在真核细胞中表达的幽门螺杆菌UreC,OipA抗原性的检测及其对细胞的作用  

Detection antigenicity of UreC,OipA of Helicobacter pylori expressed in gastric epithelial cell and effect of its on epithelial cell

作　　者：张静[1] 佘菲菲[1] 陈豪[1] 陈月秀[1] 

机构地区：[1]福建医科大学病原生物学系分子医学微生物学实验室,福州350004

出　　处：《中国免疫学杂志》2005年第9期682-686,共5页Chinese Journal of Immunology

基　　金：福建省自然科学基金重大项目(2001F003)资助

摘　　要：目的:研究幽门螺杆菌(Hp)UreC、OipA基因重组子转染胃上皮细胞后表达产物的抗原性及其对胃上皮细胞的作用,为两种毒力因子DNA疫苗的研制提供可行性依据及安全性指标。方法:用RTPCR方法从国际标准株NCTC11637中获取UreC、OipA全长基因,克隆入pGEMTEasy载体并测序,以重组T载体为模板,将两种基因的开放读码框架分别定向克隆入pcDNA3.1载体;获得的重组子转染SGC7901细胞,筛选耐潮霉素的细胞克隆,用RTPCR及Immunoblot方法检测细胞内UreC、OipA蛋白的表达和抗原性;用荧光染色技术、MTT、流式细胞术分别检测UreC、OipA对细胞表型、增殖及凋亡的影响。结果:SoipA、SureC细胞(分别转染OipA、UreC重组子)表达相应的产物且具有抗原性。荧光显微镜下观察SoipA、SureC细胞未见明显形态学改变;用MTT法检测细胞增殖:SoipA、SureC细胞与SpcDNA3.1细胞(转染pcDNA3.1)比较,生长增殖无显著性差异(P>0.05),流式细胞技术检测细胞凋亡结果:SoipA和SureC的凋亡率与SpcDNA3.1比较无显著性差异(P>0.05)。结论:OipA、UreC在培养细胞内的表达产物具有抗原性且对细胞的功能无影响,可考虑用于制备DNA疫苗。Objective：To investigate the effect of recombinant plasmid of Helicobacter pylofi UreC, OipA gene transfected into SGC-7901 on epithelial cell and offer security index for developing DNA vaccine of two virulence factors.Methods：The whole sequences of UreC, OipA were amplified by RT-PCR from standard strain NCTC11637, inserted into pGEM-T Easy vector and sequenced. The ORFs of UreC, OipA were inserted directionally into the eukaryotie expression vector pcDNA3.1, SGG-7901 cell was transfected with recombinant plasmid. The cloned cell resisting Hygromacine were picked out. Gene expression in cell were detected by RT-PCR and Immuno-blot. Effects of UreC, OipA on cell phenotypo, proliferation, apoptosis and cell cycles were detected respectivly by fluorescence straining, MTr assay and flow eytometyr. Results：Corresponding products which have antigenicity were expressed in SoipA and SureC. No obvious morphology change were observed in SoipA and SureC. There was no statistic significance（P〉0.05） between the growth of SpcDNA3.1 and that of SoipA, SureC. There was no statistic significance（ P 〉 0.05 ） between the apoptosis rate of SpcDNA3.1 and flint of SoipA, SureC. Conclusion： Expression product of UreC and OipA that have antigenicity had no effect on cell function, so they could be prepared for DNA vaccine.

关 键 词：幽门螺杆菌 UreC OIPA 细胞增殖 细胞凋亡 基因转染 

分 类 号：R373.33[医药卫生—病原生物学]