摘要
利用RT-PCR方法扩增猪传染性胃肠炎病毒(TGEV)核衣壳蛋白N基因,将其克隆入杆状病毒供体质粒pFastBacTMDual,pFastBacTMDual-N转入DH10Bac菌中进行同源重组,经三重抗性和蓝白斑筛选,得到重组质粒Bacmid-N,将其转染昆虫细胞sf9,获得重组杆状病毒rBac-N.通过间接免疫荧光试验检测显示,重组蛋白在昆虫细胞中得到表达.体外组装TGEV病毒样颗粒(VLPs)试验表明,rBac-N单独感染sf9细胞未见形成明显的病毒样颗粒,昆虫细胞内只观察到杆状病毒粒子.试验结果为探讨TGEV核衣壳蛋白在病毒装配过程中的作用提供了依据.
The N gene of transmissible gastroenteritis was amplified with RT-PCR and then cloned into the donor plasmid of baculovirus pFastBacTM Dual.Subsequently pFastBacTM Dual-N were transformed into E.coli DH10Bac competent cells,and screened by three antibiotics and blu-white patch.The resulting white colony was proved to be recombinant Bacmid-N.Then the Bacmid-N was transfected into sf9cells, and recombinant baculovirus rBac-N was obtained.The expressed product was detected by indirect immunofluorescence assay（IFA）,and the results showed that the recombinant N protein was expressed successfully,for the recombinant proteins had a positive reaction with the antiserum.An experiment of assembly of virus-like particles of transmissible gastroenteritis virus in vitro was carried by infection of insect cells with rBac-N.The results demonstrated that the expression of N protein in the insect cell sf9did not generate any similar structure to virion of TGEV.This work is considered to provide useful information for the study of the role of TGEV nuclecapsid proteins in virion assembly.
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第5期7-12,共6页
Journal of Southwest University(Natural Science Edition)
基金
新疆维吾尔自治区教育厅科研计划项目资助(20120984)
中央高校基本科研业务基金重点项目资助(XDJK2014B039)
