteurella multocidain goat was highly homologous with porcine,bovine and avian-derived Pasteurella multocida,but not highly homologous to Pasteurella multocidain rabbit.The phylogenetic tree result showed that the relationship was the closest between ompW gene of Pasteurella multocida in goat and porcine.The bioinformatics analysis results showed that the molecular formula of ompW protein was C1007H1567N257O283S3,the molecular weight was 21.90 ku,the theoretical isoelectric point(pI)was 9.16,which was a basic protein with a hydrophobic index of 96.57 and a total average hydrophobicity(GRAVY)of 0.173(>0),it belonged to a hydrophobic protein.The first 21 amino acids were signal peptides,the 5th to 27 th amino acid regions had a transmembrane region,there were N-glycosylation and phosphorylation sites,no O-glycosylation site,and there were multiple B cell,CTL cell and Th cell epitopes.The alpha helix,extended chain,beta turn and random coil of the secondary structure accounts for 17.65%,35.29%,3.92% and 43.14%,respectively.The tertiary structure was a beta barrel monomer belonging to one of the members of the outer membrane protein family.These results provided a theoretical basis for further elucidating the mechanism of anti-host immune stress and the development of vaccines in the process of infecting host of Pasteurella multocida.
引文
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[19]BENDER B S,ULMER J B,DEWITT C M,et al.Immunogenicity and efficacy of DNA vaccines encoding influenza A proteins in aged mice[J].Vaccine,1998,16(18):1748-1755.
[20]周宏展.简析基因突变对生物变异与进化的意义[J].科学大众(科学教育),2016,12:51.ZHOU H Z.A brief analysis of the significance of gene mutations to biological variation and evolution[J].Scientific Public(Scientific Education),2016,12:51.(in Chinese)
[21]张西燕,付玉荣,伊正君.结核分枝杆菌eis基因及其编码蛋白的生物信息学分析[J].中国病原生物学杂志,2018,13(2):140-151.ZHANG X Y,FU Y R,YI Z J.Bioinformatic analysis of the eis gene of Mycobacterium tuberculosis and the protein it codes for[J].Journal of Pathogen Biology,2018,13(2):140-151.(in Chinese)
[22]MORALES E H,CALDERN I L,COLLAO B,et al.Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella entericaserovar Typhimurium OmpW by the response regulator ArcA[J].BMC Microbiology,2012,12(1):63.
[23]QIAN R H,XIAO Z H,ZHANG C W,et al.Expression and purification of two major outer membrane proteins fromVibrio alginolyticus[J].World Journal of Microbiology and Biotechnology,2008,24(2):245-251.
[2]王娉,张富春.多杀性巴氏杆菌毒力因子的研究进展[J].地方病通报,2006,21(6):95-99.WANG P,ZHANG F C.Research progress on virulence factors of Pasteurella multocida[J].Endemic Diseases Bulletin,2006,21(6):95-99.(in Chinese)
[3]蔡广强.多杀性巴氏杆菌毒力因子研究进展[J].上海畜牧兽医通讯,2013,6:7-9.CAI G Q.Research progress on virulence factors of Pasteurella multocida[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2013,6:7-9.(in Chinese)
[4]王帅涛,宫强,秦翠丽.多杀性巴氏杆菌毒力因子、免疫原及重要基因研究进展[J].中国畜牧兽医,2010,37(3):186-188.WANG S T,GONG Q,QIN C L.The research advance of virulence factors immunogen and main genes of Pasteurella multocida[J].China Animal Husbandry&Veterinary Medicine,2010,37(3):186-188.(in Chinese)
[5]彭忠,梁婉,吴斌.多杀性巴氏杆菌分子分型方法简述[J].微生物学报,2016,56(10):1521-1529.PENG Z,LIANG W,WU B.Molecular typing methods for Pasteurella multocida:A review[J].Acta Microbiologica Sinica,2016,56(10):1521-1529.(in Chinese)
[6]王凤阳,范泉水.兽医微生物学[M].北京:科学出版社,2007.WANG F Y,FAN Q S.Veterinary Microbiology[M].Beijing:Science Press,2007.(in Chinese)
[7]EWERS C,LUBKE-BECKER A,BETHE A,et al.Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J].Veterinar Microbiology,2006,8114:304-317.
