摘要
试验研究铜绿假单胞菌外膜蛋白编码基因oprD在大肠杆菌中的克隆及异源表达与纯化。试验根据GenBank中已发表的铜绿假单胞菌oprD基因序列,设计合成了一对特异性引物,由铜绿假单胞菌基因组DNA中扩增获得了该目的基因,随后将其克隆于原核表达载体pET32a中,转化入大肠杆菌BL21(DE3),以异丙基硫代半乳糖苷(IPTG)诱导表达,并优化了表达条件,同时对表达的蛋白进行纯化。结果显示,试验成功构建了重组载体pET32a-oprD,oprD与组氨酸(His)标签形成的融合蛋白约为65 kD。最佳表达条件为菌液培养5 h时加入0.7 mmol/L的IPTG,37℃下诱导4.5 h,纯化的蛋白经SDS-PAGE分析获得了较高纯度的OPRD重组蛋白。研究结果可以为铜绿假单胞菌OPRD蛋白功能研究及其相关诊断试剂和疫苗的研制提供参考。
The experiment studied the clone,express and purify the outer membrane protein gene oprD of Pseudomonas aeruginosa in E.coli.A pair of specific primers were designed and synthesized according to the oprD gene sequence in GenBank.The target gene was amplified from the genomic DNA of Pseudomonas aeruginosa,and then cloned into the prokaryotic expression vector pET32a.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.The expression conditions were optimized and the expressed protein was purified.The results showed that the recombinant vector pET32a-oprD was successfully constructed.The fusion protein was about 65 kD.The best expression conditions were that 0.7 mmol/L IPTG was added to the bacterial culture for 5 h,and the purified protein was induced at 37℃for 4.5 h.The purified protein was analyzed by SDS-PAGE to obtain a higher purity OPRD recombinant protein.The research results can provide reference for the research on the function of Pseudomonas aeruginosa OPRD protein and the development of related diagnostic reagents and vaccines.
作者
翟文汉
宫强
李雅静
Zhai Wenhan;Gong Qiang;Li Yajing(Henan University of Science and Technology,Henan Luoyang 471023)
出处
《现代畜牧兽医》
2021年第9期1-4,共4页
Modern Journal of Animal Husbandry and Veterinary Medicine
基金
河南省自然科学基金项目(162300410068)。
关键词
铜绿假单胞菌
oprD基因
表达
纯化
Pseudomonas aeruginosa
OprD gene
Expression
Purification
