鼠疫耶尔森菌YopT突变体对干扰素β表达的调控  

Regulation of expressions of interferon-β by Yersinia pestis YopT mutants

作　　者：焉秋林 李靖飞 张小鹃 董颖 常桂娟 李同据 魏从文 YAN Qiu-lin;LI Jing-fei;ZHANG Xiao-juan;DONG Ying;CHANG Gui-juan;LI Tong-ju;WEI Cong-wen(Institute of Physical Science and Information Technology,Anhui University,Hefei 230601,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;EnnovaBioMedTec Co.,Ltd.,Langfang,Hebei 065001,China)

机构地区：[1]安徽大学物质科学与信息技术研究院,合肥230601 [2]军事科学院军事医学研究院生物工程研究所,北京100850 [3]新绎生物医学科技有限公司,河北廊坊065001

出　　处：《军事医学》2022年第10期758-762,共5页Military Medical Sciences

基　　金：国家自然科学基金(81671973)。

摘　　要：目的构建鼠疫耶尔森菌效应蛋白YopT-F296A、YopT-G298A、YopT-H303A、YopT-Y304F、YopT-F296AG298A和YopT-H303A-Y304F 6种突变体的真核表达载体,并检测6种突变体对干扰素β(IFN-β)表达的调控。方法以pCMV-Myc-YopT为模板,通过PCR技术分别扩增含有6种突变位点在内的yopT片段。扩增片段经核酸内切酶切割后连接到pCMV-Myc载体中构成重组质粒。重组质粒分别转染HEK293T细胞,免疫印迹检测表达水平,同时通过荧光素酶报告基因技术检测YopT及其6种突变体对IFN-β表达的影响。结果6种yopT突变体重组质粒均在真核细胞中成功表达,并发现YopT及其6种突变体均能抑制IFN-β报告基因表达。YopT-C139R对IFN-β的表达没有明显抑制作用。结论YopT 139位是影响IFN-β表达的关键位点,而YopT的296、298、303和304位不是影响IFN-β表达的关键位点。Objective To construct YopT mutants including F296A,G298A,H303A,Y304F,F296A-G298A and H303A-Y304F and to detect their regulation of expressions of interferon-β(IFN-β)was. Methods Using pCMV-MycYopT as the template,the fragments of six mutants of yopT were amplified by PCR before being digested by endonuclease and ligated into the pCMV-Myc vector to form recombinant plasmids that were transfected into HEK293T cells respectively,and the expression level of YopT mutants was detected by immunoblotting and the effect of wild type YopT and its six mutants on the expression of IFN-β was detected by luciferase reporter gene assay. Results Six yopT mutated recombinant plasmids were successfully expressed in HEK293T cells. It was found that YopT-F296A,G298A,H303A,Y304F,F296AG298A and H303A-Y304F could inhibit the expression of IFN-β reporter gene. YopT-C139R had no significant inhibitory effect on the expression of IFN-β. Conclusion YopT 139 is the key site affecting the expression of IFN-β,while the sites of 296,298,303,304 are not the key site affecting the expression of IFN-β,and its virulence function is of vital importance in studies on the innate immune evasion strategy adopted by Yersinia pestis.

关 键 词：鼠疫耶尔森菌 YopT 突变体 干扰素Β 真核表达 荧光素酶报告基因 

分 类 号：R378.61[医药卫生—病原生物学]