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目的:制备CD80-链亲和素(streptavidin,SA)融合蛋白,研究CD80-SA锚定的鼻咽癌CNE2细胞对T细胞杀伤活性的影响。方法:pET21a-CD80-SA-6His质粒转化大肠杆菌BL21(DE3),IPTG诱导CD80-SA融合蛋白的表达,经Ni-NTA亲和层析纯化后透析复性。流式细胞仪检测CD80-SA融合蛋白在CNE2细胞表面的锚定效率;LDH释放法检测CD80-SA锚定的CNE2细胞对T细胞杀伤活性的影响。结果:成功制备和纯化了CD80-SA融合蛋白。CNE2细胞表面低表达CD80分子[(2.2±0.18)%]。CD80-SA融合蛋白可有效地锚定于生物素化的CEN2细胞表面,锚定效率可达73%。CD80-SA锚定的CNE2细胞可有效激活T细胞的杀伤作用,效靶比在1∶1、1∶20、1∶40时T细胞的杀伤率分别约为(37±3.12)%、(51±2.63)%和(58±2.47)%,均显著高于对照CNE2细胞激活的T细胞(均P<0.01)。结论:CD80-SA融合蛋白可有效锚定于生物素化的CNE2细胞表面,从而增强T细胞对CNE2细胞的杀伤活性。
OBJECTIVE: To prepare CD80-streptavidin (SA) fusion protein to study the effect of CD80-SA-anchored CNE2 cells on T cell killing activity. Methods: The plasmid pET21a-CD80-SA-6His was transformed into E.coli BL21 (DE3). The expression of CD80-SA fusion protein was induced by IPTG and purified by Ni-NTA affinity chromatography. The effect of CD80-SA fusion protein on the surface of CNE2 cells was detected by flow cytometry. The effect of CD80-SA-anchored CNE2 cells on T cell cytotoxicity was tested by LDH release assay. Results: The CD80-SA fusion protein was successfully prepared and purified. CNE2 cell surface expression of CD80 molecules [(2.2 ± 0.18)%]. The CD80-SA fusion protein is effectively anchored to the surface of biotinylated CEN2 cells with an anchoring efficiency of up to 73%. CD80-SA-anchored CNE2 cells can effectively activate the killing effect of T cells. The killing rates of T cells were (37 ± 3.12)% and (51 ± 2.63)% and (58 ± 2.47)%, respectively, which were significantly higher than that of control T cells activated by CNE2 cells (all P <0.01). Conclusion: CD80-SA fusion protein can effectively anchor the surface of biotinylated CNE2 cells and enhance the killing activity of T cells on CNE2 cells.