重组巴氏毕赤酵母高密度发酵表达人乳铁蛋白的研究  

Producing human lactoferrin by high-density fermentation recombinant Pichia pastoris

作　　者：应革[1] 吴淑华[1] 王静[2] 赵小东[1] 陈建明[1] 张晓光[3] 侯云德[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [2]中国农业大学食品科学与营养工程学院生物工程系 [3]中国疾病预防控制中心病毒病预防控制所肿瘤病毒室,北京100052

出　　处：《中华实验和临床病毒学杂志》2004年第2期181-185,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的　在保持生物学活性的前提下 ,实现人乳铁蛋白基因在巴氏毕赤酵母中的高密度发酵表达。方法　从人外周血中分离中性粒细胞 ,提取总RNA ,RT PCR方法获得人乳铁蛋白前体全长cDNA序列 ,适当改造后克隆至巴氏毕赤酵母表达载体pPIC 3 5K上并转化酵母宿主KM71,双层滤膜法筛选高表达株进行补料分批发酵 ,对发酵产物的理化及生物学活性进行初步检测。结果　筛选到一株人乳铁蛋白表达量较高的重组巴氏毕赤酵母p3 5 k 7并对其进行了补料分批发酵。整个发酵历时约 9d ,菌浓吸光度A6 0 0 值最高达到 2 6 0以上 ,重组人乳铁蛋白最高表达量为 115mg L ,是摇瓶培养条件下的 7 6 7倍。结论　成功实现了人乳铁蛋白基因在巴氏毕赤酵母中的高密度发酵表达 ,并对发酵参数的优化进行了初步探索。Objective To evaluate expression of human lactoferrin gene by high-density fermentation in recombinant Pichia pastoris on the premise of maintaining its biological activities. Methods The neutrophil was isolated from human peripheral blood and its total RNA was prepared. Full-length cDNA of human lactoferrin gene was then obtained by RT-PCR,cloned into expression vector pPIC 3.5 K and transformed into Pichia pastoris strain KM71. With two-layer filter method,the transformants with high-productivity of human lactoferrin were screened out into fed-batch high-density fermentation. And later,the physical,chemical and biological activities of fermentation product were detected preliminarily. Results The strain p3.5-k-7 with better productivity of human lactoferrin was screened out into fed-batch high-density fermentation. The fermentation lasted nearly for nine days, with A 600 of culture once above 260 and the highest productivity of human lactoferrin being 115 mg/L,7.67 times the amount of that in shake flask cultivation. Conclusion The authors successfully realized high-density fermentation expression of human lactoferrin gene in recombinant Pichia pastoris .

关 键 词：重组巴氏毕赤酵母 高密度发酵 人乳铁蛋白 基因表达 发酵参数 

分 类 号：R346[医药卫生—基础医学]