巴斯德毕赤酵母高密度发酵表达rHCMVp的研究

目的：提高人巨细胞病毒嵌合肽(rHCMVp)在巴斯德毕赤酵母中的表达量，并进行发酵罐高密度发酵。方法：将生长培养基的菌体离心后等量接入诱导培养基中(包括不同体积分数的甲醇、pH值)培养，通过菌体密度检测和发酵液上清的SDS—PAGE结果分析不同条件、不同诱导时间对人巨细胞病毒嵌合肽表达的影响，并依据优化条件进行高密度发酵。结果：摇瓶培养时，转化子在体积分数1％的甲醇pH6．0的条件下诱导72h，A600(菌体密度)为45．2，目的蛋白表达量达到10．2mg／L。2L发酵罐进行了高密度发酵，经体积分数1％甲醇诱导48h，最终A600(菌体密度)达到124，每升发酵液中含目的蛋白57．21mg。结论：通过条件优化，进行高密度发酵，获得大量的rHCMVp。

The expression of recombinant human cytomegalovirus peptide in Pichia pastoris in high-density fermentation

Aim: To enhance the expression level of recombinant human cytomegalovirus peptide (rHCMVp) in Pichia pastoris. Methods: The recombinant yeasts growing in growth medium were transfered equally to inducing medium, which contained different methanol concentration and with different pH. The optimized methanol concentration, pH and induced time for produce of rHCMVp were determined by A_(600) of cells and the SDS-PAGE results of supernatants in shake flasks. Then the conditions were applied to the production of rHCMVp in high-density fermentation. Results: The highest expression of rHCMVp was obtained when the Pichia pastoris were induced with 1.0% methanol in pH 6.0 culture medium for 72 h in shake flasks. The yield of interested protein reached 10.2 mg/L with A_(600) 45.2. After the genetically engineered yeast were induced in the 2-liter fermentor for 48 h, the A_(600) of cells was 124 that was 3 times more than that induced in the shake flasks for 72 h. The concentration of rHCMVp in the supernatant was 57.21 mg/L that was 6.4 times more than that induced in the shake-flask for 72 h.Conclusion: The expression of rHCMVp in Pichia pastoris was heightened by high-density fermentation.