羊源多杀性巴氏杆菌toxA-N基因的克隆、原核表达及生物信息学分析
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摘要
试验旨在对羊源多杀性巴氏杆菌toxA-N基因进行克隆、原核表达及纯化,并对表达蛋白toxA-N进行生物信息学分析,为探索羊源多杀性巴氏杆菌毒素基因的相关特性提供参考依据。以羊源多杀性巴氏杆菌基因组为模板,参考GenBank中公布的多杀性巴氏杆菌HN06中toxA基因序列(登录号:CP003313.1)设计引物,通过PCR技术扩增出目的片段,构建重组质粒pET28a(+)-toxA-N,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达后,经考马斯亮蓝染色及Western blotting鉴定,纯化蛋白并运用生物信息学软件对表达蛋白进行特性分析。结果显示,试验成功扩增出大小为1 515bp的toxA-N基因片段,经BamHⅠ和NotⅠ双酶切得到大小约为5 369和1 515bp两条片段,表明成功构建了pET28a(+)-toxA-N重组质粒,IPTG诱导表达的菌株经考马斯亮蓝染色及Western blotting鉴定后,成功表达出大小约为60ku的toxA-N蛋白;生物信息学分析表明,toxA-N蛋白为包涵体,其分子式为C2635H4002N664O797S17,原子总个数为8 115,消光系数为84 480,不稳定指数为43.50,亲水性平均值为-0.381。在toxA-N蛋白二级结构中,α-螺旋、β-折叠、延伸链和无规则卷曲分别占53.47%、2.77%、11.28%和32.48%,与三级结构预测结果一致。本试验通过对toxA-N基因的初步研究,揭示了多杀性巴氏杆菌毒素的相关特性,对家畜的疾病预防、诊断、治疗具有重要意义。
This study was aimed to clone,prokaryotically express and purify toxA-N gene of Pasteurella multocida in goat,and analyze the expressed protein toxA-N by bioinformatics,which provided a reference for exploring the relevant characteristics of the Pasteurella multocida toxingene in goat.The genome of the above-mentioned bacteria was used as a template,and the primer was designed with reference to the Pasteurella multocida HN06 toxAgene sequence published in GenBank(accession No.:CP003313.1),and the target fragment was amplified by PCR.The recombinant plasmid pET28 a(+)-toxA-N was constructed and then transferred to E.coli BL21(DE3)competent cells.After inducted by IPTG,the expressed proteins were characterized by Coomassie blue staining,Western blotting,protein purification and bioinformatics softwares.The results showed that the 1 515 bp gene fragment was successfully amplified,and the two fragments of 5 369 and 1 515 bp were digested by BamH Ⅰ and Not Ⅰ,indicating that the recombinant plasmid pET28 a(+)-toxA-N was successfully constructed.The Coomassie blue staining and Western blotting results showed that a size of about 60 ku toxA-N protein was successfully expressed by IPTG induction.Bioinformatics analysis results showed that toxA-N protein was an inclusion body with a molecular formula of C2635 H4002 N664 O797 S17,the total number of atoms was8 115,the extinction coefficient was 84 480,the instability index was 43.50,and the average hydrophilicity was-0.381.In the secondary structure of toxA-N protein,α-helix,β-turn,extended chain and random coil accounted for 53.47%,2.77%,11.28% and 32.48%,respectively,which were consistent with the prediction results of the tertiary structure.In summary,this study revealed the relevant properties of the Pasteurellamultocidatoxin by preliminary study of toxA-N gene,which was of great significance for disease prevention,diagnosis and treatment of livestock.
This study was aimed to clone,prokaryotically express and purify toxA-N gene of Pasteurella multocida in goat,and analyze the expressed protein toxA-N by bioinformatics,which provided a reference for exploring the relevant characteristics of the Pasteurella multocida toxingene in goat.The genome of the above-mentioned bacteria was used as a template,and the primer was designed with reference to the Pasteurella multocida HN06 toxAgene sequence published in GenBank(accession No.:CP003313.1),and the target fragment was amplified by PCR.The recombinant plasmid pET28 a(+)-toxA-N was constructed and then transferred to E.coli BL21(DE3)competent cells.After inducted by IPTG,the expressed proteins were characterized by Coomassie blue staining,Western blotting,protein purification and bioinformatics softwares.The results showed that the 1 515 bp gene fragment was successfully amplified,and the two fragments of 5 369 and 1 515 bp were digested by BamH Ⅰ and Not Ⅰ,indicating that the recombinant plasmid pET28 a(+)-toxA-N was successfully constructed.The Coomassie blue staining and Western blotting results showed that a size of about 60 ku toxA-N protein was successfully expressed by IPTG induction.Bioinformatics analysis results showed that toxA-N protein was an inclusion body with a molecular formula of C2635 H4002 N664 O797 S17,the total number of atoms was8 115,the extinction coefficient was 84 480,the instability index was 43.50,and the average hydrophilicity was-0.381.In the secondary structure of toxA-N protein,α-helix,β-turn,extended chain and random coil accounted for 53.47%,2.77%,11.28% and 32.48%,respectively,which were consistent with the prediction results of the tertiary structure.In summary,this study revealed the relevant properties of the Pasteurellamultocidatoxin by preliminary study of toxA-N gene,which was of great significance for disease prevention,diagnosis and treatment of livestock.