[8]WILKIE I W,HARPER M,BOYCE J D,et al.Pasteurella multocida:Diseases and pathogenesis[J].Current Topics in Microbiology and Immunology,2012,361:1-22.
[9]NIKAIDO H.Molecular basis of bacterial outer membrane permeability revisited[J].Microbiology and Molecular Biology Reviews,2003,67:593-656.
[10]MAY B J,ZHANG Q,LI L L,et al.Complete genomic sequence of Pasteurella multocida,Pm70[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(6):3460-3465.
[11]HORST R,STANCZAK P,WTHRICH K.NMRpolypeptide backbone conformation of the E.coli outer membrane protein W[J].Structure,2014,22(8):1204-1209.
[12]HONG H,PATEL D R,TAMM L K,et al.The outer membrane protein OmpW forms an eight-strandedβ-barrel with a hydrophobic channel[J].The Journal of Biological Chemistry,2006,281(11):7568-7577.
[13]DEVI L B,BORA D P,DAS S K,et al.Virulence gene profiling of porcine Pasteurella multocida isolates of Assam[J].Veterinary World,2018,11(3):348-354.
[14]WILSON B A,HO M.Pasteurella multocida:Form zoonosis to cellular microbiology[J].Clinical Microbiology Reviews,2013,26(3):631-655.
[15]GONG Q,QU N,NIU M F,et al.Immune responses and protective efficacy of a novel DNA vaccine encoding outer membrane protein of avian Pasteurella multocida[J].Veterinary Immunology and Immunopathology,2013,152(3-4):317-324.
[16]ADLER B,BULACH D,CHUNG J,et al.Candidate vaccine antigens and genes in Pasteurella multocida[J].Journal of Biotechnology,1999,73(2-3):83-90.
[17]REN W K,ZOU L X,RUAN Z,et al.Dietary L-proline supplementation confers immunostimulatory effects on inactivated Pasteurella multocida vaccine immunized mice[J].Amino Acids,2013,45(3):555-561.
[18]SHIVACHANDRA S B,YOGISHARADHYA R,AHUJA A,et al.Expression and purification of recombinant typeⅣfimbrial subunit protein of Pasteurella multocidaserogroup B:2in Escherichia coli[J].Research in Veterinary Science,2012,93(3):1128-1131.
[19]BENDER B S,ULMER J B,DEWITT C M,et al.Immunogenicity and efficacy of DNA vaccines encoding influenza A proteins in aged mice[J].Vaccine,1998,16(18):1748-1755.
[20]周宏展.简析基因突变对生物变异与进化的意义[J].科学大众(科学教育),2016,12:51.ZHOU H Z.A brief analysis of the significance of gene mutations to biological variation and evolution[J].Scientific Public(Scientific Education),2016,12:51.(in Chinese)
[21]张西燕,付玉荣,伊正君.结核分枝杆菌eis基因及其编码蛋白的生物信息学分析[J].中国病原生物学杂志,2018,13(2):140-151.ZHANG X Y,FU Y R,YI Z J.Bioinformatic analysis of the eis gene of Mycobacterium tuberculosis and the protein it codes for[J].Journal of Pathogen Biology,2018,13(2):140-151.(in Chinese)
[22]MORALES E H,CALDERN I L,COLLAO B,et al.Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella entericaserovar Typhimurium OmpW by the response regulator ArcA[J].BMC Microbiology,2012,12(1):63.
[23]QIAN R H,XIAO Z H,ZHANG C W,et al.Expression and purification of two major outer membrane proteins fromVibrio alginolyticus[J].World Journal of Microbiology and Biotechnology,2008,24(2):245-251.