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Published on: January 16, 2021
[Expression, purification and identification of fusion protein mTSLPR-Ig]
Fang Zhang1, Yi Shi, Gang Huang
1Nanjing General Hospital of Nanjing Military Region, Department of Respiratory Medicine, Nanjing 210002, China. Zhangfanglab@yahoo.com.cn
Aim:
To construct an adenovirus vector (Ad-mTSLPR-Ig) expressing the fusion protein of the extracellular domain of mouse TSLPR and the Fc fragment of mouse Ig, further purify and assess the soluble fusion protein mTSLPR-Ig.
Methods:
The gene of mTSLPR extracellular domain was amplified from cDNA library of mouse thymus by PCR. Then an adenovirus vector (Ad-mTSLPR-Ig) expressing the fusion protein of mTSLPR and the Fc fragment of mouse Ig was constructed. After Ad-mTSLPR-Ig was transfected into COS-7 cells, the supernatants were collected. The fusion proteins in the supernatants were detected by double antibody sandwich ELISA. The soluble fusion proteins mTSLPR-Ig were purified by protein A affinity chromatography.
Results:
The gene sequence of Ad-mTSLPR-Ig was confirmed by DNA sequencing, and the recombinat adenoviruses harboring mTSLPR-Ig were successfully obtained after the infection of Ad-mTSLPR-Ig. Fusion proteins mTSLPR-Ig in the supernatants were detected by ELISA assay. The purified fusion protein were obtained by affinity chromatography, and identified by Western blot.
Conclusion:
The soluble fusion proteins mTSLPR-Ig have been obtained successfully, which enables further study of its biological activity.
