链亲和素标记的人粒细胞巨噬细胞-集落刺激因子融合蛋白的制备

Abstract

Abstract: Objective To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF)
fusion protein and evaluate its bioactivity. MethodsPET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids
were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins. The two fusion proteins were
refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion
exchange chromatography. MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the
proliferation of human erythroleukemia cells (TF-1). The efficiency of the fusion proteins for surface modification of
biotinylated MB49tumor cells was evaluated by flow cytometry. ResultsThe recombinant fusion proteins SA-hGM-CSF and
hGM-CSF-SA were highly expressed in Rosetta (DE3) at about20% of the total bacterial proteins, with a purity of about96%
after purification. The two fusion proteins exhibited bifunctional activities, namely the pro-proliferation effect on human
erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified
rate of about 99% ).ConclusionSA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the
development of cancer cell vaccines with surface modification by hGM-CSF.

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