摘要
AIM:To construct a prokaryotic expression system of a Helicobacter py/ori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody,so as to understand the manner in which the infection of CagA-expressing Hpylori (CagA^+ Hpylori) isolates cause diseases. METHODS:Hpyloristrains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated.PCR was used to detect the frequency of cagA gene in the 109 Hpyloriisolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06.A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE.Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and ant igenicity of rCagA1,respectively. Two ELISAs were established to detect CagA expression in 109 Hpyloriisolates and the presence of CagA antibody in the corresponding patients'sera,and the correlations between infection with CagA+ Hpyloriand gastritis as well as 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.In comparison with the reported data,the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively.The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein,rCagA1 was able to bind to the commercial antibody against the whole-cells of Hpyloriand to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4.A proportion 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 biopsy specimens of peptic
To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays-(ELISA) for detectin.g Ca.gA.and its antibody, so as to understand the manner in which the infection of CagA-expressing Hpylori(CagA+ Hpylori) isolates cause diseases.
基金
Supported by the Outstanding Young Teacher Fund of Education Ministry of China and the General Research Plan of Zhejiang Provincial Science and Technology Commission,No.001110438
