【摘要】 目的克隆和表达人可溶性OX40L重组蛋白(sOX40L),探讨OX40L信号在T细胞对乳腺癌细胞反应中的协同刺激效应。方法运用基因工程技术在大肠杆菌中表达人重组融合蛋白GSTsOX40L,Westernblot分析表达蛋白的特异性;采用体外T细胞和乳腺癌细胞混合培养体系,观察对T细胞生长和增殖的影响。结果构建了GSTsOX40L原核表达体系,从而进行重组蛋白的有效表达;纯化的重组蛋白通过免疫印迹分析能被特异性单抗识别,并在体外培养体系中显示出协同刺激T细胞对乳腺肿瘤细胞的反应性促进T细胞生长、增殖以及IL2的分泌。结论成功地研制了具有生物学活性的sOX40L,为体外激发肿瘤特异性T细胞提供良好的物质基础。更多还原

【Abstract】 Objective:To clone and express human soluble OX40L in BL-21 E.coli to investigate the costimulate effect of OX40 signal to the biological activity of T cells cultured with breast tumor cell in vitro.Methods:DNA fragment encoding OX40L was obtained from total RNA of human mature Dendritic cells by reverse-transcriptase polymerase chain reaction(RT-PCR). The cDNA fragment encoding extra-cellular domain of OX40L was cloned into GST fusion protein expression vector pGEX-5X-3 by endonuclease digesting. The fusion protein GST-sOX40L was expressed and purified step by step according to manufactory’s protocol and verified by Western Blotting method. The effect of this human recombinant protein to the biological activity of T cells was investigate by 3H-TdR incorporation method and ELISA.Results:The human recombinant protein GST-sOX40L was verified specially by Western Blotting method and could promote T cell proliferation and IL-2 secretion cultured with breast tumor cell in vitro.Conclusion:The fusion protein was expressed successfully and functional. It provided an effective way to stimulate tumor specific T cell in vitro. [更多还原