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FMDV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.
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.
[1] Baranowski E,Molina N,Nunez J I,et al.Recovery of infectious foot-and-mouth disease virus from suckling mice after direct inoculation with in vitro-transcribed RNA[J].Journal of virology,2003,77(20):11290-11295.
[2] Abrams C C,King A M Q,Belsham G J.Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system[J].Journal of General Virology,1995,76:3089-3098.
[3] 周建华,丛国正,高闪电,等.口蹄疫OA/58 病毒株VP2 蛋白结构模拟与B 细胞抗原表位的分析[J].华北农学报,2008,23(3):1-4.
[4] Belsham G L,Brangwyn J K,Ryan M D,et al.Intracellular expression and processing of foot-and-mouth disease virus capsid precursors using vaccinia virus vectors influence of the L protease[J].Virology,1990,176:524-530.
[5] 周建华,丛国正,高闪电,等.口蹄疫病毒株OA/583C 蛋白酶的结构模拟和功能分析[J].华北农学报,2007,22(6):9-13.
[6] Brown F.Vaccination against foot and mouth disease virus[J].Vaccine,1992,10:1022-1026.
[7] Mayr G A,Chinsangaram J,Grubman M J.Development of replication defective adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate[J].Virology,1999,263(2):496-506.
[8] Leticia C B,Mildred F C,Graham J,et al.Induction of a protective response in swine vaccinated with DNA cncoding foot-and-mouth disease virus empty capsid proteins and the 3D RNA polymerase[J].Journal of General Virology,2001,82:1713-1724.
[9] Doel T R,Baccarini P J.Thermal stability of foot-and-mouth disease virus[J].Archives of Virology,1981,70:21-32.
[10] Lewis S A,Morgan D O,Grubman M J.Expression,processing and assembly of foot-and-mouth disease virus capsid structures in heterologous systems:induction of a neutralizing antibody response in guinea pigs[J].Journal of Virology,1991,65(12):6572-6580.
[11] 兰丽盼,吴小锋.昆虫杆状病毒研究和应用新进展[J].农业生物技术学报,2008,16(6):1056-1060.
[12] Urakawa T,Ferguson M,Minor P D,et al.Synthesis of immunogenic,but non-infectious,poliovirus particles in insect cells by a baculovirus expression vector[J].Journal of General Virology,1989,70:1453-1463.
[13] Cao Y M,Lu Z J,Sun J C,et al.Synthesis of empty capsid-like particles of Asia I foot-and-mouth disease virus in insect cells and their immunogenicity in guinea pigs[J].Veterinary Microbiology,2009,137(1-2):10-17.
[14] Chiang Y W,Wu J C,Wang K C,et al.Varied properties of hepatitis-delta virus-like particles produced by baculovirus-transduced mammalian Cells[J].The Open Biotechnology Journal,2007,1:34-40.
[15] Gilbert L,Toivola J,Lehtomaki E,et al.Assembly of fuorescent chimeric virus-like particles of canine parvovirus in insect cells[J].Biochemical and Biophysical Research Communications,2004,313:878-887.
[16] Oka T,Yamamoto M,Miyashita K,et al.Self-assembly of sapovirus recombinant virus-like particles from polyprotein in mammalian cells[J].Microbiology and Immunology,2009,53(1):49-52.
[17] 张韵,易咏竹,李志勇,等.O型口蹄疫病毒P1-2A3C基因在家蚕杆状病毒表达系统中的表达[J].蚕业科学,2008,34(1):148-153.