[Helicobacter pylori VacA up-regulates secretion of macrophages by transfection of VacA eukaryotic expression vector].
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 sequencing. Then the recombinant plasmids pDsRed-Monomer-C1/vacA were transfected into macrophages. Their expression 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-alpha or IL-1beta in the culture medium was tested quantitatively with Enzyme Linked Immunosorbent Assay (ELISA) kit, respectively. The effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the secretion of macrophages transfected with the recombinant 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 developed in some of macrophages transfected with the recombinant plasmids. VacA over-expressed increased the level of TNF-alpha and IL-1beta. PDTC decreased the production of TNF-alpha and IL-1beta induced by VacA. In conclusion, we have successfully constructed the eukaryotic expression plasmid encoding VacA. The over-expression of VacA fusion protein can up-regulate secretion of macrophages. Activation of NF-kappaB is probably involved in VacA induced cytokines production.
Li
Cunyan
C
Institute of Pathogenic Biology, University of South China, Hengyang 421001, China. lcunyan8@yahoo.com.cn
Zhang
Yan
Y
Liu
Zhijie
Z
Yu
Minjun
M
Yu
Wen
W
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000378:metabolism; D055032:Electron Microscope Tomography; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D009499:Neutral Red / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; C074689:VacA protein, Helicobacter pylori; D009499:Neutral Red