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[目的]克隆铜绿假单胞菌OprF基因,并进行原核表达,以期进一步开展铜绿假单胞菌基因工程疫苗的研究。[方法]利用PCR技术从铜绿假单胞菌基因组中扩增出OprF基因。采用T-A克隆构建pMD18-T-OprF重组质粒,测序正确后经BamHⅠ、HindⅢ双酶切获得OprF片段,插入到表达载体pET32a,获得pET32a-OprF重组表达质粒并在表达宿主菌E.ColiBL21中诱导表达,利用Ni-NTA柱对目的蛋白进行纯化,通过SDS-PAGE电泳和蛋白质印迹进行鉴定。[结果]克隆OprF基因(459bp)并经DNA测序证实,表达重组蛋白OprF并获得纯化蛋白,经SDS-PAGE电泳和蛋白质印迹鉴定正确。[结论]成功克隆了OprF基因并获得原核表达物,为铜绿假单胞菌的致病性研究和开展基因工程疫苗的研制奠定了基础。
[Objective] The research aimed to clone the gene of OprF of Pseudomonas aeruginosa and to carry out prokaryotic expression in order to further carry out the research of Pseudomonas aeruginosa genetic engineering vaccine. [Method] OprF gene was amplified from Pseudomonas aeruginosa genome by PCR. The recombinant plasmid pMD18-T-OprF was constructed by TA cloning. After sequencing correctly, OprF fragment was digested with BamHⅠand HindⅢ and inserted into the expression vector pET32a to obtain the recombinant expression plasmid pET32a-OprF. The recombinant plasmid pET32a-OprF was induced to express in E.coli BL21 The target protein was purified by Ni-NTA column and identified by SDS-PAGE electrophoresis and Western blotting. [Result] The OprF gene (459bp) was cloned and confirmed by DNA sequencing. The recombinant protein OprF was expressed and purified, which was identified by SDS-PAGE electrophoresis and Western blot. [Conclusion] OprF gene was successfully cloned and prokaryotic expression was obtained, which laid the foundation for the pathogenicity of Pseudomonas aeruginosa and the development of gene engineering vaccine.