副猪嗜血杆菌辽宁株tbpA基因鉴定与分析
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摘要
副猪嗜血杆菌(HPS)是感染猪产生浆膜炎、关节炎、脑膜炎的病原菌,为明确辽宁省副猪嗜血杆菌tbp A基因的保守性、遗传变异情况以及功能,以复苏培养后的菌株为研究对象,利用细菌学常规技术和PCR方法对HPS及其血清型进行验证,将目的基因连接于T5载体上,并转化到大肠杆菌感受态细胞中,获得克隆质粒,对不同血清型该基因序列进行PCR鉴定。通过NCBI网站中BLAST进行序列比对,并且运用DNA star软件进行核苷酸同源性分析和遗传变异规律分析。通过在线软件对tbp A编码蛋白的氨基酸组成、分子量、等电点、亲疏水性以及二级结构等进行分析。结果表明:从保存菌的HPS中成功复苏了4型、5型、9型和14型菌株;经PCR扩增这4种血清型HPS的tbp A基因长度均为2700bp;tbp A基因与参考菌株核苷酸同源性范围为86.5%~97.9%,4型和5型tbp A基因与84~(-1)5995、84~(-1)7975、H425亲缘性较近,9型tbp A基因与SW124和174亲缘性较近;14型tbp A基因与NO.4亲缘性较近;4种血清型tbp A基因与瑞典菌株D74在两个不同分支上,具有较远的亲缘关系。4种血清型tbp A基因编码蛋白分别有874,871,918,915个氨基酸组成;等电点分别为9.51,9.30,8.65,8.76;该蛋白属于亲水性蛋白;该蛋白的二级结构主要由α螺旋、无规则卷曲和伸展链构成。tbp A基因与美国株84~(-1)5995(KT588784.1)、德国株H425(KT588778.1)、日本株NO.4(KT588774.1)同源性相对较高,亲缘关系较近,但是也存在一定的差异性,该结果可为建立HPS最新基因突变跟踪提供参考依据;同时,通过对tbp A蛋白进行生物学信息分析,为进一步研究tbp A基因的功能奠定了分子基础。
Haemophilus parasuis(HPS) is the pathogen of serovars, arthritis and meningitis in pigs. The clarification of the conservation and genetic variation and function of the tbp A gene of Haemophilus parasuis in Liaoning province is of great significance for the prevention and control of this disease. In this study, the resuscitated and cultured strains were the research objects. HPS and its serotypes were verified by bacteriology conventional techniques and PCR methods. The target genes were connected to T5 vector and transformed into E. coli competent cells. The sequences of different serotypes were identified by PCR. The sequences were compared with other strains by using BLAST on the NCBI website and DNA star software was used for nucleotide homology analysis and genetic variation analysis. The amino acid composition, molecular weight, isoelectric point,hydrophobicity and secondary structure of tbp A-encoded proteins were analyzed by using online software. Type 4, 5, 9 and 14 strains were successfully resuscitated from HPS, and the tbp A gene length of these four serotypes of HPS was 2700 bp by PCR.The nucleotide homology between tbp A genes and reference strains ranged from 86.5% to 97.9%. The tbp A gene of type 4 and type 5 was close to that of 84~(-1)5995, 84~(-1)7975 and H425, and the tbp A gene of type 9 was close to that of SW124 and SW174. Type 14 tbp A gene was close to NO.4 gene. The four serotype tbp A genes and the Swedish strain D74 had distant phylogenetic relationships on two different branches. The four serotype tbp A genes encoding proteins had 874, 871, 918 and 915 amino acids, respectively. The isoelectric points were 9.51, 9.30, 8.65 and 8.76, respectively. The protein was a hydrophilic protein with a secondary structure of Alpha helix mainly, random curl, and stretch chain. The tbp A gene had higher homology and closer relatives to the American strain 84-15995(KT588784.1), German strain H425(KT588778.1), Japanese strain NO.4(KT588774.1), however, there were also some differences. The results could provide a reference for the establishment of HPS latest gene mutation tracking. The bioinformatics analysis of tbp A protein laid a molecular foundation for further study of tbp A gene function.
Haemophilus parasuis(HPS) is the pathogen of serovars, arthritis and meningitis in pigs. The clarification of the conservation and genetic variation and function of the tbp A gene of Haemophilus parasuis in Liaoning province is of great significance for the prevention and control of this disease. In this study, the resuscitated and cultured strains were the research objects. HPS and its serotypes were verified by bacteriology conventional techniques and PCR methods. The target genes were connected to T5 vector and transformed into E. coli competent cells. The sequences of different serotypes were identified by PCR. The sequences were compared with other strains by using BLAST on the NCBI website and DNA star software was used for nucleotide homology analysis and genetic variation analysis. The amino acid composition, molecular weight, isoelectric point,hydrophobicity and secondary structure of tbp A-encoded proteins were analyzed by using online software. Type 4, 5, 9 and 14 strains were successfully resuscitated from HPS, and the tbp A gene length of these four serotypes of HPS was 2700 bp by PCR.The nucleotide homology between tbp A genes and reference strains ranged from 86.5% to 97.9%. The tbp A gene of type 4 and type 5 was close to that of 84~(-1)5995, 84~(-1)7975 and H425, and the tbp A gene of type 9 was close to that of SW124 and SW174. Type 14 tbp A gene was close to NO.4 gene. The four serotype tbp A genes and the Swedish strain D74 had distant phylogenetic relationships on two different branches. The four serotype tbp A genes encoding proteins had 874, 871, 918 and 915 amino acids, respectively. The isoelectric points were 9.51, 9.30, 8.65 and 8.76, respectively. The protein was a hydrophilic protein with a secondary structure of Alpha helix mainly, random curl, and stretch chain. The tbp A gene had higher homology and closer relatives to the American strain 84-15995(KT588784.1), German strain H425(KT588778.1), Japanese strain NO.4(KT588774.1), however, there were also some differences. The results could provide a reference for the establishment of HPS latest gene mutation tracking. The bioinformatics analysis of tbp A protein laid a molecular foundation for further study of tbp A gene function.
