铜绿假单胞菌外膜蛋白H原核表达、多克隆抗体制备及免疫保护作用  

Prokaryotic expression,polyclonal antibody preparation and immunoprotection potential of Pseudomonas aeruginosa outer membrane protein OprH

作　　者：刘祥[1] 

机构地区：[1]陕西理工学院生物科学与工程学院生物化学与分子生物学教研室,汉中723001

出　　处：《第二军医大学学报》2015年第10期1092-1096,共5页Academic Journal of Second Military Medical University

基　　金：陕西省教育厅科学研究计划(2013JK0723)~~

摘　　要：目的为铜绿假单胞菌外膜蛋白H(OprH)工业发酵与疫苗开发研究奠定基础。方法采用分子克隆技术构建OprH蛋白的表达菌株,通过正交试验设计筛选OprH菌株的最佳培养条件与表达条件。利用切胶纯化获得OprH蛋白并免疫小鼠,制备OprH蛋白多克隆抗体,采用ELISA法检测抗体滴度,蛋白质印迹法检测抗血清特异性;对小鼠免疫OprH蛋白,攻毒铜绿假单胞菌,检测蛋白的免疫保护作用。结果 OprH重组载体双酶切、DNA测序鉴定结果,以及蛋白表达、纯化条带大小与预测一致。OprH菌株最佳培养条件为转速230r/min,葡萄糖浓度0%,装液量50mL;最佳诱导表达条件为异丙基-β-D-硫代半乳糖苷终浓度0.3mmol/L,诱导时菌液D600值0.8,温度32℃,诱导时间3h。ELISA法获得OprH抗血清滴度达1∶1 600,蛋白质印迹法证实抗血清具有很好的特异性。OprH免疫小鼠激活的特异性免疫对小鼠铜绿假单胞菌感染的保护率达到46.15%,与对照相比较差异有统计学意义(P<0.05)。结论成功构建OprH表达载体,纯化获得OprH蛋白,制备OprH蛋白多克隆抗体,实验结果表明OprH蛋白对小鼠铜绿假单胞菌感染具有免疫保护作用,并获得OprH蛋白菌株的最佳培养条件与表达条件。Objective To lay a foundation for the industrial fermentation and vaccine development of Pseudomonas aeruginosa（P.oeruginosa）outer membrane protein OprH.Methods The OprH expression strain was obtained by molecular cloning.The culture condition and optimal expression of the experiment was obtained by the method of orthogonal design.OprH was purified by gel slice strategy and was used to immunize mice to prepare the polyclonal antibody.The antibody titer and specificity were detected by ELISA and Western blotting analysis,respectively.Mice were immunized by OprH protein and infected with P.aeruginosa,and the immune protection of OprH was detected.Results OprH recombinant vector were digested and sequenced,and the results confirmed the correct construction;and OprH expression and purification of strip size agreed with the prediction.The optimal culture condition was as follows：rotation rate was 230r/min,glucose concentration was 0%,and the medium volume was 50 mL.The optimal inducing expression condition of OprH was as follows：isopropy-β-Dthiogalactoside final concentration was 0.3mmol/L,strain D600 value was 0.8,inducing temperature was 32℃,and inducing time was 3h.The OprH antibody titer was 1∶1 600 as detected by ELISA,and Western blotting analysis proved that the antiserum had good specificity.Mice specific immune was activated by OprH,and immune protection rate for mice against P.aeruginosainfection was 46.15 %,which had significant differences compared with the control（P〈0.05）.Conclusion We have successfully cloned OprH expression vector,purified OprH,prepared the polyclonal antibodies of OprH.It is confirmed that OprH protein has significant immune protection against P.aeruginosa,and the culture and induction conditions of the recombinant OprH have been obtained.

关 键 词：铜绿假单胞菌 外膜蛋白H 正交试验 多克隆抗体 

分 类 号：Q81[生物学—生物工程] R349.8[医药卫生—基础医学]