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[Preparation and bioactivity evaluation of SA-mIL15 fusing protein]
Yan-li Chen1, Xiao-ling Xu, Jia Tang
1Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou, China. chenyanli198402@163.com
Aim:
To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins.
Methods:
pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding. The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer. MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA.
Results:
Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta (DE3) at about 20% of total bacterial proteins. They exhibited the bi-functionality: proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1x10(6); IU/mg for SA-mIL15 or 2 x 10(5); IU/mg for mIL15-SA, and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency.
Conclusion:
SA/mIL15 bi-functional fusion proteins were generated, which made feasible the development of mIL15-surface-modified cancer cell vaccine.
