Abstract: Objective To construct and identify a Helicobacter pylori(H.pylori) GroEL eukaryotic expression vector pcDNA3.0-GroEL for the development of gene vaccine of H.pylori.Methods Genomic DNA of H.pylori was extracted from H.pylori ATCC 26695.The GroEL gene fragment was amplified from the genomic DNA of H.pylori by PCR.The purified PCR fragment was ligated into the pMD19-T simple vector.The positive clones were screened and identified by PCR from bacteria directly and the PCR products were digested via double enzymes following sequencing of the recombinant plasmid.The GroEL gene fragments from TA clones were cloned into the eukaryotic expression vector pcDNA3.0 using restriction enzymes.The recombinant plasmid pcDNA3.0-GroEL was identified by PCR,enzyme digestion and sequencing.Results The GroEL gene fragment was amplified correctly,with a gene of about 1 640 bp,and a H.pylori GroEL eukaryotic expression vector pcDNA3.0-GroEL was successfully constructed.This indicated that GroEL gene was successfully inserted into pcDNA3.0 plasmid.Conclusion Construction of the H.pylori GroEL eukaryotic expression vector pcDNA3.0-GroEL lays a foundation for the development of H.pylori gene vaccine.