口蹄疫病毒A型AF/72株衣壳蛋白在昆虫细胞中的表达及鉴定

Expression and Identification of Capsid Protein of AF /72 Strain of Foot-and-mouth Disease Virus Type A in Insect Cells

通过限制性酶切位点将P12A和3C基因插入到带有双启动子(Pp10和PPH)的杆状病毒表达载体p Fast BacTMDual,转化E.coli DH10感受态细胞进行蓝白斑筛选,将鉴定正确的重组杆状病毒质粒转染Sf9细胞后收获重组杆状病毒r Bac-Dual-P12A3C,通过间接免疫荧光(IFA)和蛋白质印迹(Western-blot)检测衣壳蛋白的表达。IFA结果表明表达产物能够被A型FMDV猪抗阳性血清所识别,鉴定正确并具有良好反应原性。Western-blot检测到81 k D(P12A)、57 k D(VP0+VP3)、47 k D(VP3+VP1)、33 k D(VP0)和24 k D(VP1/VP3)5条蛋白条带,与预期相符。该研究为进一步研究A型口蹄疫病毒空衣壳的体外组装提供了试验依据。

The P12 A and 3C genes were cloned into the baculovirus expression vector pFastBacTM Dual which simultaneously expressed the target genes by two individual promoters（ Pp10 and PPH） by restriction enzyme cutting sites. The recombinant baculovirus expression vectors,which carried the target genes,were transformed into E. coli DH10 Bac competent cells to make white and blue screening. After transfecting the sf9 cells,the recombinant baculovirus（ r Bacmid-Dual-P12A3C） were obtained and the detection of the recombinant protein was analyzed by the immunouorescent assay and Western-blot. The result of immunouorescent assay indicated that the recombinant protein could react with the positive porcine antiserum against FMDV type A. As shown by Western-blot,the recombinant baculovirus（ r Bacmid-Dual-P12A3C） appeared five protein bands that represented the P12 A protein（ 81 kD）,VP0 and VP3 protein（ 57 kD）,VP3 and VP1 protein（ 47 kD）,VP0 protein（ 33 kD）,and VP1 or VP3 protein（ 24 kD）,respectively. The study provides experimental basis for the research of the construction of virus-like particles of FMDV Type A in vitro.