AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA...AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.展开更多
基金Supported by the National Natural Science Foundation of China,No. 30270078
文摘AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.
