Cloning, Expression, Purification And Identification Of CagA And VacA Proteins Of Helicobacter Pylori

Objective:To construct the recombinant plasmid of protein cagA (cytotoxin-associated gene A) and VacA (Vacuolating cytotoxin A)of Helicobacter pylori and to express the fusion protein in E. coli, for the purpose of detection of wild pathogenic species and patients infected with Hp.Methods:The NCTC11639 strains of H. pylori were collected, DNA were extracted,and the gene segments were amplified by PCR.The gene segments were cloned to T vecter and sequenced, then the objective genes were cloned into pET16b, followed by constructing recombination of expression vector pET16b/CagA and pET16b/VacA. After induced with IPTG, the expression of CagA and VacA protein were identifed by SDS-PAGE, purified with nickel affinity chromatograph, and detected by ELISA.Results:The sequencing analysis demonstrated the inserted CagA gene segment was 1962bp,VacA gene segment was 2256bp, and relative molecule mass(Mr) of expressed product were 75KDa and 87KDa respectively. The detecfion of SDS-PAGE showed the soluble expression product accounted for 20% of total bacterial protein and the the purity of recombinant fusion protein was about 90%. ELISA analysis showed these recombinant fusion proteins have good antigenicity.Conclusion:The gene coding for H. pylori CagA and VacA are cloned,expressed and protein were purified successfully. The results were helpful for the research on development of H. pylori protein vaccine and a quick diagnostic kit applying to detection of H. pylori infection.