Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients

AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori (H py/ori) isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori. METHODS: H pylori strains in biopsy specimens from 157 patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected. The target recombinant proteins rUreB, rVacA, rCagAl, rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography. Rabbit antisera against rUreB, rVacA, rCagAl, rHpaA, rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody, expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed. RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagAl, HpaA, NapA, FlaA and FlaB were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2% respectively. In the 125 serum samples from the H pylori infected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FIaA and FlaB were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4% and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P>0.05, x2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P<0.01, x2= 13.19; P<0.05, x2= 6.13). When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P<0.05, x2 = 5.93; P<0.01,x2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P<0.01, x2 = 8.88; P<0.05, X2=5.00). CONCLUSION: The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB, FlaA, HpaA, FlaB, NapA and CagAl, but not VacA.

Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins

AIM: To clone flagellin genes A (flaA) and B (flaB) from a clinical strain of Helicobacter pylori (H pylori) and to construct prokaryotic expression systems of the genes and identify immunity of the fusion proteins.METHODS: The flaA and flaBgenes from a clinical H pylori isolate Y06 were amplified by high fidelity PCR. The nucleotide sequences of target DNA amplification fragments from the two genes were sequenced after T-A cloning. The recombinant expression vector pET32a inserted with flaA and flaB genes was constructed, respectively. The expressions of FlaA and FlaB fusion proteins in E. Coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG)at different concentrations were examined by SDS-PAGE.Western blot using commercial antibodies against whole cell of H pylori and immunodiffusion assay using self-prepared rabbit antiserum against FlaA (rFlaA) or FlaB (rFlaB)recombinant proteins were applied to the determination of the fusion proteins immunity. ELISA was used to detect the antibodies against rFlaA and rFlaB in sera of 125 H pylori infected patients and to examine rFlaA and rFlaB expression in 98 clinical isolates of H pylori, respectively.RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homologies of the cloned flaA and flaB genes were from 96.28-97.13% and 96.31-97.73%, and their putative amino acid sequence homologies were 99.61-99.80% and 99.41-100% for the two genes, respectively. The output of rFlaA and rFlaB expressed by pET32a-flaA-BL21DE3 and pET32a-flaBBL21DE3 systems was as high as 40-50% of the total bacterial proteins. Both rFlaA and rFlaB were able to combine with the commercial antibodies against whole cell of H pylori and to induce rabbits to produce specific antibodies with the same 1:2 immunodiffusion titers after the animals were immunized with the two recombinant proteins. Ninety-eight and zero point 4 and 92.80% of the serum samples from 125 patients infected with H pylori were positive for rFlaA and rFlaB antibodies, respectively.One hundred percent and 98.98% of the 98 tested isolates of H pylori were detectable for rFlaA and rFlaB epitopes,respectively.CONCLUSION: Two prokaryotic expression systems with high efficiency of H pylori flaA and flaB genes were successfully established. The expressed rFlaA and rFlaB showed satisfactory immunoreactivity and antigenicity. High frequencies of FlaA and FlaB expression in different H pylori clinical strains and the general existence of specific antibodies against FlaA and FlaB in H pylori infected patients strongly indicate that FlaA and FlaB are excellent antigen candidates for developing H pylori vaccine.

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.

A Convenient Method for Synthesis of Novel Cyclic Ethers (1R,2R,3R,5S,7S,9R,12R)-3-(t-Butyldimethylsilyl)oxy-7-methoxymethyl-oxy-2,10-dimethyl-12-oxatricyclo [7.2.1.0^(5,12)] dodecane

Novel cyclic esters (1R, 2R, 3R, 5S, 7S, 9R, 12R)-3-(t-butyldimethylsilyl)oxy-7- methoxymethyloxy-2, 10-dimethyl-12-oxatricyclo [7.2.1.05,12] dodecane were prepared when their precursor 1 was treated with SOCl2/pyridine. A plausible mechanism was hypothesized.