High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells
High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells作者机构：Department of Basic Veterinary Medicine School of Animal Science and Technology Hebei Agricultural University Baoding 071001 Hebei Province China Department of Biology College of Life Science Hebei University Baoding 071002 Hebei Province China
出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)
年 卷 期：2006年第12卷第9期
页 面：1452-1457页
核心收录：
学科分类：1002[医学-临床医学] 100201[医学-内科学(含：心血管病、血液病、呼吸系病、消化系病、内分泌与代谢病、肾病、风湿病、传染病)] 10[医学]
主 题：SARS-CoV S-protein Expression
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.
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