RESUMO
OBJECTIVE: To construct the recombinant Bb-pGEX-OprI vaccine of Pseudomonas aeruginosa (Pa) outer membrane protein I (OprI) and study its protection effect in mice against Pa. METHODS: The OprI gene was amplified by PCR,and cloned into pGEX-1λT to generate pGEX-OprI. The pGEX-OprI was transformed into Bifidobacterium bifidum(Bb) to construct recombinant Bb-pGEX-OprI vaccine by electroporation. After identification with double enzyme digestion,PCR and sequencing,the vaccine was then induced with IPTG,and its expression was analyzed and identified by SDS-PAGE and Western blot respectively. Twenty-one mice were randomly divided into 3 groups and vaccinated by intragastric administration with Bb-pGEX-OprI,Bb-pGEX-1λT and Bb respectively. All mice were challenged with PA01 strain at 4 weeks after the first vaccination. At 2 weeks after the challenge,mice were sacrificed to separate their lungs,and the numbers of bacterial colonies in lungs were counted. Venous blood was collected before vaccination,at 4 weeks after the first vaccination and 2 weeks after the challenge of PA01 strain. The serum IgG,IgG subclasses and IgE were detected by routine ELISA. RESULTS: The OprI gene of 194 bp was successfully amplified by PCR. Double enzyme digestion,PCR and sequencing confirmed that the OprI gene was successfully cloned into pGEX-1λT and pGEX-OprI was successfully transformed into Bb,constructing the Bb-pGEX-OprI vaccine. SDS-PAGE indicated that Bb-pGEX-OprI vaccine expressed an OprI-GST fusion protein with the relative molecular mass of approximately 32×103. Western blot verified that the fusion protein could be specifically identified by the sera of mice infected with Pa. The number of bacterial colonies in lung of Bb-pGEX-OprI vaccine group was lower than that of Bb-pGEX-1λT or Bb control ( P<0.01). The levels of serum IgG,IgG2b,IgG3 and IgE in Bb-pGEX-OprI vaccine group rose at 4 weeks after the first vaccination and 2 weeks after the challenge successively. The levels of serum antibodies in Bb-pGEX-OprI vaccine group were higher than those in Bb-pGEX-1λT or Bb control at the same time point ( P<0.01 or P<0.05). CONCLUSION: The recombinant Bb-pGEX-OprI vaccine was successfully constructed and produced an effective humoral immune response against the Pa infection.