Li Cunyan, Zhang Yan, Liu Zhijie, Yu Minjun, Yu Wen
Institute of Pathogenic Biology, University of South China, Hengyang 421001, China.
Wei Sheng Wu Xue Bao. 2008 Mar;48(3):349-54.
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.
我们构建了一个包含幽门螺杆菌vacA基因N端基因的重组质粒，并研究了VacA作为单一毒力决定因素对巨噬细胞分泌的影响。通过聚合酶链反应（PCR）从幽门螺杆菌扩增的VacA基因被克隆到真核表达载体pDsRed-Monomer-C1中。重组质粒通过限制性内切酶分析和核苷酸测序进行验证。然后将重组质粒pDsRed-Monomer-C1/vacA转染到巨噬细胞中。通过蛋白质免疫印迹法和荧光显微镜检查其在巨噬细胞中的表达。通过电子显微镜和中性红摄取观察巨噬细胞中的空泡化表型。分别使用酶联免疫吸附测定（ELISA）试剂盒定量检测培养基中TNF-α或IL-1β的细胞因子含量。还研究了核因子κB（NF-κB）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对转染重组质粒的巨噬细胞分泌的影响。限制性内切酶分析和核苷酸测序表明成功构建了真核表达重组体pDsRed-Monomer-C1/vacA。转染重组质粒的一些巨噬细胞出现了明显的空泡化表型。VacA过表达增加了TNF-α和IL-1β的水平。PDTC降低了VacA诱导的TNF-α和IL-1β的产生。总之，我们成功构建了编码VacA的真核表达质粒。VacA融合蛋白的过表达可上调巨噬细胞的分泌。NF-κB的激活可能参与了VacA诱导的细胞因子产生。
