ABSTRACT
Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.
ABSTRACT
Objective To construct a recombinant fusion protein with pneumococcal surface pro-tein A (PspA) of Stretococcus pneumonia (SPN) familyⅠclade 1 and 2, and to analyze the immunogenici-ty of the fusion protein.Methods The gene fragments encoding theα-helix of PspA of the two clades were amplified by PCR and then inserted into the expression vector pET-27b(+) to construct the recombinant ex-pression plasmid.The transformed Escherichia coli BL21 strains carrying expression plasmid were induced by IPTG to express the recombinant protein.The titers and affinity of antibodies against PspA protein were measured by ELISA.An opsonophagocytic assay and an animal experiment were performed to evaluate the immunogenicity of the recombinant protein.Results Double enzyme cutting and gene sequencing confirmed the two purpose gene fragments were correctly expressed in the expression vector pET-27b(+).The titers of anti-PspA antibody in the serum of Kunming ( KM) mice immunized with the fusion protein were 1 ×104 . The affinity of anti-PspA antibody reached to 2×105 .The rates of recombinant PspA6B-PspA05 protein me-diated phagocytosis for SPN6B, SPN05 and SPN01 strains were 20%, 15% and 8.8%, respectively.No SPN23F strain was engulfed by macrophages upon the stimulation with PspA6B-PspA05 protein.The survival rates of mice injected with SPN05, SPN6B, SPN01 and SPN23F strains were respectively 75%, 92%, 75%and 33%upon the immunization of PspA6B-PspA05 protein.Conclusion The recombinant fusion protein PspA6B-PspA05, constructed with the PspA proteins of Stretococcus pneumonia familyⅠclade 1 and 2, was successfully expressed in the E.coli prokaryotic system with the advantage of high immunogenicity.High ti-ters of anti-PspA antibodies with high specificity were induced in KM mice upon the stimulation with Ps-pA6B-PspA05 protein.Moreover, a cross-protective immunity was induced in KM mice upon the immuniza-tion with PspA6B-PspA05 protein.