Abstract: 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.