人内皮抑素在毕赤酵母中的表达、纯化及其对小鼠肺腺癌LA795细胞生长的抑制

Expression and purification of human endostatin in Pichia pastoris and its inhibition on the growth of mouse pulmonary adenocarcinoma cell line LA795

目的通过基因重组技术获得能高效分泌表达人内皮抑素的毕赤酵母菌株;观察纯化的重组人内皮抑素(rhES)对小鼠肺腺癌LA795生长的抑制作用。方法 利用氯化锂转化法将人内皮抑素基因整合入毕赤酵母基因组,获得可分泌表达人内皮抑素(endostatin)的菌株。用肝素亲和层析的方法纯化目的蛋白。观察人内皮抑素对碱性纤维生长因子(bFGF)刺激的人血管内皮细胞系ECV－304细胞增殖的作用。将接种LA795肺腺癌细胞的T739小鼠随机分成2组,分别给予rhES和磷酸缓冲盐液(PBS)皮下注射,每日1次,连续14d。观察2组小鼠肿瘤生长情况。结果经筛选获得表达量较高的转化菌株,其表达的rhES能明显抑制bFGF刺激的人血符内皮细胞系ECV－304细胞的增殖,与对照组比较具有显苫差异(P＜0．001),动物实验表明其有效抑制小鼠肺腺癌LA795的生长(P＜0．001)。结论用毕亦酵母作为宿主分泌表达的rhES具有良好的生物学活性,能有效抑制小鼠肺腺癌LA795的生长。

Objective To obtain yeast strain Pichia pus torts that highly expresses human endostatin by gene recombination technology, and to evaluate the inhibitory effect of recombinant human endostatin (rhES) on the growth of mouse pulmonary adenocarcinoma cell line LA795. Methods The gene coding for human endostatin was cloned into the genome of Pichia pusLoris through LiCI transformation method, and the clones with high soluble rhES expression were selected. Purification of rhES was perfonned with heparin affinity chromatography. Effects of rhES on bFGF-induced proliferation of human endothehal cell line ECV-304 cells were observed. T739 mice with subcutaneous inoculation of LA795 cells were treated with injections of either rhES or PBS for 14 consecutive days, and the volume of the tumors were measured. Results Clones with high rhES expression were obtained and purified rhES potently inhibited the proliferation of ECV-304 cells and the growth of LA795 cells in T739 mice. Conclusion rhES produced by Pichia pastors possesses good biological activities and conspicuously inhibits the growth of LA795 cells.