猪脑心肌炎病毒非结构蛋白3AB基因在重组杆状病毒中的表达与鉴定

Expression and identification of non-structural protein 3AB gene of Encephalomyocarditis virus in recombinant baculovirus

利用PCR方法扩增获得EMCV非结构蛋白3AB基因,并将其克隆至杆状病毒转座载体pFastBacTM中,提取阳性克隆质粒转化至DH10Bac感受态细菌,通过蓝白斑筛选和PCR鉴定,得到重组杆状病毒穿梭载体Bacm id-3AB,经脂质体介导转染sf9细胞获得重组杆状病毒,并IFA和W estern b lotting鉴定其抗原性。结果表明,EMCV 3AB基因在昆虫细胞中获得成功表达,分子量约16 ku,具有良好的EMCV抗原性。因此利用杆状病毒表达系统成功表达了EMCV 3AB基因,为该病毒抗原表位和诊断方法研究奠定了重要基础。

The 3AB gene of Encephalomyocarditis virus （EMCV） was amplified by PCR, cloned into baeulovirus expression vector pFast- BacT, and confirmed by enzyme digestion. Then, the recombinant plasmid was transformed to DH10 Bac competent cells, and the recombinant Bacmid-3AB plasmid was screened by blue-white colony and identified by PCR. After the purified Bacmid-3AB plasmid was transfection into Si9 cells, the recombinant baculovirus was obtained. Expression of the 3AB gene was confirmed by indirect immunofluorescent assay （IFA） , SDS-PAGE and Western-blotting. The expressed 3AB gene product had a molecular weight of 16kD and could be recognized by the antibody to 3AB protein of EMCV. The results showed that the 3AB protein was efficiently expressed in the recombinant baculovirus. The expressed protein could be used to develop the method for detecting the antibody to EMCV.