口蹄疫病毒非结构蛋白2C基因在昆虫细胞中的表达及应用  

Expression of Non-Structural Protein 2C of Foot-and-mouth Disease Virus in Insect Cells and Its Application

作　　者：张小丽[1,2] 卢曾军[2] 马小军[1] 曹轶梅[2] 付元芳[2] 刘在新[2] 谢庆阁[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,中国农业科学院兰州兽医研究所,兰州730046

出　　处：《畜牧兽医学报》2011年第12期1724-1731,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家支撑计划(2006BAD06A12);国家“973”计划(2005CB523201)

摘　　要：研究口蹄疫病毒(FMDV)非结构蛋白(NSP)2C在区分灭活疫苗免疫动物与自然感染动物方面的意义。本研究将FMDV NSP 2C基因,克隆到穿梭载体pFast-bac-HT-B,将其转入含骨架载体Bacmid的DH10Bac,经蓝白斑筛选得到重组骨架质粒Bacmid-2C。将Bacmid-2C转染昆虫细胞Sf9,鉴定正确后,经3次增殖获得高滴度的P-3代病毒后,在High Five细胞中进行目的蛋白的表达。SDS-PAGE结果显示在High Five细胞中得到了相对分子质量约为38.93ku的目的蛋白2C,Western blotting及Dot-ELISA结果显示,该表达产物对FMDV感染动物阳性血清有良好的反应性。以电泳纯化的2C蛋白为抗原建立间接ELISA,检测健康非免疫动物、免疫动物及FM-DV试验感染动物血清,结果表明2C-ELISA不但能区分免疫动物和感染动物的血清,而且还能检测FMDV感染早期动物血清中的2C抗体。说明昆虫细胞表达的2C蛋白可作为FMDV疫苗免疫动物与自然感染动物鉴别诊断的良好抗原。2C基因在Bac-to-Bac系统中的成功表达为建立通过检测几种NSP抗体,筛查感染及隐性带毒动物,净化畜群的方法奠定了基础。In recent years,the potential value of nonstructural protein（NSP） 2C was well documented for distinguishing foot-and-mouth disease virus（FMDV） infected animals from vaccinated animals.In this study,2C gene of FMDV was cloned into baculovirus transfer vector pFast-bac-HT-B,and then the recombinant vectors were transformed into DH10Bac which contain backbone Bacmid.The white colonies were selected and Bacmid-2C recombinant vectors were used for transfection of Sf9 cells.After three times amplification of the viral stocks,the NSP-2C was expressed in High Five cells.The SDS-PAGE showed that a 38.93 kD fusion protein 2C was successfully expressed,and the products showed a specific reactivity with serum from FMDV infected animal using Western-blot analysis and Dot-ELISA.An indirect ELISA based on fusion protein 2C was developed,the results showed that this 2C-ELISA not only could be applied to distinguish FMDV infected animals from vaccinated animals,but also could be used to detect early anti-2C antibodies in cattle and pigs which had been infected with FMDV.The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.This study make a good foundation for development of diagnosis method for detection of antibodies against multiple NSPs to discriminate the FMDV infected animals from vaccinated ones,and this will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.

关 键 词：口蹄疫病毒 2C基因 昆虫杆状病毒 表达 应用 

分 类 号：S852.659.6[农业科学—基础兽医学]