幽门螺杆菌VacA N端真核表达载体的构建、鉴定及其诱导巨噬细胞空泡化和凋亡的观察

目的构建幽门螺杆菌(Helicobacter pylori,HP)空泡毒素(Vacuolating cytotoxin,VacA)基因的真核表达载体pDsRed-Monomer-C1/vacA,并在THP-1巨噬细胞中表达,为研究VacA单一毒力决定簇的致病性奠定实验基础。方法用Primer 5.0软件设计引物,以HP基因组为模板,PCR扩增vacA目的基因片段,克隆入真核表达载体pDsRed-Monomer-C1中,经酶切、PCR鉴定及测序鉴定后,转染THP-1巨噬细胞中,荧光显微镜和Western-blot检测VacA蛋白在细胞中的表达;电镜观察巨噬细胞(MΦ)的空泡样变和凋亡;流式细胞仪检测细胞的凋亡率。结果PCR扩增得到了大小约为1 428bp的目的片段,双酶切及测序鉴定证明成功构建了HP真核表达重组载体;转染后24h,重组质粒组部分细胞中有聚集的荧光颗粒,部分细胞发生空泡样变和凋亡改变;细胞凋亡率明显高于空质粒组和阴性对照组(P<0.001),细胞核因子-κB(nuclear factorkappaB,NF-κB)的抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocarbamate,PDTC)抑制细胞的凋亡。结论VacA蛋白瞬时高表达促进THP-1巨噬细胞空泡样变和凋亡。NF-κB可能参与调节VacA诱导的巨噬细胞凋亡。

Construction and identification of the eukaryotic expression vector for gene encoding the vacuolating cytotoxin of Helicobacter pylori and observations on the induction of macrophage vacuole formation and apoptosis

To construct the eukaryotic expression vector for gene encoding the vacuolating cyto-toxin(VacA)of Helicobacter pylori(Hp)and to investigate the effect of VacA on macrophages as an individual virulence determinant,the vacA gene was amplified using the primers designed by Promer 5.0 software and the Hp-genome as template and then cloned to eukaryotic expression plasmid pDs-Red-Monomer-C1.After endonuclease digestion,PCR identification and sequencing,this gene was transfected into THP-1 macropages.The expression of protein VacA was determined by examination with fluorescence microscopy and Western blotting.Meanwhile,the vacuole-like lesion and apoptosis were observed with electron microscopy,and the apoptotic rate was determined by flow cytometry analysis.It was demonstrated that a target gene fragment with a molecular mass of 1428 bps was obtained through PCR amplification.As demonstrated by restriction endonucleases analysis and sequencing,it was proved that the eukaryotic expression recombinant pDs-Red-Monomer-C1/vacA was successfully constructed.When this recombinant plasmids were transfected into macrophages,a clear vacuolated phenotype and VacA-over-expresson-induced apoptotic changes in some of macrophages could be demonstrated,in which the apoptotic rate was significantly higher than those the plasmid-free and negative control groups(P<0.001)and the nuclear factor NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)could suppress the cell apoptosis.From the above observations,it is concluded that the transient high expression of VacA protein may induce the vacuole-like lesion and apoptosis in THP-1 macro-phages,in which NF-κB may participate the regulation of cell apoptosis induced by VacA protein.