共表达O型口蹄疫病毒P12A+3C基因的重组杆状病毒的构建

构建共表达O型口蹄疫病毒(Foot and mouth disease virus,FMDV)衣壳蛋白前体P12A和蛋白酶3C的重组杆状病毒,为进一步研究FMDV空衣壳抗原和基因工程亚单位疫苗奠定基础。从质粒T-OP1中扩增出编码O型FM-DV衣壳蛋白前体的P12A基因,并将其插入到杆状病毒转移载体pFastDual-3C的PH启动子之下,构建重组杆状病毒转移载体pD-P12A3C。通过在大肠杆菌内转座重组,获得重组杆粒B-P12A3C,转染Sf9细胞,获得表达O型FMDV衣壳蛋白的重组杆状病毒。重组杆状病毒经增殖并感染Sf9细胞后,通过双抗体夹心ELISA方法及间接免疫荧光来检测目的蛋白的表达。结果表明,表达产物能被O型FMDV阳性血清识别,具有一定的反应原性,表明重组杆状病毒构建成功,该研究为O型FMDV空衣壳的体外组装及基因工程亚单位疫苗的研究提供了前期材料。

Construction of Recombinant Raculovirus Containing the P12A and 3C Genes of Foot and mouth Disease Virus Type O

The capsid protein precursor P12A gene of foot-and-mouth disease virus(FMDV) type O were amplified from the plasmid T-OP1,the P12A were inserted into the baculovirus transfer vector pFast Dual-3C to construct recombinant transfer vector pD-P12A3C,in which the P12A and 3C were under the control of PH promoter and P10 promoter respectively.The recombinant plasmids were transformed into Escherichia coli DH10Bac(Invitrogen) to construct the recombinant bacmid B-P12A3C,and then,transfected into Sf9 cells,the recombinant baculovirus was harvested.After amplied,the recombinant baculovirus were infected into Sf9 cells.The expressed proteins were analyzed by an indirect sandwich-ELISA and by immuno uorescent assay.These results indicated that the expressed proteins were accurately expressed in Sf9 cells,and displayed specificity to FMDV type O antisera and biologic activation.From this study,The recombinant baculovirus containing the the capsid(P1) and 3C protease coding regions of FMDV type O were successfully obtained,thus providing a basis for the research of FMDV type O empty capsid assembly in vitro and empty capsid vaccine.