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 .

Objective To study the effect of hyperbaric oxygen preconditioning management on the expression of MMP-2,MMP-9 and Cx43 in a rat abdominal skin flap model of ischemia-reperfusion injury.Methods Eighteen male adult SD rats,weight ranged from 220-250 g,were randomly divided into three groups:sham group (SH),ischemia-reperfusion group (IR) and hyperbaric oxygen preconditioning group (HBO).All the rats in HBO group received hyperbaric oxygen treatment twice each day,apart 12 hours,during the last three days before operation.Abdominal skin flap with superficial epigastric artery as pedicle was established.In HBO and IR group,3 hours of ischemia was performed.On the 3rd postoperative day,samples were taken to assess the expression of MMP-9,MMP-2 and Cx43 by immunohistochemical staining and Western blot.Results Compared with IR group,the expression of Cx43 was significantly increased (IR 15.03±3.66;HBO 36.01±4.12) and the ex pression of MMP-9 and MMP-2 was decreased (MMP 2:IR 12.01±1.23;HBO 5.98±1.48;MMP9:IR 16.77±2.01;HBO 11.48±1.77).Conclusions Hyperbaric oxygen preconditioning for the rat abdominal skin flap model of ischemia-reperfusion injury has inhibitory effect on the expression of MMP-9 and MMP-2,and stimulative effect on the expression of Cx43.