GFP拆分方法示踪幽门螺杆菌CagA的转运研究  

Visualization of Helicobacter pylori CagA deliveringby Split-GFP

作　　者：吴倩文 刘书振 张建辉 曹新颖 孙泽坤 贾钰刚 张莹[1] 李波清[1] 赵慧琳[1] 季晓飞[1] WU Qianwen;LIU Shuzhen;ZHANG Jianhui;CAO Xinying;SUN Zekun;JIA Yugang;ZHANG Ying;LI Boqing;ZHAO Huilin;JI Xiaofei(Department of Pathogen Biology,Binzhou Medical University,Yantai,Shandong 264003,China;The Second Clinical Medical College,Binzhou Medical University)

机构地区：[1]滨州医学院病原生物学教研室,山东烟台264003 [2]滨州医学院第二临床医学院

出　　处：《中国病原生物学杂志》2024年第5期502-506,513,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81702054);山东省自然科学基金(No.ZR2020MH297,ZR2023MH101);山东省高校青年创新科技支持计划(No.2020KJK006)。

摘　　要：目的用GFP拆分方法示踪幽门螺杆菌(Helicobacter pylori,Hp)感染过程中CagA向胃上皮细胞内的转运过程。方法(1)PCR扩增CagA基因的上下游同源臂,连入敲除质粒pSJHK4中,将质粒通过电击转化的方法转入Hp中,利用卡那霉素筛选CagA敲除突变株ΔCagA。(2)PCR扩增绿色荧光蛋白GFP第11β折叠(GFP11)的基因序列并与CagA基因序列的3′端相连,将融合序列连入Hp-大肠埃希菌穿梭质粒pCHFHp中,并通过电击转化的方法转入ΔCagA中,利用氯霉素筛选CagA与GFP-11的融合表达突变株。(3)PCR扩增GFP第1-10β折叠(GFP1-10)的基因序列,包装慢病毒,利用病毒感染的方法将其转入胃上皮细胞GES-1中。将(2)中构建的Hp突变株以100∶1的感染复数感染(3)构建的表达GFP(1-10)的GES-1细胞体系,利用荧光显微镜观察细胞内的荧光情况,通过Western blot检测细胞内的CagA蛋白含量。结果构建了CagA敲除质粒pSJHK4-cagA,转入Hp中得到CagA敲除突变株ΔCagA。构建了CagA与GFP-11的融合表达质粒pCHFHp-cagAgfp11,转入ΔCagA中获得CagA与GFP-11的融合表达突变株HpG27-cagAgfp11。构建了含有GFP1-10基因序列的慢病毒,感染GES-1细胞后获得GFP1-10稳定表达的细胞株。在HpG27-cagAgfp11感染GES-1模型中,观察到细胞中的绿色荧光,说明CagA被转运到GES-1细胞内部,其融合表达的GFP-11与细胞内表达的GFP1-10结合发出荧光,Western blot结果也证实CagA被转运至胞内。结论在Hp感染GES-1细胞模型中,GFP拆分方法能够示踪CagA向细胞的转运过程,这为CagA的转运研究提供了新方法,有助于HpIV型分泌系统(T4SS)作用机制的深入研究。Objective Visualization of CagA delivering from Helicobacter pylori(Hp)into infected gastric epithelial cells by Split-GFP method.Methods(1)The upstream and downstream homologous arms of cagA gene were amplified by PCR and inserted into knockout vector pSJHK4.The resulting plasmid was transformed into Hp G27 strain by electroporation.The CagA-knockout mutantΔcag Awas selected by kanamycin resistance.(2)Sequence of the 11~(th)strand of GFPβ-barrel(GFP-11)was amplified by PCR and linked into 3′teminal of cagA sequence.The fusion sequence was then inserted into the Hp-E.coli shuttle plasmid pCHFHp.The resulting plasmid was transformed intoΔCagA strain by electroporationand transformants expressing CagA-GFP11 fusion protein were selected by chloramphenicol resistance.(3)Gene sequence of the first to tenth strands of GFP(GFP1-10)was amplified by PCR and packaged into lentivirus,and then transfected into GES-1 cells by viral infection.Hp mutant expressing fused CagA-GFP11 constructed in(2)was co-cultured with this cell line expressing GFP1-10 at an MOI(multiplicity of infection)of 100.The fluorescence intensityof infected GES-1 cells was observed by fluorescence microscope,and the amount of CagA transferred into GES-1 cells was detected by Western blot.Results ACagA-knockout mutant,ΔCagA,was obtained by transforming a CagA knockout plasmid pSJHK4-cagA into Hp G27 strain.An expression plasmid pCHFHp constructed in our previous work was then inserted with CagA-GFP11 gene sequence and transferred intoΔCagA to obtain the mutant Hp G27-cagAgfp11 expressing CagA-GFP11 fusion protein.Lentivirus packaging GFP 1-10βstrands encoding genes was constructed to infect GES-1 cells and generate a cell line expression GFP1-10βstrands stably.After Hp G27-cagAgfp11 infection,fluorescence was observed in the cytoplasm of infected GES-1 cells,indicating that CagA entered into the recipient cells,and the fused GFP-11βstrand combined with the intracellular GFP1-10 part.Meanwhile,the presence of intracellular CagA was also confirmed

关 键 词：幽门螺杆菌 CAGA GFP拆分 T4SS 

分 类 号：R573.1[医药卫生—消化系统]