重组铜绿假单胞菌外膜蛋白D的原核表达与多克隆抗体制备研究
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摘要
为获得铜绿假单胞菌外膜蛋白OprD并研究其分子功能。本研究采用分子克隆构建OprD蛋白重组表达菌株,用正交试验设计方法获得OprD菌株的最优表达条件和培养条件,通过SDS-PAGE切胶的方法纯化OprD蛋白,小鼠免疫制备OprD多克隆抗血清,蛋白质印迹法检测抗血清的特异性。使用DNAMan和MEGA软件对OprD蛋白进行同源性和系统发生分析。结果表明,OprD重组载体双酶切、DNA测序鉴定结果、OprD蛋白表达与纯化条带大小分别与预测相符。正交试验获得OprD菌株的最优培养条件为:葡萄糖浓度为0,转速230r/min,装液量为50mL;最优表达条件为:菌液OD600=0.8时加入终浓度为0.3mmol/L的异丙基β-D-1-硫代吡喃半乳糖苷(IPTG),37℃诱导12h。SDS-PAGE电泳切胶纯化获得纯度较高的OprD蛋白。蛋白质印迹法证实OprD蛋白抗血清具有较好的特异性。OprD蛋白序列系统发生分析发现,同菌属细菌存在较高的同源性,并且亲缘关系较近的细菌中OprD蛋白可能具有相似的分子功能。
To obtain Pseudomonas aeruginosa outer membrane protein(OprD),and to study its molecular function,the recombinant OprD expression strain was obtained by molecular clone,and the optimal expression conditions and culture conditions of the OprD strain were obtained by orthogonal design method.OprD was then purified by SDS-PAGE gel slices,and the polyclonal antibody was prepared by immunized mice.Western blotting was used to test the specificity of antiserum.DNAMan and MEGA software were used to perform homology and phylogenetic analysis on OprD.The results showed that double enzyme digestion and DNA sequencing of OprD recombinant vector,as well as the size of express and purified strip were consistent with the prediction.By orthogonal experiment,the optimal culture condition of OprD protein was:glucose concentration as 0,rotation rate as 230 r/min,and medium volume as 50 mL.The optimal expressing condition was:adding 0.3 mmol/L IPTG and inducing for 12 hat 37℃ when the strain OD600 value was 0.8.The high-purity OprD protein was obtained by SDS-PAGE gel purification.Western blotting proved that the antiserum had good specificity.OprD sequence phylogenetic analysis showed that bacteria of same genus had high homology.OprD might have the similar molecular function in bacteria of close relationship.This work laid a foundation for the research of drug resistance function of OprD.
To obtain Pseudomonas aeruginosa outer membrane protein(OprD),and to study its molecular function,the recombinant OprD expression strain was obtained by molecular clone,and the optimal expression conditions and culture conditions of the OprD strain were obtained by orthogonal design method.OprD was then purified by SDS-PAGE gel slices,and the polyclonal antibody was prepared by immunized mice.Western blotting was used to test the specificity of antiserum.DNAMan and MEGA software were used to perform homology and phylogenetic analysis on OprD.The results showed that double enzyme digestion and DNA sequencing of OprD recombinant vector,as well as the size of express and purified strip were consistent with the prediction.By orthogonal experiment,the optimal culture condition of OprD protein was:glucose concentration as 0,rotation rate as 230 r/min,and medium volume as 50 mL.The optimal expressing condition was:adding 0.3 mmol/L IPTG and inducing for 12 hat 37℃ when the strain OD600 value was 0.8.The high-purity OprD protein was obtained by SDS-PAGE gel purification.Western blotting proved that the antiserum had good specificity.OprD sequence phylogenetic analysis showed that bacteria of same genus had high homology.OprD might have the similar molecular function in bacteria of close relationship.This work laid a foundation for the research of drug resistance function of OprD.
引文
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[10]Versace R E,Lazaridis T.Modelling Protein-Micelle Systems in Implicit Water[J].Biophysical Journal,2015,108(2):249a
[11]刘祥,俱雄,陈春琳.重组大肠埃希菌外膜蛋白OmpT的载体构建和表达条件优化及多克隆抗体制备[J].湖南农业大学学报:自然科学版,2015,41(4):350-355.
[12]刘祥.溶藻弧菌附着定植因子ACFA蛋白的原核表达、抗原性鉴定及生物信息学分析[J].西南农业学报,2016,29(7):1755-1760.
[13]刘祥.重组铜绿假单胞菌外毒素A的原核表达和多克隆抗体制备及免疫保护功能分析[J].湖南农业大学学报:自然科学版,2016,42(1):33-38.
[14]朱小兰,许文林,吕旭晶,等.汉防己甲素预防K562细胞获得性耐药机制的研究[J].中国实验血液学杂志,2011,19(2):363-366.
