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目的:制备链亲合素标记的鼠白细胞介素21融合蛋白(mIL21-SA),治疗小鼠表浅膀胱癌。方法:构建mIL21-SA-pET21质粒,BL21表达,Ni-NTA纯化,透析复性,Western blot鉴定,MTT法检测其对小鼠胸腺细胞的增殖作用,流式检测其对生物素化的MB49锚定修饰率。建立小鼠MB49表浅膀胱癌模型,将mIL21-SA锚定在小鼠膀胱表面进行治疗并观察小鼠存活时间。60d后对mIL21-SA治疗后存活小鼠进行二次攻击。结果:mIL21-SA可以促进T细胞增殖,且具有SA介导的高效结合已生物素化的MB49表面的功能(修饰率98.74%)。膀胱灌注60d后,mIL21-SA组有10只(10/25)存活,二次攻击后,仍有6只(6/10)存活。与对照组比较均有统计学意义(P<0.05)。结论:该实验研制了具有双重活性的mIL21-SA,mIL21-SA锚定修饰治疗表浅膀胱癌是一种有效的免疫治疗方法。
Objective: To prepare strept avidin-labeled murine interleukin-21 fusion protein (mIL21-SA) for the treatment of mouse superficial bladder cancer. Methods: The mIL21-SA-pET21 plasmid was constructed, BL21 expression was purified by Ni-NTA, dialyzed and refolded, and identified by Western blot. MTT assay was used to detect the proliferation of mouse thymocytes. Flow cytometry was used to detect the biotinylated MB49 anchor Fixed rate. The mouse MB49 superficial bladder cancer model was established, the mIL21-SA was anchored on the bladder surface of mice and the survival time was observed. After 60 days, mIL21-SA survived mice were challenged twice. Results: mIL21-SA promoted T cell proliferation and had the function of SA-mediated efficient binding to the surface of biotinylated MB49 (modification rate: 98.74%). After 60 days of bladder perfusion, 10 (10/25) survived in the mIL21-SA group and 6 (6/10) survived after the second challenge. Compared with the control group were statistically significant (P <0.05). Conclusion: This experiment developed a dual active mIL21-SA, mIL21-SA anchor modified superficial bladder cancer is an effective immunotherapy.