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  

Prokaryotic expression and polyclonal antibody preparation of recombinant Pseudomonas aeruginosa outer membrane protein D

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作  者:伍娜娜 荣娜 康超 刘祥[1,2] 陈琛 陈春琳[1,2] 马心怡 吴三桥[1,2] WU Nana;RONG Na;KANG Chao;LIU Xiang;CHEN Chen;CHEN Chunlin;MA Xinyi;WU Sanqiao(Chinese-German Joint Institute for Natural Product Research,/School of Biological Science&Engineering,Shaanxi University of Technology,Hanzhong 723001,China;Shaanxi Engineering Research Center for Tall Gastrodia Tuber and Medical Dogwood,Hanzhong 723001,China)

机构地区:[1]陕西理工大学生物科学与工程学院/中德天然产物研究所,陕西汉中723001 [2]陕西省天麻山茱萸工程技术研究中心,陕西汉中723001

出  处:《河北农业大学学报》2019年第1期99-105,共7页Journal of Agricultural University of Hebei

基  金:陕西省教育厅科学研究计划项目(17JK0137);陕西理工大学人才项目(SLGQD1803);国家外国专家局项目(GDT20186100426).

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 h at 37℃when the strain OD 600 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.

关 键 词:铜绿假单胞菌 外膜蛋白OprD 多克隆抗体 诱导条件 

分 类 号:Q816[生物学—生物工程]

 

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