[15]Richardot C,Plésiat P,Fournier D,et al.Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosaas a result of a mino acid substitutions in porin OprD[J].International journal of antimicrobial agents,2015,45(5):529-532.
[16]Epp S F,Pechère J C,Kok M.Raising antibodies against OprD,an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE[J].Journal of microbiological methods,2001,46(1):1-8.
[17]刘祥.溶藻弧菌外膜蛋白OmpU的克隆、表达条件优化及多克隆抗体制备[J].西南农业学报,2016,29(3):699-705.
[18]陈伟,刘文恩,李艳华,等.艰难梭菌毒素B受体结合区CDB3融合蛋白诱导表达条件优化及其纯化[J].检验医学与临床,2014,11(6):725-728.
[19]王昭,王席,杨威,等.金属硫蛋白基因MT1A在大肠杆菌中的自诱导表达条件优化及其抗镉性[J].河南农业科学,2016,45(5):66-70.
[20]热依汗古丽·阿里木,毛新芳,刘忠渊.黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析[J].生物工程学报,2013,29(6):836-847.
[2]Paulsson M,Singh B,Al-Jubair T,et al.Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa[J].Journal of Cystic Fibrosis,2015,14(5):600-607.
[3]Dosunmu E,Chaudhari A A,Singh S R,et al.Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes:a potential mechanism for their antimicrobial effect[J].International journal of nanomedicine,2015,10:5025-5035.
[4]Ducret V,Gonzalez M R,Scrignari T,et al.OprDrepression upon metal treatment requires the RNAchaperone Hfq in Pseudomonas aeruginosa[J].Genes,2016,7(10):82-96.
[5]Kucharska I,Liang B,Ursini N,et al.Molecular interactions of lipopolysaccharide with an outer membrane protein from Pseudomonas aeruginosa probed by solution NMR[J].Biochemistry,2016,55(36):5061-5072.
[6]Shen J,Pan Y,Fang Y.Role of the outer membrane protein OprD2in carbapenem-resistance mechanisms of Pseudomonas aeruginosa[J].PloS one,2015,10(10):e0139995.
[7]Shu J C,Kuo A J,Su L H,et al.Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms,particularly the a mino acid substitution at codon 170[J].Journal of Antimicrobial Chemotherapy,2017,72(9):2489-2495.
[8]Choudhury D,Talukdar A D,Choudhury M D,et al.Carbapenem nonsusceptibility with modified OprDin clinical isolates of Pseudomonas aeruginosa from India[J].Indian journal of medical microbiology,2017,35(1):137-139.
[9]Richardot C,Plésiat P,Fournier D,et al.Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosaas a result of a mino acid substitutions in porin OprD[J].International journal of antimicrobial agents,2015,45(5):529-532.
[10]Versace R E,Lazaridis T.Modelling Protein-Micelle Systems in Implicit Water[J].Biophysical Journal,2015,108(2):249a
[11]刘祥,俱雄,陈春琳.重组大肠埃希菌外膜蛋白OmpT的载体构建和表达条件优化及多克隆抗体制备[J].湖南农业大学学报:自然科学版,2015,41(4):350-355.
[12]刘祥.溶藻弧菌附着定植因子ACFA蛋白的原核表达、抗原性鉴定及生物信息学分析[J].西南农业学报,2016,29(7):1755-1760.
[13]刘祥.重组铜绿假单胞菌外毒素A的原核表达和多克隆抗体制备及免疫保护功能分析[J].湖南农业大学学报:自然科学版,2016,42(1):33-38.
[14]朱小兰,许文林,吕旭晶,等.汉防己甲素预防K562细胞获得性耐药机制的研究[J].中国实验血液学杂志,2011,19(2):363-366.
[15]Richardot C,Plésiat P,Fournier D,et al.Carbapenem resistance in cystic fibrosis strains of Pseudomonas aeruginosaas a result of a mino acid substitutions in porin OprD[J].International journal of antimicrobial agents,2015,45(5):529-532.
[16]Epp S F,Pechère J C,Kok M.Raising antibodies against OprD,an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE[J].Journal of microbiological methods,2001,46(1):1-8.
[17]刘祥.溶藻弧菌外膜蛋白OmpU的克隆、表达条件优化及多克隆抗体制备[J].西南农业学报,2016,29(3):699-705.
[18]陈伟,刘文恩,李艳华,等.艰难梭菌毒素B受体结合区CDB3融合蛋白诱导表达条件优化及其纯化[J].检验医学与临床,2014,11(6):725-728.
[19]王昭,王席,杨威,等.金属硫蛋白基因MT1A在大肠杆菌中的自诱导表达条件优化及其抗镉性[J].河南农业科学,2016,45(5):66-70.
[20]热依汗古丽·阿里木,毛新芳,刘忠渊.黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析[J].生物工程学报,2013,29(6):836-847.