Study On Expression,Purification And Immunogenicity Of Adhesin Protein From Pseudomonas Aeruginosa

Objective:This article intends to analyze the biological properties of P.aeruginosa Adhesin protein by bioinformatics method,and use genetic engineering to purify the recombinant protein and test its immune activity,in order to study the functions of P.aeruginosa Adhesin protein and provide experimental basis for its function in pathogenesis.Methods:1.The biological characteristics of P.aeruginosa Adhesin protein were analyzed by bioinformatics.2.The Adhesin gene was amplified by PCR through specific primers and using P.aeruginosa DNA as template.The Adhesin gene and pET28a were double digested with BamHI and HindIII,which were harvested and then ligated by T₄ DNA ligase to obtain the recombinant plasmid pET28a-Adhesin.The pET28a-Adhesin was transferred into expression E.coli BL21 to obtain the recombinant strain BL21/pET28a-Adhesin.3.Adhesin protein was induced and purified by affinity chromatography,endotoxin was removed,and the protein was quantified by BCA method.4.The sera were isolated form BALB/C mice immunized with Adhesin protein,and the antibody titer and its subtype in sera were determined.5.The proliferation of lymphocytes and the secreted cytokines were measured in isolated spleen cells.Results:1.The adhesin protein contains 317 amino acids.Its molecular weight is about 34.3 kDa,and its isoelectric point is 9.2.It has a half-life of more than 10 h in bacteria and is a very stable protein.Adhesin is a transmembrane protein located on the cell membrane.2.Adhesin has an alpha-helix content of 46.37%,a random coil content of 35.96%,and a beta sheet content of 17.67%.And the three-level structure of adhesin was obtained by simulation.3.Adhesin has multiple phosphorylation sites and glycosylation sites.4.Adhesin protein contains many B cell epitopes.5.The expression plasmid pET28a ferritin was successfully obtained.After induction by IPTG,37.9 kDa of Adhesin was expressed,and the high purity of Adhesin was obtained.6.Adhesin can induce mice to secrete high titer antibodies,but the content of IgA and IgM was low..However,the content of IgG1 and IgG2a in the sera of mice inoculated with Adhesin protein was significantly higher than that of the control.7.The spleen lymphocytes of mice inoculated with Adhesin protein can proliferate obviously.8.Adhesin protein can induce spleen lymphocyte to secrete Th2cytokines IL-4 and IL-10,but has no effect on Th2 cytokines TNF-αand IFN-γ.Conclusion:1.P.aeruginosa Adhesin protein can be purified by genetic engineering.2.The adhesin protein of P.aeruginosa has good immunogenicity,it may play a protective role through humoral immune response.