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High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells
Fei Zhong1, Zhen-Yu Zhong, Shuang Liang
1Department of Basic Veterinary Medicine, School of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, Hebei Province, China. feizhong2000@yahoo.com
Aim:
To develop a highly efficacious method for preparation of soluble SARS 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 approximately 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 microg/10(6) 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.
