口蹄疫病毒融合表位基因在原核及真核表达系统中的正确表达

Correct expression of fusion epitopes cDNA of foot-and-mouth disease virus in eukaryotic and proeukaryotic expression systems

目的:在体外检测口蹄疫病毒融合表位基因的表达。方法:计算机辅助设计(CAD)口蹄疫病毒融合表位基因SG,采用Western印迹分析和双抗体夹心ELISA法对该基因在原核系统和真核系统的表达进行检测。结果:得到不同表位组合的融合表达克隆,37℃培养2h后用IPTG诱导5h,融合蛋白在2×YT培养液中得到高效表达;全菌中相应蛋白条带与阳性抗体呈阳性反应,阳性反应强弱基本与表位特性及数量有关;得到4个P815细胞阳性克隆,培养上清、细胞膜和细胞质的抗原均为阳性,其中培养上清中浓度较低,细胞膜和细胞质中浓度较高。结论:CAD的融合表位基因在原核和真核系统均得到正确表达;CAD对于表位研究的设计很有帮助。

Objective: To detect the expression of the fusion epitopes gene of foot-and-mouth disease virus (FMDV) in vitro. Methods: The fusion epitopes gene of FMDV was obtained by the computer aided design (CAD) and its expression in eukaryotic and proeukaryotic expression systems were detected by Western blot and Sandwich ELISA. Results: The clones composed of different epitopes produced the large yield of fusion protein expression when incubated in 2×YT culture medium for 2 h and then induced by isopropyl-β-D-thiogalactopyranoside(IPTG) for 5 h. The corresponding bands of fusion proteins positively reacted with positive serum. The reactive strength was basically corresponding to the number of fused epitopes. Four positive transfectants of P815 cells were got and there was more antigen protein detected on cell membrane and in plasma than in culture supernatant. Conclusion: The constructed epitope genes designed by CAD are correctly expressed in eukaryotic and proeukaryotic system. CAD is very useful in epitope study.