人免疫缺陷病毒II型gag基因在毕赤酵母中表达条件的研究

目的 :提高人免疫缺陷病毒 II型 ( HIV- 2 ROD) gag基因在毕赤酵母中的表达量。方法 :研究了转化子生长的培养条件 ,包括不同的甲醇诱导浓度、诱导时间、溶解氧、摇瓶转速及不同 p H值对人 HIV- 2 gag表达的影响。表达产物进一步通过盐析和 Sephadex G- 1 0 0分子筛层析柱层析分离纯化。结果 :最佳表达条件是 1 .0 %甲醇诱导 ,诱导时间 3d,大于 85 %的溶解氧 ,摇瓶转速2 5 0 r·min-1,p H6.0～ 6.5 ,目的蛋白表达量达到 2 1 mg· L-1。同时 ,经纯化后得到了纯度大于95 %的目的蛋白。结论 :获得了 HIV- 2 RODgag重组蛋白在巴斯德毕赤酵母系统中分泌表达的最佳表达条件。

Study on the Expression Conditions of HIV-2 gag Gene in Pichia pastoris

Objective:To enhance the expression level of HIV 2 gag gene in Pichia pastoris. Methods:The transformant growth conditions in shake flasks including methanol concentration, induction time, aeration, rotation speeds and pH were optimized. Expressed gag was isolated and purified by sating out and Sephadex G 50 chromatography. Results:More than 85% aeration,3 days for induction, the initial pH of 6.0~6.5, the final methanol concentration of 1.0% and agitation speed of 250 r·min -1 was the optimum inductive condition for gag expression.With this condition, the expressed gag was secreted into fermentation broth and reached a yield approximately 21 mg·L -1 . Conclusion:The optimum inductive condition for gag expression in methanol trophic yeast expression system had been obtained.