Preparation and bioactivity evaluation of streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion protein
10.3969/j.issn.1673-4254.2015.12.10
- VernacularTitle:SA/hI-TAC双功能融合蛋白的制备及其生物学功能鉴定
- Author:
Xiaoling XU
1
;
Ying LIU
;
Qingge CHEN
;
Tongliang HUANG
;
Jimin GAO
Author Information
1. 温州医科大学//浙江省模式生物技术与应用重点实验室
- Keywords:
human interferon-inducible T cell alpha chemoattractant;
streptavidin;
fusion protein;
surface modification
- From:
Journal of Southern Medical University
2015;(12):1715-1720
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity. Methods pET24a-SA-hI-TAC/pET21a-hI-TAC-SA plasmids were constructed and expressed in BL21. SA-hI-TAC and hI-TAC-SA fusion proteins were purified by Ni-NTA affinity chromatography, refolded by dialysis and identified by Western blotting. The bifunctionality of the fusion proteins (biotin-binding function and hI-TAC activity) was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively. Results SA-hI-TAC/hI-TAC-SA fusion proteins were expressed at about 12% and 25% of the total bacterial protein, respectively. The two fusion proteins had a purity of about 85%and 90%after purification, and their purity reached 98%after purification with S-100 gel filtration chromatography. Both of the fusion proteins were efficiently immobilized on the surface of biotinylated mouse bladder cancer MB49 cells (91.3% for SA-hI-TAC and 98.8% for hI-TAC-SA). SA/hI-TAC induced lymphocyte chemotaxis in a dose-dependent manner, and hI-TAC-SA showed a stronger chemotactic effect than SA-hI-TAC. Conclusion We successfully obtained SA/hI- TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.
