摘要
设计合成特异引物,扩增O型口蹄疫病毒(O/FMDV)P1-2A基因,将其克隆至T载体上,通过HindⅢ和NotⅠ双酶切P1-2A基因和真核转座载体pFastBacTMDual,构建重组转座质粒pFastBac-P12A,再将pFastBac-P12A转化入含穿梭载体Bacmid的受体菌DH10Bac,经重组筛选获得杆状病毒重组质粒Bacmid-P12A。将Bacmid-P12A质粒转染Sf9昆虫细胞,出现典型CPE。病变细胞经Dot blotting和SDS-PAGE检测和分析,结果表明,O/FMDV P1-2A蛋白在Sf9细胞中获得表达,为O型FMDV特异性蛋白。
Based on the known nucleotide sequence of FMDV gene, P1-2A gene primer was designed and synthesized. P1- 2A gene was amplified by RT-PCR. The gene was then cloned into pMD18-T plasmid. The recombinant plasmids were sequenced and cloned into transfer vector pFastBacTM Dual. The transfer plasmid pFastBac-P12A was constructed, pFastBac- P12A was further transferred into receptor DH10Bac bacteria containing a shuttle vector Bacmid. The recombinant plasmid Bacmid-P12A was obtained by selection, then was trans-infected into Sf9 insect cells. The recombinant baculovirus which expressing serotype O FMDV P1-2A gene was harvested. The Sf9 cells were trans-infected with recombinant baculovirus expressing serotype O FMDV P1-2A gene and showed typical CPE. The cells were harvested and tested by FMDV Dot blotting and SDS-PAGE analysis. Results indicated that the serotype O FMDV P1-2A gene expressed in Sf9 cells and the P1-2A protein antigen was specific to serotype O FMDV.
出处
《中国畜牧兽医》
CAS
北大核心
2011年第10期81-85,共5页
China Animal Husbandry & Veterinary Medicine
基金
云南省应用基础研究面上项目(2009CD138)
国家公益性科研(农业)专项(201103008)
云南省农业科技创新工程项目(2008LA019)
