Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene,CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

AIM:To construct a prokaryotic expression system of aHelicobacter py/ori ( H pylori) cagA gene fragment andestablish enzyme-linked immunosorbent assays (ELISA) fordetecting CagA and its antibody,so as to understand themanner in which the infection of CagA-expressing Hpylori(CagA~+ Hpylori) isolates cause diseases.METHODS:Hpyloristrains in gastric biopsy specimens from156 patients with positive results in rapid urease test wereisolated.PCR was used to detect the frequency of cagAgene in the 109 Hpyloriisolates and to amplify a 2 148-bpfragment (cagA1) of cagA gene from a clinical strain Y06.Aprokaryotic expression system of cagA1 gene was constructed,and the expression of the target recombinant protein(rCagA1) was examined by SDS-PAGE.Western blotting andimmunodiffusion assay were employed to determine theimmunoreactivity and ant igenicity of rCagA1,respectively.Two ELISAs were established to detect CagA expression in109 Hpyloriisolates and the presence of CagA antibody inthe corresponding patients’sera,and the correlationsbetween infection with CagA+ Hpyloriand gastritis as wellas peptic ulcer were analyzed.RESULTS:Of all the clinical specimens obtained,80.8%(126/156) were found to have Hpyloriisolates and 97.2%of the isolates (106/109) were positive for cagA gene.Incomparison with the reported data,the cloned cagA1fragment possessed 94.83% and 93.30% homologies withthe nucleotide and putative amino acid sequences,respectively.The output of rCagA1 produced by theconstructed recombinant prokaryotic expression system wasapproximately 30% of the total bacterial protein,rCagA1was able to bind to the commercial antibody against thewhole-cells of Hpyloriand to induce the immunized rabbitsto produce antibody with an immunodiffusion titer of 1:4.Aproportion as high as 92.6% of the Hpyloriisolates (101/109)expressed CagA and 88.1% of the patients’serum samples(96/109) were CagA antibody-positive.The percentage of CagA~+ H pylori strains(97.9%)isolated from the biopsyspecimens of peptic ulcer appeared to be higher than thatfrom gastritis(88.5%),but the difference was not statisticallysignificant(x~2=3.48,P>0.05).CONCLUSION:rCagAl produced by the prokaryoticexpression system constructed in this study possesses goodimmunoreactivity and antigenicity,and the establishedELISAs can be used to detect CagA of Hpyloriand its antibody.H pylori isolates show high frequencies of cagA gene andCagA expression,but the infections by CagA~+ H pylori strainsare not the most decisive factors to cause gastric diseases.