RESUMO
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.
RESUMO
Objective To screen and identify the predominant T-and B-cell (T-B) combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori (H.pylori).Methods The entire groEL gene of H.pylori strain NCTC11637 was amplified by PCR,and then a prokaryotic expression system for groEL gene was constructed.The expressed target recombinant protein rGroEL was analyzed by SDSPAGE and purified by Ni-NTA affinity chromatography.Laser confocal microscopy was applied to determine the distribution of GroEL in H.pyloristrains of NCTC11637 and SS1.The outer membrane protein (OMP) samples were extracted from the two strains using Triton X-114 method.GroEL in OMP samples was detected by Western blot assay.Special bioinformatic softwares were used to predict T-B combined antigenic epitopes on GroEL molecule,and phage display systems for every T-B combined antigenic epitope were established.Western blot assay and ELISA were used to detect the immunoreactivity of recombinant T-B combined antigenic epitope-containing phage PⅢ proteins and artificially synthesized T-B combined antigenic epitope peptides,respectively.Results The groEL genes from H.pylori strains of NCTC11637 and SS1 showed 97.68％ ～99.63％ identities in their nucleotide and amino acid sequences.The established prokaryotic expression system could efficiently express soluble rGroEL.GroEL located on the outer membrane of H.pylori strains of NCTC11637 and SS1.Among the six major predicted T-B combined epitopes (GroEL168,GroEL258,GroEL288,GroEL365,GroEL396 and GroEL438),GroEL168 showed the strongest positive immunoblotting signal.The results of ELISA showed that the immunoreactivity of GroEL168 was also the strongest (P＜0.01),followed by GroEL258 and GroEL288 (P＜0.05).Conclusion GroEL is an outer membrane protein of H.pylori.GroEL168 being the predominant T-B combined antigenic epitope of GroEL can be used to develop multiple antigenic peptide vaccines of H.pylori.