Study On The Improvement Of The Antiimmunization Of Pseudomonas Aeruginosa And Staphylococcus Aureus

Pseudomonas aeruginosa and Staphylococcus aureus are important conditioned pathogens causing pneumonia in humans and mammals.The pneumonia caused by them has a high fatality rate and is difficult to treat.In recent years,people have used a lot of antibiotics,which has led to the emergence of corresponding drug-resistant strains,which has increased the difficulty of treatment.Vaccination may be one of the effective ways to prevent related infections,but the current immune effect of some vaccines is not ideal.To overcome the shortcomings of single immune response and weak immunogenicity caused by the existing subunit vaccines,more supplements are needed.Effective immune adjuvant to enhance the immune response of the vaccine.Therefore,the development of new safe and effective vaccine adjuvants to stimulate the body to produce efficient natural and acquired immune responses,and to improve the level of humoral and cellular immune responses,maybe a new idea for the development of effective P.aeruginosa and S.aureus vaccines.In this study,safe and non-toxic lactic acid bacteria extracellular polysaccharides were used as injection and mucosal adjuvants to observe the immunoprotective effects of P.aeruginosa and S.aureus recombinant subunit vaccines.This study will lay an experimental foundation for the molecular design and adjuvant compatibility optimization of novel and effective P.aeruginosa and S.aureus genetic engineering polyvalent subunit vaccines.The main results as following:1.The target genes oprF,oprH,oprI,pcrV were cloned using the standard strain of P.aeruginosa as the template,and the target genes gap C and mnt C were cloned using the standard strain of S.aureus as the template.The expression vectors of p EASYBlunt E1-oprF,p EASY-Blunt E1-oprH,p EASY-Blunt E1-oprI,p EASY-Blunt E1-pcr V,p EASY-Blunt E1-gap C,p EASY-Blunt E1-mnt C were successfully constructed.Transform the recombinant vector into E.coli BL21(DE3)competent cells to induce the expression of the target protein.Purified by Ni-NTA on a nickel column,the target protein is obtained.2.Mice were immunized with P.aeruginosa antigens and EPS30 adjuvant by subcutaneous injection,and the lungs of the mice after two immunizations were infected with Pseudomonas aeruginosa.The temperature changes of mice in each group were detected,the survival rate and lung bacterial load of mice in each group were counted,and the lung pathological changes of mice in each group were observed.The results showed that the experimental group could raise the level of Ig G antibody in mice serum,reduce the amount of P.aeruginosa colonization in mice lungs,and improve the survival rate of mice after challenge.The protective rate of pcrv + eps30 group was 70%;compared with other experimental groups after challenge,the amount of bacterial colonization in the lungs is lower and the time required for body temperature to return to normal is the shortest;Pcr V+EPS30 is injected subcutaneously After twice,the highest level of antigen-specific antibodies produced in mouse serum was measured.Using EPS30 and P.aeruginosa antigens to immunize mice through the nasal route,it was found that the titers of antigen-specific Ig G antibodies in the serum of mice in the immunized group and antigen-specific SIg A antibodies in the lung lavage fluid were significantly higher than those in the control group.The Pcr V+EPS30immune protection rate in the experimental group was up to 80%;the Pcr V+EPS30group compared with the Pcr V+CT adjuvant group,there was no significant difference in the detection of antigen-specific Ig G antibodies and SIg A in the mouse serum after the three immunizations.The results showed that EPS30 could be used as a subunit vaccine of P.aeruginosa Mucosal adjuvants.3.Mice were immunized with S.aureus antigens and EPS30 adjuvant by subcutaneous injection.After two immunizations,the mice were challenged by nasal drip.It was found that the experimental group could significantly increase the level of specific Ig G antibodies in the mouse serum and reduce S.aureus in mice The amount of colonization in the lungs improves the survival rate of mice after challenge.The results showed that the serum antigen-specific Ig G antibody and the survival rate of mice in the immunized group were significantly higher than those in the control group.After the challenge,the colonization amount and pulmonary injury of S.aureus in the immunized group were significantly reduced.In summary,the research of this subject found that EPS30 of Lactobacillus exopolysaccharide has good immune adjuvant properties,and it with P.aeruginosa and S.aureus antigens can improve the protection rate of mice;At the same time,we found that EPS30 has the potential of mucosal immune adjuvant,and we will continue to carry out further research as mucosal immune adjuvant in the future,so as to provide theoretical basis for clinical research.