铜绿假单胞菌外膜蛋白F原核载体构建、表达条件优化及免疫保护作用研究  

Construction of prokaryotic vector and optimization of expression conditions of outer membrane protein F of Pseudomonas aeruginosa and its immunoprotection potential

作　　者：刘祥[1] 

机构地区：[1]陕西理工学院维生素D生理与应用研究所,陕西汉中723001

出　　处：《中国现代医学杂志》2015年第11期12-16,共5页China Journal of Modern Medicine

基　　金：陕西省教育厅科学研究计划项目(No:2013JK0723)

摘　　要：目的构建并鉴定铜绿假单胞菌(PA)外膜蛋白F(Opr F)原核表达载体,分析Opr F蛋白免疫保护作用,优化Opr F蛋白诱导表达条件。方法通过分子克隆获得Opr F蛋白的表达菌株。利用切胶纯化获得Opr F蛋白,免疫小鼠,通过PA攻毒实验分析Opr F蛋白免疫保护作用。采用正交试验,获得Opr F菌株的最佳表达条件与培养条件。结果 Opr F重组载体双酶切、DNA测序鉴定结果与预测一致。Western blot检测表明抗体能与Opr F蛋白结合。Opr F蛋白对小鼠保护率达64.29%。Opr F菌株最佳诱导表达条件为:加IPTG终浓度0.1 mmol/L,诱导时菌液OD600值1.0,温度32℃,诱导时间8 h;最佳培养条件为:转速230 r/min,葡萄糖浓度为0%,装液量50 ml。结论获得Opr F表达菌株最佳的表达条件与培养条件;验证Opr F蛋白对小鼠PA感染有免疫保护作用。[Objective] To construct and identify prokaryotic expression vector for outer membrane protein F （OprF） of Pseudomonas aeruginosa （P. aeruginosa）, study the immunoprotection of OprF and optimize its expression condition. [Methods] The OprF expression strain was obtained by molecular clone. OprF was purified by the way of the gel slices. The mice were immunized and attacked with P. aeruginosa to analyze the immunoprotection effect. The optimal inducing and culture conditions were gained by the method of orthogonal experiment. [Results] OprF recombinant vector was digested and sequenced, and the result agreed with the prediction result. Western blot showed that the antibody could bind to OprF, and OprF hold significant immunoprotection effect to 64.29%. The optimal induction expressing conditions of OprF were： IPTG final concentration 0.1 mmol/L, strain OD600 value 1.0, inducing temperature 32℃ and inducing time 8 h; the optimal culture conditions were rotation rate 230 r/min, glucose concentration 0% and medium volume 50 ml. [ Conclusion] The induction and culture conditions of OprF have been optimized, and it is confirmed that OprF provides immune protection to mice from P. aeruginosa infection.

关 键 词：外膜蛋白F 免疫保护 正交试验 诱导条件 

分 类 号：Q819[生物学—生物工程]