Analysis Of The Immune Effect Induced By Pneumococcal Surface Protein A(PspA)and Complexes

Construction and expression of recombinant protein from CDR regions of clades 3 S.pneumonia surface protein A(PspA)and mouse IgG Fc fragment gene with gene recombination technology,researching the immune response induced by fusion protein Fc-PspA23F and the immunogenicity of antigens improve by fusion protein.The research contents are summarized as follows:1、Construction and verification of PspA23F-pET27b(+)recombinant plasmidPrimers for CDR region gene of clade 3 PspA(AF071816)was designed according to the published sequence in GenBank and amplified CDR region gene of PspA,the product was cloned into the vector pET-27b(+),Obtain the recombinant plasmid PspA23F-pET27b(+).The recombinant plasmid was PCR and double digestion verification,we can found that the position of 330bp we saw a very bright strip,similar with known CDR region gene of clade 3 PspA;the recombinant plasmid was sent to sequence,The results showd that the homology up to 99%compared with PspA gene,they are meet the verification requirements of positive clones.2、Construction and verification of PspA23F-Fc-pET27b(+)recombinant plasmidPrimers for the mouse IgG Fc fragment gene(V00798)was designed according to the published sequence in GenBank and amplified mouse IgG Fc gene,the product was cloned into the constructed vector PspA23F-pET27b(＋),Obtain the recombinant plasmid PspA23F-Fc-pET27b(+).The recombinant plasmid was PCR and double digestion verification,we can found that the position of 680bp we saw a very bright strip,similar with known mouse IgG Fc gene;the recombinant plasmid was sent to sequence,The results showd that the homology up to 99%compared with mouse IgG Fc gene,they are meet the verification requirements of positive clones.3、Expression and purification of PspA23F-Fc fusion proteinThe recombinant plasmid PspA23F-Fc-pET27b(+)transformed into E.coli BL21,inducing by IPTG and expressing recombinant protein,we can found fusion protein was in bacteria supernatant and molecular weight was about 42KDa.The recombinant protein PspA23F-Fc was separated and purified by the nickel affinity chromatography column.4.Immunoassays of PspA23F-Fc fusion proteinAfter immuned mice with the recombinant protein PspA23F-Fc,collected mice blood,using ELISA and flow cytometry method,we can found that the titer of antibodies is 100 induced by fusion protein,possible reason is that immune time is too short or direct ELISA method is less sensitive,the specific reasons for the need to continue experiments.Different cross-immunity caused by different clades,different strains affinity from branches are also difference.Fusion protein can binding to immune cells mediated by Fc,and antigen can target binding to antigen presenting cells.In summary,PspA protein as an important antigen ingredient and heterogeneity exists in a different clade of bacteria,the fusion protein can enhance the efficiency of delivery of antigen present by the Fc region react with the immune cells,provide a new ideas for the development of similar vaccines.