铜绿假单胞菌外膜蛋白I的原核表达、纯化及免疫保护作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.032

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 207-213.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.032

铜绿假单胞菌外膜蛋白I的原核表达、纯化及免疫保护作用研究

陈春琳¹, 刘祥¹, 王成祥², 张晓娟¹, 俱雄¹, 张涛¹, 吴三桥¹

(1.陕西理工学院生物科学与工程学院，汉中 723001；2. 安徽省肥西县特殊教育学校，合肥 231200)

收稿日期:2014-11-18 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:陈春琳，女，硕士研究生，研究方向：分子生物学；E-mail：chen2362505579@163.com
基金资助:
陕西省教育厅科学研究计划(14JK1152)，陕西理工学院人才启动项目(SLGQD13-15)

Expression, Purification and Immunoprotection Potential of Pseudomonas aeruginosa Outer Membrane Lipoprotein I

Chen Chunlin¹, Liu Xiang¹, Wang Chengxiang², Zhang Xiaojuan¹, Ju Xiong¹, Zhang Tao¹, Wu Sanqiao¹

(1. College of Biological Sciences and Engineering，Shaanxi University of Technology，Hanzhong 723001；2. Special Education School in Feixi County of Anhui Province，Hefei 231200)

Received:2014-11-18 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要： 铜绿假单胞菌外膜蛋白OprI具有较强的免疫原性，在疫苗上具有开发前景。利用分子方法获得OprI蛋白的表达菌株。Western blotting 验证表明抗体能与OprI蛋白特异性结合。SDS-PAGE电泳切胶纯化获得OprI蛋白。将OprI蛋白免疫小鼠并攻毒铜绿假单胞菌，发现OprI蛋白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57.14%，与对照组相比较达到显著性。采用正交试验设计，获得OprI菌株最佳诱导表达条件为：加IPTG菌夜OD₆₀₀值0.8，加IPTG终浓度 0.3 mmol/L，诱导时间 8 h，诱导温度 28℃；菌株最佳培养条件为转速230 r/min，葡萄糖浓度0％，装液量50 mL。

关键词: 铜绿假单胞菌, OprI蛋白, 免疫保护, 正交试验, 诱导条件

Abstract: Outer membrane lipoprotein I(OprI)in Pseudomonas aeruginosa has solid immunogenicity and holds prospects in the development of vaccine. The OprI-expressing strain was obtained by molecular cloning. Western blotting verified that antibody specifically combined with OprI. The purified OprI was gained by the SDS-PAGE gel slices. Immunizing mice with OprI, specific immunity activated by OprI protected 57. 14% mice from P. aeruginosa infection, which reached significant protection compared with the control group. The orthogonal experiment was employed to obtain the optimal inducing expressing condition of OprI strain：strain OD₆₀₀ value，IPTG final concentration，inducing time and temperature，were 0. 8，0. 3 mmol/L，8 h，and 28℃，respectively；the optimal culture condition was that the rotation rate，glucose concentration，and medium volume were 230 r/min，0% and 50 mL，respectively. This work laid the groundwork for the researches on industrial fermentation，protein function and vaccine development of OprI.

Key words: Pseudomonas aeruginosa, OprI protein, immunoprotection, orthogonal experiment, induction conditions

陈春琳, 刘祥, 王成祥, 张晓娟, 俱雄, 张涛, 吴三桥. 铜绿假单胞菌外膜蛋白I的原核表达、纯化及免疫保护作用研究[J]. 生物技术通报, 2015, 31(7): 207-213.

Chen Chunlin, Liu Xiang, Wang Chengxiang, Zhang Xiaojuan, Ju Xiong, Zhang Tao, Wu Sanqiao. Expression, Purification and Immunoprotection Potential of Pseudomonas aeruginosa Outer Membrane Lipoprotein I[J]. Biotechnology Bulletin, 2015, 31(7): 207-213.

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铜绿假单胞菌外膜蛋白I的原核表达、纯化及免疫保护作用研究

Expression, Purification and Immunoprotection Potential of Pseudomonas aeruginosa Outer Membrane Lipoprotein I

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