Construction and eukaryotic expression of a recombinant plasmid encoding Pseudomonas aeruginosa toxA and typeⅢsecretion system pcrV / 医学研究生学报

ABSTRACT

Objective Exotoxin A ( encoded by gene toxA ) , one of the most toxic protein secreted by pseudomonas aerugi-nosa(P.a.), and PcrV (encoded by gene pcrV), key component to type Ⅲsecretion system of P.a., both matter significantly to the virulence of P.a.The article was to construct a novel DNA vaccine encoding a mutated toxA gene and the pcrV gene of P .a.and i-dentify gene expressions in eukaryotic cells . Methods The genes of toxA and pcrV were amplified by PCR , and the toxA gene was mutated to reduce the toxicity of Exotoxin A .Then gene fragments toxA m and pcrV were inserted into eukaryotic expression plasmid pIRES simultaneously to construct a recombinant DNA vaccine pIRES-toxAm-pcrV.The novel plasmid was transfected into HEK-293 cells by lipofectamine 2000 .The expressions of toxA m and pcrV were detected by Western blot . Results Gel electrophoresis demon-strated the target gene fragments encoding Exotoxin A and PcrV .Western blot exhibited proteins encoded toxA and pcrV expressed by HEK 293 cells. Conclusion The recombinant plasmid pIRES-toxAm-pcrV was successfully constructed .Western blot analysis indi-cated the expressions of toxA m and pcrV in HEK-293 cells.It may be used as a potential candidate of preventive vaccine of Pseudo-monas aeruginosa .