Helicobacter pylori VacA up-regulates secretion of macrophages by Transfec-tion of vacA eukaryotic expression vector

Abstract:

We constructed a recombinant plasmid containing the N-terminus gene of vacA gene of Helicobacter pylori and studied the effect of VacA on the secretion of macrophages as an individual virulence determinant. VacA gene amplified by Polymerase Chain Reaction (PCR) from Helicobacter pylori was cloned into eukaryotic expression vector pDsRed-Monomer-C1. The recombinant plasmids were verified by restriction endonucleases analysis and nucleotide se-quencing. Then the recombinant plasmids pDsRed-Monomer-C1/vacA were transfected into macrophages. Their expres-sion in macrophages was examined by Western blot and fluorescence microscope. Vacuolated phenotype in macrophages was observed by electron microscopy and neutral red uptake. The cytokine content of TNF-α or IL-1β in the culture me-dium was tested quantitatively with Enzyme Linked Immunosorbent Assay( ELISA) kit, respectively. The effect of pyr-rolidine dithiocarbamate (PDTC), an inhibitor of NF-kB, on the secretion of macrophages transfected with the recombi-nant plasmids, was also studied. Restriction endonucleases analysis and nucleotide sequencing showed that the eukaryotic expression recombinant pDsRed-Monomer-C1/vacA was successfully constructed. A clear vacuolated phenotype devel-oped in some of macrophages transfected with the recombinant plasmids. VacA over-expressed increased the level of TNF-a and IL-1b. PDTC decreased the production of TNF-a and IL-1b induced by VacA. In conclusion, we have suc-cessfully constructed the eukaryotic expression plasmid encoding VacA. The over-expression of VacA fusion protein can up-regulate secretion of macrophages. Activation of NF-kB is probably involved in VacA induced cytokines production.