引文
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[13]MELNIKOWA E,DOMAN S,SARGENT C,et al.Microarray analysis of Haemophilus parasuis gene expression under in vitro growth condotion mimicking the in vivo environment[J].Vet Microbiol,2005,110:255-123.
[14]贾爱卿,王贵平,宣华,等.副猪嗜血杆菌毒力因子的研究进展[J].中国动物传染病学报,2015,23(4):72-80.
[15]OLVERA A,PINA S,PEREZ-SIMO M,et al.Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo[J].Vet Res,2010,41(3):26.
[16]李建军,胡明明,朱晓凯,等.副猪嗜血杆菌血清5型Tbp A蛋白的表达及其免疫原性分析[J].中国预防兽医学报,2012,34(3):239-240.
[17]DELRIO ML,GUTIERREZ-MARTIN CB,RODRIGUEZ-BARBOSA JI,et a I.Indentification and characterization of the Ton B region and its role in transferring-mediated iron acquisition in Haemophilus parasuis[J].Fems Immunol Med Microbiol,2005,45(1):75-86.
[18]李郁,陈申秒,王桂军,等.安徽PRSS发病猪副猪嗜血杆菌的分离鉴定及其耐药性分析[J].中国微生物学杂志,2009,21(8):682-684.
[19]KIM TJ,LEE JI.Cloning and expression of genes encoding transferring-binding protein A and B from Actinobacillus pleuropneumoruae serotype 5[J].Protein Expr Purif,2006,45(1):235-240.
[20]黄晓慧.副猪嗜血杆菌安徽分离株的耐药性与转铁结合蛋白A免疫原性研究[D].合肥:安徽农业大学,2012.
[2]DAI K,HE L,CHANG Y F,et al.Basic characterization of natural transformation in a highly transformable Haemophilus parasuis strain SC1401[J].Front Cell Infect Microbiol,2018,8:32.
[3]HUANG J,WANG X,CAO Q,et al.Clp P participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis[J].Vet Microbiol,2016,182:141-149.
[4]OLIVEIRA S,BLAEKALL P J,POJOAN C.Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping[J].American Journal of Veterinary Research,2003,64:435-442.
[5]CASTILLA K S,DE GOBBI D D,MORENO L Z,et al.Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping,AFLP and PFGE[J].Research in Veterinary Science,2012,92:366-371.
[6]OLVERA A,BALLESTER M,NOFRARIAS M,et al.differences in phagocyosis susceptibility in Haemophilus parasuis strains[J].Vet Res,2009,40(3):24.
[7]LIU S,LI W,WANG Y,et al.Coinfection with Haemophilus parasuis serovar 4 increases the virulence of porcine circovirus type 2 in piglets[J].Virol J,2017,14(1):227.
[8]KAVANOV魣L,PRODE姚LALOV魣J,NEDBALCOV魣K,et al.Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro[J].Vet Microbiol,2015,180(1-2):28-35.
[9]李静怡,张建民,徐成刚,等.副猪嗜血杆菌主要毒力因子和致病机理的研究进展[J].中国畜牧兽医,2010,37(12):173-176.
[10]HUANG X,LI Y,FU Y,et al.Cross-protective efficacy of recombinant transferrin-binding protein A of Haemophilus parasuis in guinea pigs[J].Clin Vaccine Immunol,2013,20(6):912-919.
[11]CHARLAND N,D`SILVA C G,DUMONT R A,et al.Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis[J].Can J Microbiol,1995,41(1):70-74.
[12]黄润标,李小军,叶广胜,等.副猪嗜血杆菌的流行病学及预防保护研究发展[J].广东农业科学,2010,37(8):175-177.
[13]MELNIKOWA E,DOMAN S,SARGENT C,et al.Microarray analysis of Haemophilus parasuis gene expression under in vitro growth condotion mimicking the in vivo environment[J].Vet Microbiol,2005,110:255-123.
[14]贾爱卿,王贵平,宣华,等.副猪嗜血杆菌毒力因子的研究进展[J].中国动物传染病学报,2015,23(4):72-80.
[15]OLVERA A,PINA S,PEREZ-SIMO M,et al.Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo[J].Vet Res,2010,41(3):26.
[16]李建军,胡明明,朱晓凯,等.副猪嗜血杆菌血清5型Tbp A蛋白的表达及其免疫原性分析[J].中国预防兽医学报,2012,34(3):239-240.
[17]DELRIO ML,GUTIERREZ-MARTIN CB,RODRIGUEZ-BARBOSA JI,et a I.Indentification and characterization of the Ton B region and its role in transferring-mediated iron acquisition in Haemophilus parasuis[J].Fems Immunol Med Microbiol,2005,45(1):75-86.
[18]李郁,陈申秒,王桂军,等.安徽PRSS发病猪副猪嗜血杆菌的分离鉴定及其耐药性分析[J].中国微生物学杂志,2009,21(8):682-684.
[19]KIM TJ,LEE JI.Cloning and expression of genes encoding transferring-binding protein A and B from Actinobacillus pleuropneumoruae serotype 5[J].Protein Expr Purif,2006,45(1):235-240.
[20]黄晓慧.副猪嗜血杆菌安徽分离株的耐药性与转铁结合蛋白A免疫原性研究[D].合肥:安徽农业大学,2012.