Identification Of Predominant T-and B-combined Antigenic Epitopes In Outer Membrane Protein GroEL Of Helicobacter Pylori

Objective To screen as well as 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 NCTC11637was amplified by PCR, and then a prokaryotic expression system of groEL gene was constructed. Expression of the target recombinant protein rGroEL was detected by SDS-PAGE and the expressed rGroEL was extracted by Ni-NTA affinity chromatography. Laser confocal microscopy was applied to determine the distribution of GroEL in H. pylori NCTC11637and SSI strains. The outer membrane protein (OMP) samples of the two strains were extracted using Triton X-114method, and GroEL in the OMP samples was detected by Western Blot assay. Special bioinformatic softwares were used to predict the T-B combined antigenic epitopes in GroEL molecule, and the phage display system of each of the T-B combined antigenic epitopes was generated. Western Blot assay and ELISA were used to detect the immunoreactivity of both the recombinant T-B combined antigenic epitope-containing phage P8Ⅲ proteins and the artificial synthesized T-B combined antigenic epitope peptides, respectively. Results The groEL genes from H. pylori NCTC11637and SSI strains displayed97.68-99.63%identities in their nucleotide and amino acid sequences. The constructed prokaryotic expression system could efficiently express soluble rGroEL. GroEL was confirmed to locate in the outer membrane of H. pylori NCTC11637or SS1strain. Among the six major predicted T-B combined epitopes (GroEL168, GroEL258, GroEL288, GroEL365, GroEL396and GroEL438), GroEL168showed strong positive immunoblotting signal. The results of ELISA showed that the immunoreactivity of GroEL168was strongest (P<0.01), followed by GroEL258and GroEL288(P<0.05).Conclusions GroEL is an outer membrane protein of H. pylori. GroEL168acts as the predominant T-B combined antigenic epitope of GroEL that can be used to develop multiple antigenic peptide vaccine of H. pylori.