Background: Helicobacter pylori is the most common bacteria causing chronic infections worldwide. An important virulence factor of H. pylori is a vacuolating cytotoxin (VacA) that induces the formation of acidic vacuoles in cytoplasm and damage to epithelial cells. The aim of this study was to examine the antigenic properties of the recombinant VacA of H. pylori in infected sera of mice and human.

Materials and Methods: In this experimental study, the highly antigenic region of VacA gene (1233 bp) was detected by bioinformatics methods, and it was amplified by PCR method and cloned into the pET32a expression vector. After expression and purification of the target protein, its antigenicity was studied by Western Blotting using human sera infected with H. pylori and sera from immunized mice infected with purified recombinant VacA.

Results: PCR and sequencing results showed that the target gene was correctly cloned into the recombinant vector. Antibodies used in Western Blotting indicated the production and expression of the recombinant protein (65kDa) with concentration of 2.1 mg/ml.

Conclusion: Recombinant VacA protein has antigenic and immunogenic properties thus, it is a proper candidate for designing H. pylori vaccine and diagnostic kits

Background: Helicobacter pylori (H. pylori) is a gram negative bacilli that causes the stomach and duodenum diseases in human. An important virulence factor of H. pylori is a CagA gene that increases of colonization it in stomach epithelial cells and lead to inflammation and peptic ulcers. The aim of the present study was to production of recombinant protein containing highly antigenic region of CagA in E. coli.

Materials and Methods: In this experimental study, the antigenic region (1245 base pair) of CagA gene was detected by bioinformatics methods, proliferated by PCR method, digested by BamHI and XhoI restriction enzymes and cloned into pET32a plasmid and was expressed in the E. coli BL21 (DE3) pLysS with induced by IPTG. The expressed protein was purified with Ni-NTA kit and its antigenicity was studied by western blotting method.

Results: Data showed the successful cloning and expression of the target gene. Recombinant CagA protein purified by Ni-NTA kit and dialysis with concentration of 1.5 mg/ml. In western blotting, the produced protein was interacted with infected human and mice sera.

Conclusion: Results indicated that recombinant CagA protein (65 KDa) maintains its antigenicity, so could be used for serological diagnosis of H. pylori diseases and production of vaccine.

Background: Streptokinase is one of the most common and cost effective fibrinolytic drugs for treatment of heart attacks and vein thrombosis. Unlike many advantages over other thrombolytic drugs, administration of streptokinase can produce some complications such as immunologic reactions, hemorrhage and incomplete treatment due to relative short half life. Pegylation is one of the most common methods for improving of these shortcomings.

Materials and Methods: In this study, designing a proper candidate for specific pegylation with cysteine was done by means of SPDBviewer software. After a meaning ful mutation by SOEing PCR method, mutated (sk45cys) and intact SK (ski) genes were cloned in pET26-b vector and the structures were transformed in E.coli. Clones, Afrer growing, were expressed by IpTG and exptression of proteins was confirmed by SDS-PAGE and western blotting. The proteins were purified by affinity chromatography with NiNTA columns and amidolytic activity of purified proteins was assayed using chromogenic method and different concentrations of S2251 substrate.

Results: Results of activity assays showed that amidolytic activity of SK45cys had about 10% increase in comparison to Ski, after 30 minutes of complex formation with plasminogen.

Conclusion: Generally, it was concluded that, considering cys45 as a superficial aminoacid and also relative increase of activity, SK45cys can be considered a suitable protein for specific pegylation.