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

LUO Bao-zheng; BO Qing-ru; CHEN Jin-ding; WANG Wei-yi; CHENG Gang; HE Yun-shao

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June 2005

LUO Bao-zheng, BO Qing-ru, CHEN Jin-ding, WANG Wei-yi, CHENG Gang and HE Yun-shao. Expression of FMDV 3ABC Gene in Bac-to-Bac Baculovirus Expression System[J]. Virologica Sinica, 2005, 20(3): 303-306.

Citation: LUO Bao-zheng, BO Qing-ru, CHEN Jin-ding, WANG Wei-yi, CHENG Gang, HE Yun-shao. Expression of FMDV 3ABC Gene in Bac-to-Bac Baculovirus Expression System .VIROLOGICA SINICA, 2005, 20(3) : 303-306.

昆虫杆状病毒系统表达口蹄疫病毒3ABC基因

罗宝正 ¹ ,
薄清如 ² ,
陈金顶 ³ ,
王伟毅 ⁴ ,
程钢 ⁴ ,
何蕴韶 ^4*

1.
1.广东广州中山大学达安基因股份有限公司 2.珠海出入境检验检疫局广东珠海 3.华南农业大学兽医学院 4.中山大学中山医学院

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。
- 棉铃虫核多角体病毒
- , 泛素
- , 克隆
- , 原核表达
- , 抗血清

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

1.
1. Inst itute of Entomology , Central China Normal University , Wuhan 430079 , China

Abstract

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.
- HaSNPV
- , Ubiquitin
- , Gene clone
- , Prokaryotic expressing
- , Antiserum

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

1. 1. Inst itute of Entomology , Central China Normal University , Wuhan 430079 , China

Abstract: 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.