AsiaⅠ型口蹄疫病毒VP1基因在昆虫细胞/杆状病毒中的表达

Expression of Foot-and-mouth Disease Virus AsiaⅠ VP1 Gene in Insect Cell/Baculovirus

利用杆状病毒表达系统对AsiaⅠ型口蹄疫病毒(foot-and-mouth disease virus,FMDV)VP1基因在Sf9昆虫细胞中进行表达,为研究AsiaⅠ型FMDVVP1蛋白功能及建立AsiaⅠ型FMDV血清学诊断方法奠定基础。采用PCR方法从pGEM-T-Easy-AsiaⅠ型VP1质粒中扩增VP1基因,将其插入杆状病毒转座载体pFastBacHTA,构建的重组质粒pFast-BacHTA-VP1再转入DH10Bac感受态细胞,经三重抗性与蓝白斑筛选,获得杆状病毒重组质粒Bacmid-VP1,然后转染Sf9昆虫细胞。PCR鉴定证实VP1基因正确地插入到Bacmid中,成功构建了杆状病毒重组质粒Bacmid-VP1,SDS-PAGE和Western-blotting检测结果表明,VP1基因在Sf9昆虫细胞中表达出约26.5ku的VP1蛋白。将可溶性表达的融合蛋白用Ni-NTA亲和层析方法进行纯化,通过ELISA分析,能特异性地检测出AsiaⅠ型口蹄疫病毒阳性血清。AsiaⅠ型FMDV VP1基因在杆状病毒表达系统中的成功表达为AsiaⅠ型FMDV VP1蛋白的抗原性及血清学抗体水平检测研究奠定了基础。

VP1 gene of foot-and-mouth virus （FMI）V） Asia Ⅰ was expressed in insect cell/Baculovirus expression system to lay a foundation for research of FMDV Asia Ⅰ VP1 protein function and serology diagnostic method. The gene VP1 was amplified from the recombinant plasmid of PGEM-FEasy-VP1 of FMDV Asia Ⅰ by PCR and inserted into Baculovirus vector pFastBacHTA to get recombinant plasmid pFastBacHTA-VP1. The recombinant plasmid pFastBacHTA-VP1 was transformed into DH10Bac and the VP 1 gene was integrated into Bacmid by site-specific transposition. Subsequently, the recombinant Bacmid-VP1 was transfected into Sf9 insect cells. The expressed product was identified by SDS-PAGE and Western blotting. The soluble fusion protein was purified by Ni-NTA affinity chromatography. ELISA analysis showed that the protein purified was reactive with the positive serum against FMDV AsiaI The successful expression of FMDV AsiaⅠ VP1 gene in insect celt/Baeutovirus expression system provide material for research of antigenicity of Asia Ⅰ VP1 protein as well as the surveillance of serology antibody.