亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和蛋白酶3C基因在昆虫细胞中的共表达

目的在昆虫细胞中表达亚洲1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)衣壳蛋白前体P1-2A基因和蛋白酶3C基因,为进一步研究FMDV空衣壳抗原和疫苗奠定基础。方法从质粒pTOP-P1-2A和pTOP-3C中分别扩增出编码亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和3C蛋白酶基因,构建转移载体pDual-P1-2A-3C并与大肠杆菌(Escherichia coli)DH10Bac中的Bacmid质粒重组,构建重组转座质粒rBacmid-P1-3C。在脂质体介导下将rBacmid-P1-3C转染Sf9昆虫细胞获得重组杆状病毒。在Sf9昆虫细胞中共表达P1-2A和3C蛋白,通过SDS-PAGE、Western blot和Dot-ELISA试验鉴定重组蛋白。结果目的基因在昆虫细胞中得到了正确的表达,表达蛋白与抗亚洲1型FMDV血清发生特异性反应,有良好的抗原性。结论成功在昆虫细胞中表达亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和蛋白酶3C基因。

Co-expression of genes encoding the capsid protein precusor P1-2A and protease 3C of foot-and-mouth disease virus Asia type 1 in insect cells

The capsid protein precursor P1-2A gene and protease 3C gene of foot-and-mouth disease virus(FMDV) Asia type 1 were amplified from the plasmid pTOP-P1-2A and pTOP-3C,respectively.P1-2A and 3C gene were inserted into Baculovirus transfer plasmid pFastBacTM Dual to construct recombinant transfer vector pDual-P1-2A-3C.The pDual-P1-2A-3C was then transformed into Escherichia coli DH10Bac competent cells,transposited with Baculovirus shuttle vector(Bacmid) and constructed recombinant transposition rBacmid-P1-3C.After the rBacmid-P1-3C transfected into Sf9 cells,the recombinant Baculovirus was harvested.The expressed proteins were analyzed by SDS-PAGE,Western blotting and Dot-ELISA.It was found that the expressed proteins could react with rabbit sera against FMDV Asia type 1.These results indicated that the expressed proteins were accurately expressed in Sf9 cells,and displayed nice specificity to FMDV Asia type 1 antisera and biologic activation.