巨噬细胞激活剂Polytuflsin在毕赤酵母中的表达及其活性检测

Expression of macrophage activator Polytuftsin in Pichia pastoris and analysis of its biological activity

目的利用毕赤酵母表达系统分泌表达巨噬细胞激活剂Polytuftsin。方法化学法将编码Polytuftsin蛋白的重组质粒转入毕赤酵母菌中，甲醇诱导阳性转化子分泌表达Polytufisin目的蛋白；分别于体内外水平上检测纯化后的eolytuflsin对巨噬细胞功能的影响。结果经SDS—PAGE、PCR法验证该Polytufisin重组蛋白是正确的目的产物，相对分子质量约为20×10^3；该重组蛋白能够体外刺激巨噬细胞分泌NO、TNF—α因子；提高巨噬细胞杀死L1210白血病细胞的能力，最大杀伤率达到40％；经小鼠白血病模型治疗实验发现：给予Polytuftsin后小鼠出瘤时间延迟，小鼠生存期延长，平均达到21d。结论所得到的Polytuflsin重组蛋白可激活巨噬细胞，发挥抗肿瘤作用。

Objective To investigate the secrection of Polytuftsin, a macrophage activator in Pichia pastoris expression system. Methods The recombinant plasmid encoding polytuftsin was transformed into the yeast by chemical method, and the positive clones were screened and expressed under the induction of methanod. It was observed that the influence of polytuftsin on macrophage in vivo and in vitro. Results It was verified that the recombinant products was confirmed to be our target protein by SDS-PAGE analysis and PCR assay. The expressed product could activate macrophage to secret cytokine and enhance the capacity of macrophages to kill L1210 tumor cells, the maximum killing rate is up to 40 %. In vivo the leukemia therapeutic trial, the time of tumor arise was delayed; the survival time of L1210 tumor-bear mice treated with Polytuftsin was extended, which is about 20 day. Conclusion The recombination protein polytuftsin, which could activate macrophage and has anti-tumor activity.