引文
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[5]陈品安,汪义金.羊巴氏杆菌病的流行特点及综合防治措施[J].畜牧与饲料学,2013,1:110-111.CHEN P A,WANG Y J.The epidemiological characteristics and integrated control measures of Pasteurellosis[J].Animal Husbandry&Feed Science,2013,1:110-111.(in Chinese)
[6]唐先春,吴斌,索绪峰,等.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[J].畜牧兽医学报,2005,36(6):590-595.TANG X C,WU B,SUO X F,et al.Isolation,identification and biological characteristics of Pasteurella multocida[J].Journal of Animal Husbandry and Veterinary Medicine,2005,36(6):590-595.(in Chinese)
[7]唐先春.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[D].武汉:华中农业大学,2004.TANG X C.Isolation,identification and biological characteristics of Pasteurella multocida[D].Wuhan:Huazhong Agricultural University,2004.(in Chinese)
[8] WANGMAN M,CHAIVISUTHANGKURA P,SRITUNYALUCKSANA K,et al.Development of monoclonalantibodies specific to ToxA and ToxB of vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture,2017,474:75-81.
[9]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 of LpxBgene and bioinformatics analysis of its protein[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1681-1687.(in Chinese)
[10]曹瑞勇,聂鑫,李宝宝,等.羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2017,44(8):2234-2240.CAO R Y,NIE X,LI B B,et al.Cloning,prokaryotic expression and bioinformatics of BPSS1512gene from Burkholderia sinensis analysis[J].China Animal Husbandry&Veterinary Medicine,2017,44(8):2234-2240.(in Chinese)
[11]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 Brucella Omp10 gene in sheep[J].China Animal Husbandry&Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[12]戴碧红.鸭瘟病毒UL19基因截段表达、蛋白纯化及抗体制备[D].雅安:四川农业大学,2014.DAI B H.Segmentation,protein purification and antibody preparation of duck plague virus UL19gene[D].Ya’an:Sichuan Agricultural University,2014.(in Chinese)
[13] 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.
[14] 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.
[15]孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698.SUN X J,DU X H,YANG X P,et al.Cloning and bioinformatics analysis of CDS region of yak cattle CYGBgene[J].Chinese Agricultural Sciences,2014,47(13):2690-2698.(in Chinese)
[16] El TAYEB A B,MORISHITA T Y,ANGRICK E J.Evaluation of Pasteurella multocida isolated from rabbits by capsular typing,somatic serotyping,and restriction endonuclease analysis[J].Journal of Veterinary Diagnostic Investigation,2004,16(2):121-125.
[17] MAGNE B.Multilocus sequence analysis of Pasteurella multocida demonstrates a type species under development[J].Microbiology,2013,159(Pt3):580-590.
[18]李卉.多杀性巴氏杆菌毒力因子及基因表达的研究进展[J].中国畜牧兽医文摘,2015,9:68-69.LI H.Research progress on virulence factors and gene expression of Pasteurella multocida[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2015,9:68-69.(in Chinese)
[19] SIEGERT P,SCHMIDT G,PAPATHEODOROU P,et al.Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade via the Gα(q/11)-p63RhoGEF-RhoA axis[J].PLoS Pathogens,2013,9(5):e1003385.
[20]鲍娟.猪源多杀性巴氏杆菌分离物荚膜PCR分型与毒素研究[D].贵阳:贵州大学,2009.BAO J.PCR typing and toxin study of isolates of Pasteurella multocida isolates[D].Guiyang:Guizhou University,2009.(in Chinese)
[21]许田亮.牦牛源多杀性巴氏杆菌分离鉴定及重组外膜蛋白OmpH、OmpA免疫效果的初步研究[D].石河子:石河子大学,2013.XU T L.Isolation and identification of Pasteurella multocidafrom yak and preliminary study on the immune effect of recombinant outer membrane proteins OmpH and OmpA[D].Shihezi:Shihezi University,2013.(in Chinese)
[22]江涛,谭春萍,任灵芝,等.猪源D型产毒素多杀性巴氏杆菌毒素基因的原核表达[J].动物医学进展,2010,12:64-68.JIANG T,TAN C P,REN L Z,et al.Prokaryotic expression of toxin-producing Pasteurella multocida toxin gene from pig D-type[J].Progress in Animal Medicine,2010,12:64-68.(in Chinese)
[23] GOORMAGHTUGH E,RUYSSCHAERT J M,RAUSSENS V.Evaluation of the information content in infrared spectra for protein secondary structure determination[J].Biophysical Journal,2006,90(8):2946-2957.
