摘要
AIM： To develop a highly efficacious method for preparation of soluble SAPS S-protein using adenovirus vector to meet the requirement for S-protein investigation. METHODS： The human adenovirus vector was used to express the soluble S-protein （corresponding to 1-1190 amino acids） fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells. RESULTS： Under the conditions of infection dose （MOI of 50） and expression time （48 h）, the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106 cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells, demonstrating that the soluble S-protein could remain the biologic activity in the native molecule. CONCLUSION： The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.
瞄准：用侵入人体气管粘膜的病菌向量为可溶的 SARS S 蛋白质的准备开发一个高度有效的方法为 S 蛋白质调查满足要求。方法：人的侵入人体气管粘膜的病菌向量被用来表示可溶的 S 蛋白质(相应于 1 约 1190 氨基酸) 在 HEK239 房间用优化 codon 的基因构造与 Myc/His 熔化了标签。忍受 S 蛋白质基因的 recombinant 侵入人体气管粘膜的病菌被结扎方法产生。有 Myc/His 标签的表示 S 蛋白质与统帅玫瑰祷告由分离跟随了的 Ni-NTA 从培养基被净化。S 蛋白质被西方的污点检测，它的生物学的活动被绑定分析到 Vero 房间。结果：在感染剂量(50 的 MOI ) 和表示时间(48 h ) 的条件下面， S 蛋白质的高级表示被获得。表示水平决心是在纯化以后的约 75 个 microg/10 (6 ) 房间。净化的可溶的 S 蛋白质被西方的污点乐意地与 anti-Myc 抗体检测并且显示了能力绑 Vero 房间出现，证明可溶的 S 蛋白质能仍然是在本国的分子的生物学的活动。结论：在 HEK293 房间的 S 蛋白质的高级表示由侵入人体气管粘膜的病菌调停了能在优化表示条件下面被完成。蛋白质拥有生物学的活动，它为 S 蛋白质生物功能的进一步的调查打一个基础。
