Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H pylon) strain Y06 and the ItB gene from Escherichia coli (E. coli) strain 44851 were linked into ItB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of Hpylori and a self-prepared rabbit anti-rUreB serum, respectively,and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA.RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E, coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins, rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB.CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of Hpylorigenetically engineered vaccine.

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 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 ulcer appeared to be higher than that from gastritis(88.5%),but the difference was not statistically significant(x^2=3.48,P>0.05). CONCLUSION:rCagAl produced by the prokaryotic expression system constructed in this study possesses good immunoreactivity and antigenicity,and the established ELISAs can be used to detect CagA of Hpyloriand its antibody. H pylori isolates show high frequencies of cagA gene and CagA expression,but the infections by CagA^+ H pylori strains are not the most decisive factors to cause gastric diseases.