Preliminary Study On Nasal Immunization Of Plague Recombinant F1-V Fusion With Mucosal Adjuvant

Yersinia pestis (Y.pestis) is the pathogenic bacteria of plague,which can induce bubonic, pneumonic and septicemic plague. Pneumonic plague have the characteristic of higher infectivity and mortality. In recent years, the plague especially showed a trendency to recur, and it could be used to make bio-terrorist preparation, so plague has become a potential threat to human being and national security.The vaccine is one of effective means of prevention and control infectious disease.The traditional plague vaccine can not be used widely because of their side-effect, ineffective to pneumonic plague and so on.According to the characteristics of pneumonic plague,the research of vaccine to prevent pneumonic plague focused on stimulating mucosal immune response.Therefore, it is very important to search for safe and effective mucosal adjuvants for the plague mucosal vaccine research.Proteosome are the outer memebrane protein of Neisseria meningitis , comprising three or four porin.They are strong immiune adjuvants and delivery vector.Neisserial PorB is the major outer memebrane protein expressed on the surface of Neisseria species and comprise >60% of the outer memebrane protein content. PorB protein is similar to proteosome in the immune enhance activity. In recent years, Purified PorB and proteosome have been used as mucosal adjuvant in a number of vaccine preparations,and has made breakthrough progress,but PorB recombinant protein as mucosal adjuvant has also not been reported.Many researches displayed that plague F1,V protein is the best selection for developing the new type plague vaccine.We selected different mucosal adjuvants,and mixed with the recombinant F1-V protein in different porportion, intranasally immunized the Balb/c mice to select the safe and effective plague vaccine.Firstly,the high purified proteosome was extracted from group B type 2 Neisseria meningitides.They are comprosed of PorA,PorB and Class4,three major protein with relative molecular weights 41 ku, 38 ku and 34 ku,respectively.The content of lipopolysaccharide, capsular polysaccharides and nucleic acid in proteosome have measure up to a safe standard. That the protein as mucosal adjuvant are safe.Secondly,the gene of porB was obtained from the whole genome isolated group B type 2 Neisseria meningitides by using PCR technology, the porB gene was cloned into pET-28a(+) prokaryotic expression vector, and expressed in E.coli BL21(DE3), then identified recombinant PorB protein using western blot analyse.Finally, the Balb/c mices were intranasal immunized for three times using recombinant F1-V fusion protein adjuvanted with rPorB and proteosome,only recombinant F1-V fusion protein. The IgG titer and subgroup in serum and the IgA titer in douch of nasal pharynx, alveolus, intestine and vagina were tested by ELISA,and FCM was used to detect the phenotype of splenic lymphocyte. The result showed that the PorB-adjuvanted Fl-V and the proteosome-adjuvanted Fl-V elicited significant higher titres of serum IgG(IgG1> IgG2a), sIgA in lung and nasal washes than plague F1-V alone (P<0.01). the effect of rPorB-plague F1-V vaccine was comparable with proteosome-plague F1-V vaccine and they all can induced the higher system immune response and mucosal immune response.The rPorB adjuvant is the effective mucosal adjuvant to selected for the recombinat F1-V antigen in our research.The research of prevention plague safely and effectively laid a theoretical foundation, and it also has provided technical support for the mucosal vaccine research on other infectious diseases.