Cloning and Sequence Analysis of Gene Encoding OipA from Iranian Clinical Helicobacter pylori

Abstract

Background: Outer inflammatory protein A (OipA) is one of the important adhesins of H. pylori and a valuable candidate for vaccine development. Its gene is under "on-off" switch status which correlates with OipA protein expression. Objectives: We aimed to obtain a recombinant OipA clone (with "on" status) from an Iranian clinical isolate. Materials and Methods: A clinical H. pylori-isolate demonstrating high expression for an outer membrane protein (OMP) with an apparent MW of 33-35 kDa was selected. oipA specific primer was designed according to oipA sequences from B8 strain. The purified PCR-product was sequenced and submitted to Gene Bank. The pET-28a plasmid and E. coli DH5α were used for cloning and transformation. The recombinanBackground: Outer inflammatory protein A (OipA) is one of the important adhesins of H. pylori and a valuable candidate for vaccine development. Its gene is under “on-off" switch status which correlates with OipA protein expression. Objectives: We aimed to obtain a recombinant OipA clone (with “on" status) from an Iranian clinical isolate. Materials and Methods: A clinical H. pylori-isolate demonstrating high expression for an outer membrane protein (OMP) with an apparent MW of 33-35 kDa was selected. oipA specific primer was designed according to oipA sequences from B8 strain. The purified PCR-product was sequenced and submitted to Gene Bank. The pET-28a plasmid and E. coli DH5α were used for cloning and transformation. The recombinant plasmid was transferred to E. coli BL21 (DE3). Extracted proteins were purified and presence of OipA was confirmed by western blotting using both anti His-tag monoclonal antibody and anti-OipA specific antibody. Results: The sequence of the oipA gene and the MW of the purified recombinant OipA protein consisted on 924 bp and 33-35 kDa, respectively. Its identity with other published oipA genes was 92-96%; highest identity was observed with that of a Mexican oipA clone, obtained from a H. pylori strain associated with severe symptoms. Conclusions: Recombinant oipA clone obtained in this work, may be a functional oipA gene with “on" status, associated with more severe outcomes of H. pylori infection.t plasmid was transferred to E. coli BL21 (DE3). Extracted proteins were purified and presence of OipA was confirmed by western blotting using both anti His-tag monoclonal antibody and anti-OipA specific antibody. Results: The sequence of the oipA gene and the MW of the purified recombinant OipA protein consisted on 924 bp and 33-35 kDa, respectively. Its identity with other published oipA genes was 92-96%; most identity was observed with that of a Mexican oipA clone, obtained from a H. pylori strain associated with severe symptoms. Conclusion: Recombinant oipA clone obtained in this work, may be a functional oipA gene with "on" status, associated with more severe outcomes of H. pylori infection.