【摘要】 为了研究铜绿假单胞菌外膜蛋白F和I的重要表位,根据已公布的F和I基因序列设计2对特异性引物,以分离的水貂源铜绿假单胞菌ZHDL9基因组为模板,扩增重要的表位基因F_190—342（F1）和I_21—83（I2）,并对得到的2段基因序列进行生物信息学分析。结果表明,PCR扩增所得F1和I2两段基因序列高度保守,不存在核苷酸的插入和缺失;通过融合PCR技术将得到的F1基因和I2基因无缝连接,得到651 bp的融合基因F1I2。将融合基因F1I2克隆至原核表达载体p ET-28a,成功构建了融合表达质粒p ET-28a-F1I2;将pET-28a-F1I2转化大肠杆菌BL21 （DE3）,成功构建重组菌株BL21（pET-28a-F1I2）,并对重组菌株进行IPTG诱导表达。SDS-PAGE检测表明,成功表达了融合蛋白F1I2,且融合蛋白能够被His镍柱纯化; Western Blotting检测表明,表达的融合蛋白F1I2与铜绿假单胞菌ZHDL9菌株感染的水貂血清具有较好的反应原性。更多还原

【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 F_190—342（F1） and I_21—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-28 a,and the fusion expression plasmid pET-28a-F1I2 was constructed. Then the expression plasmid pET-28a-F1I2 was transformed into E. coliBL21（ 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.更多还原