口蹄疫病毒3A蛋白和FLAG标签的融合表达与血清学分析  

Prokaryotic expression and serological analysis of FLAG-fused 3A protein of foot-and-mouth disease virus

作　　者：祁国财[1] 曹轶梅[1] 付元芳[1] 厍大亮[1] 况文东[1] 孙普[1] 卢曾军[1] 刘在新[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点实验室甘肃省生物检测工程技术研究中心国家口蹄疫参考实验室OIE口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2012年第7期690-695,共6页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2011AA10A211)

摘　　要：通过对比各短肽标签,选择由8个氨基酸组成的FLAG标签弥补口蹄疫疫苗毒株OZK 3A蛋白的93～102位10个氨基酸缺失,原核表达FLAG与3A的嵌合蛋白,并命名为3AF。同时也表达了OZK毒株原始3A蛋白。分别以3A和3AF蛋白作为免疫原免疫6～8周龄雌性BALB/c小鼠,采用ELISA和Western-blot分析融合蛋白的血清学反应活性。结果表明,3A蛋白和3AF蛋白均能在大肠杆菌中有效表达,且主要以可溶性形式表达。纯化的3AF蛋白能与FLAG单抗特异性反应而未能诱导免疫小鼠产生针对该标签的抗体,体现了体外和体内的生物反应性差异。FLAG标签对3A蛋白的表达水平、表达形式等未产生显著影响。本研究为下一步FLAG标记病毒的拯救及其应用奠定了基础。The FLAG tag,which consisted of 8 amino acids,was selected to make up the 10 amino acids deletion corresponding to residues 93 to 102 within 3A protein of foot-and-mouth disease virus（FMDV） vaccine strain OZK, and then the recombinant FLAG-fused 3A protein（3AF protein） was expressed in Escherichia coll. Meanwhile,the native 3A protein of wild type strain was also expressed as a control. Six to 8 week-old female BALB/c mice were vaccinated with 3A and 3AF as immunogens, the in vivo and in vitro serological reactivity of the proteins 3A and 3AF was also analyzed by ELISA and Western-blot,re- spectively. The results showed that both 3A and 3AF proteins were expressed effectively,primarily in solu- ble form. The anti-FLAG monoclonal antibody could specifically recognize the FLAG tag within the 3AF protein. Interestingly,both 3A and 3AF proteins could induce antibodies against 3A protein without anti- bodies against FLAG tag in mice,indicating the difference between the in vivo and in vitro reaction. The FALG tag made non significant difference in the expression level and expression form of 3A protein. The present study laid foundation for the rescue and application of FLAG-markered FMDV.

关 键 词：口蹄疫病毒 3A蛋白 FLAG标签 酶联免疫吸附试验 

分 类 号：S852.659.6[农业科学—基础兽医学]