昆虫杆状病毒系统表达口蹄疫病毒3ABC基因  

Expression of FMDV 3ABC Gene in Bac-to-Bac Baculovirus Expression System

作　　者：罗宝正[1] 薄清如[2] 陈金顶[3] 王伟毅[4] 程钢[4] 何蕴韶[4] 

机构地区：[1]中山大学中山医学院,广东广州510080 [2]珠海出入境检验检疫局,广东珠海519015 [3]华南农业大学兽医学院,广东广州510642 [4]中山大学达安基因股份有限公司,广东广州510080

出　　处：《中国病毒学》2005年第3期303-306,共4页Virologica Sinica

摘　　要：以O型口蹄疫病毒为研究对象,经过RTPCP扩增得到非结构蛋白3ABC基因,克隆到转移载体pFastbacHT,将其转入含穿梭载体Bacmid的DH10Bac,与Bacmid发生位点特异性转座作用,得到3ABC的重组穿梭载体Bacmid3ABC,再将其转染昆虫细胞HiFive。PCR鉴定证实3ABC基因正确地插入到病毒基因组的多角体蛋白基因启动子下游,经过SDSPAGE和Westernblot检测,3ABC基因在昆虫细胞中表达了大小约为50kDa的蛋白条带,3ABC基因在BactoBac系统中的成功表达为建立以基因工程产品为抗原、鉴别诊断自然感染和免疫动物的方法提供了技术条件。The nonstructural protein gene 3ABC of Foot-and-mouth disease virus (FMDV) was amplified by reverse transcription (RT) PCR and the PCR product was inserted into transfer vector pFastbacHT. Then, the recombinant vector pFastbac HT-3ABC was extracted and transfered into DH-{10}Bac containing a shuttle vector Bacmid. 3ABC gene was integrated into Bacmid by site specific transposition. Subsequently, recombinant shuttle vector Bacmid-3ABC was transfected with Hi Five insect cells. Identification by PCR amplification demonstrated that 3ABC gene was correctly inserted into baculovirus genome at the downstream of polyhedrin promoter. SDS-PAGE and Western blot analysis detected a band of about 50 kDa in the expression product of 3ABC gene in insect cells. The successful expression of 3ABC gene in Bac-to-Bac expression system should provide a new technical platform for the development of novel approaches for distinguishing the infected animals from vaccinated animals.

关 键 词：口蹄疫病毒 非结构蛋白3ABC 杆状病毒 鉴别诊断 

分 类 号：S852.65[农业科学—基础兽医学]