Detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in serum of patients with gastric diseases by a recombinant protein CagA 1

Abstract

To construct a prokaryotic expression system of a fragment from Helicobacter pylori cagA gene and to detect the CagA positive Helicobacter pylori (CagA(+) H. pylori) and its antibody with the recombinant protein cagA 1. H.pylori isolates were obtained from biopsy specimens of 156 patients with gastric diseases. PCR method was used to detect frequency of cagA gene in 109 H. pylori isolates and to amplify a 2 148 bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 was constructed. Expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blot and immunodiffusion assay were applied to determine immunoreactivity and antigenicity of rCagA1. Two ELISA protocols were established to detect CagA expression in 109 H. pylori isolates and CagA antibody in serum of patients with gastric diseases. Correlations between infection of CagA(+) H. pylori and gastric diseases were analyzed. H. pylori strains were isolated from 80.8% of the biopsy specimens (126/156) and 97.2% of the isolates (106/109) were cagA gene positive. In comparison with the reported data, homologies of nucleotide and putative amino acid sequences of the cloned cagA1 fragment were 94.83% and 93.30%, respectively. The output of rCagA1 was approximate 30.0% of the total bacterial proteins. rCagA1 was able to combine with the commercial antibody against whole cell of H. pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. 92.6% of the H. pylori isolates (101/109) expressed CagA and 88.1% of serum samples (96/109) were CagA antibody positive. The percentage of CagA(+) H. pylori strains (97.9%) of peptic ulcer trended to be higher than that of gastritis (88.5%), but there was no statistically significant difference between two groups (chi(2)=3.48, P>0.05). The recombinant rCagA1 can be used to detect CagA of H. pylori and its antibody. No association is found between CagA expression of H. pylori strains and types of gastric diseases in this study.