鼠疫耶尔森菌Ⅲ型分泌系统效应子YopT基因克隆及功能的初步研究  

Construction,expression and function assay of Yersinia T3SS effector YopT

作　　者：马胜利[1,2,3] 曹叶[1,2] 苏文莉[3] 刘京梅[3] 魏从文[2] 钟辉[2] 何湘[3] 

机构地区：[1]安徽大学生命科学学院,合肥230039 [2]军事医学科学院生物工程研究所,北京100850 [3]军事医学科学院疾病预防控制所,北京100071

出　　处：《军事医学》2014年第6期463-465,470,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31270911;31300637;31170029)

摘　　要：目的构建鼠疫耶尔森菌(Yersinia pestis)Ⅲ型分泌系统效应子YopT真核表达载体,并初步探讨其对宿主天然免疫的影响。方法提取Y.pestis 91001株基因组DNA,并以此为模板,PCR扩增yopT基因,酶切并连接入pCMV-HA载体,转化后挑选阳性克隆送测序。测序正确的克隆进行质粒扩增后,转染HEK293T细胞,免疫印迹检测其表达。通过荧光素酶报告基因分析的方法检测YopT对IFN-β及NF-κB基因的影响。结果成功克隆了yopT基因,并在真核细胞中得到表达;yopT抑制IFN-β及NF-κB报道基因活性。结论初步研究表明,yopT抑制IFN-β和NF-κB基因表达,为进一步研究yopT的功能打下了基础。Objective To clone,express and explore the function of yopT,which is an effector protein that belongs to the type Ⅲ secretion system（T3SS） of Yersinia.Methods Using genomic DNA of Yersinia pestis strain 91001 as the template,yopT gene was amplified by PCR.After digestion by restriction endonucleases,the fragments of yopT gene were ligated into pCMV-HA vector and the ligation product was transformed.The positive clone,which was verified by sequencing,was named HA-YopT.The construct,HA-YopT,was transfected into HEK 293T cell line and its expression was monitored by immunoblotting.Finally,the effect of YopT on innate immunity was analyzed by luciferase reporter gene assays.Results HA-YopT was well expressed in HEK 293T cells.YopT inhibited the promotor activity of IFN-β and NF-κB induced by polydA∶ T.Conclusion YopT inhibits host innate immune response,contributing to future study on the function of YopT.

关 键 词：耶尔森菌 鼠疫 Ⅲ型分泌系统 YopT 先天性免疫 

分 类 号：R378.2[医药卫生—病原生物学] Q785[医药卫生—基础医学]