[2]叶尔生·吐尔逊哈孜.羊巴氏杆菌病的诊断与防控[J].当代畜牧,2016,35:90.YERSIN T.Diagnosis and prevention of Pasteurellosis[J].Contemporary Animal Husbandry,2016,35:90.(in Chinese)
[3]徐朋朋.鸭源多杀性巴氏杆菌分离鉴定及感染雏鸭动态病理学观察[D].洛阳:河南科技大学,2011.XU P P.Isolation and identification of Pasteurella multocida and dynamic pathological observation of infected ducklings[D].Luoyang:Henan University of Science and Technology,2011.(in Chinese)
[4]李健.猪产毒素多杀性巴氏杆菌toxA基因的功能结构域研究[D].武汉:华中农业大学,2007.LI J.Functional domain of toxAgene of porcine toxinproducing Pasteurella multocida[D].Wuhan:Huazhong Agricultural University,2007.(in Chinese)
[5]陈品安,汪义金.羊巴氏杆菌病的流行特点及综合防治措施[J].畜牧与饲料学,2013,1:110-111.CHEN P A,WANG Y J.The epidemiological characteristics and integrated control measures of Pasteurellosis[J].Animal Husbandry&Feed Science,2013,1:110-111.(in Chinese)
[6]唐先春,吴斌,索绪峰,等.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[J].畜牧兽医学报,2005,36(6):590-595.TANG X C,WU B,SUO X F,et al.Isolation,identification and biological characteristics of Pasteurella multocida[J].Journal of Animal Husbandry and Veterinary Medicine,2005,36(6):590-595.(in Chinese)
[7]唐先春.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[D].武汉:华中农业大学,2004.TANG X C.Isolation,identification and biological characteristics of Pasteurella multocida[D].Wuhan:Huazhong Agricultural University,2004.(in Chinese)
[8] WANGMAN M,CHAIVISUTHANGKURA P,SRITUNYALUCKSANA K,et al.Development of monoclonalantibodies specific to ToxA and ToxB of vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease(AHPND)[J].Aquaculture,2017,474:75-81.
[9]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 of LpxBgene and bioinformatics analysis of its protein[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1681-1687.(in Chinese)
[10]曹瑞勇,聂鑫,李宝宝,等.羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2017,44(8):2234-2240.CAO R Y,NIE X,LI B B,et al.Cloning,prokaryotic expression and bioinformatics of BPSS1512gene from Burkholderia sinensis analysis[J].China Animal Husbandry&Veterinary Medicine,2017,44(8):2234-2240.(in Chinese)
[11]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 Brucella Omp10 gene in sheep[J].China Animal Husbandry&Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[12]戴碧红.鸭瘟病毒UL19基因截段表达、蛋白纯化及抗体制备[D].雅安:四川农业大学,2014.DAI B H.Segmentation,protein purification and antibody preparation of duck plague virus UL19gene[D].Ya’an:Sichuan Agricultural University,2014.(in Chinese)
[13] 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.
[14] 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.
[15]孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698.SUN X J,DU X H,YANG X P,et al.Cloning and bioinformatics analysis of CDS region of yak cattle CYGBgene[J].Chinese Agricultural Sciences,2014,47(13):2690-2698.(in Chinese)
[16] El TAYEB A B,MORISHITA T Y,ANGRICK E J.Evaluation of Pasteurella multocida isolated from rabbits by capsular typing,somatic serotyping,and restriction endonuclease analysis[J].Journal of Veterinary Diagnostic Investigation,2004,16(2):121-125.
[17] MAGNE B.Multilocus sequence analysis of Pasteurella multocida demonstrates a type species under development[J].Microbiology,2013,159(Pt3):580-590.
[18]李卉.多杀性巴氏杆菌毒力因子及基因表达的研究进展[J].中国畜牧兽医文摘,2015,9:68-69.LI H.Research progress on virulence factors and gene expression of Pasteurella multocida[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2015,9:68-69.(in Chinese)
[19] SIEGERT P,SCHMIDT G,PAPATHEODOROU P,et al.Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade via the Gα(q/11)-p63RhoGEF-RhoA axis[J].PLoS Pathogens,2013,9(5):e1003385.
[20]鲍娟.猪源多杀性巴氏杆菌分离物荚膜PCR分型与毒素研究[D].贵阳:贵州大学,2009.BAO J.PCR typing and toxin study of isolates of Pasteurella multocida isolates[D].Guiyang:Guizhou University,2009.(in Chinese)
[21]许田亮.牦牛源多杀性巴氏杆菌分离鉴定及重组外膜蛋白OmpH、OmpA免疫效果的初步研究[D].石河子:石河子大学,2013.XU T L.Isolation and identification of Pasteurella multocidafrom yak and preliminary study on the immune effect of recombinant outer membrane proteins OmpH and OmpA[D].Shihezi:Shihezi University,2013.(in Chinese)
[22]江涛,谭春萍,任灵芝,等.猪源D型产毒素多杀性巴氏杆菌毒素基因的原核表达[J].动物医学进展,2010,12:64-68.JIANG T,TAN C P,REN L Z,et al.Prokaryotic expression of toxin-producing Pasteurella multocida toxin gene from pig D-type[J].Progress in Animal Medicine,2010,12:64-68.(in Chinese)
[23] GOORMAGHTUGH E,RUYSSCHAERT J M,RAUSSENS V.Evaluation of the information content in infrared spectra for protein secondary structure determination[J].Biophysical Journal,2006,90(8):2946-2957.