河南农业科学2018，Vol.47Issue(12)：132-136,150,6.DOI:10.15933/j.cnki.1004-3268.2018.12.021
铜绿假单胞菌F190—342-I21—83基因的克隆及其融合蛋白的原核表达
Cloning of Pseudomonas aeruginosa F190—342-I21—83 and Prokaryotic Expression of Recombinant Protein
摘要
Abstract
To study the epitope of outer membrane protein F and I from Pseudomonas aeruginosa, two pairs of specific primers were designed according to the published F and I gene sequences, and the epitope gene F190—342(F1) and I21—83(I2) were amplified from Pseudomonas aeruginosa ZHDL9 separated in mink.Bioinformatics analysis was performed on the obtained two-stage gene sequences.The results showed that the two gene sequences were highly conserved and there was no nucleotide insertion or deletion.The F1 and I2 gene were seamlessly ligated by the overlap PCR to obtain a 651 bp fusion gene F1I2, then the fusion gene was cloned into the prokaryotic expression vector pET-28a, and the fusion expression plasmid pET-28a-F1I2 was constructed.Then the expression plasmid pET-28a-F1I2 was transformed into E.coli BL21(DE3) strain, and the recombinant strain BL21(pET-28a-F1I2) was constructed.And the recombinant strain was induced by IPTG.The result of SDS-PAGE showed that fused protein F1I2 was expressed from BL21(pET-28a-F1I2), and the fused protein F1I2 could be purified by His nickel column.And the result of Western Blotting showed that obtained expression proteins had good reactivity with serum of mink infected with Pseudomonas aeruginosa ZHDL9.关键词
铜绿假单胞菌/F基因/I基因/克隆/融合蛋白/原核表达Key words
Pseudomonas aeruginosa/F gene/I gene/Cloning/Recombinant protein/Prokaryotic expression分类
农业科技引用本文复制引用
张明亮,闫新武,孙长江,顾敬敏,崔子寅,韩文瑜..铜绿假单胞菌F190—342-I21—83基因的克隆及其融合蛋白的原核表达[J].河南农业科学,2018,47(12):132-136,150,6.基金项目
国家高技术研究发展计划项目(2011AA10A210) （2011AA10A210）
国家自然科学基金青年基金项目(31802170) （31802170）
安阳工学院博士科研启动基金项目(BSJ2016013) （BSJ2016013）
