重组人巨细胞病毒嵌合肽基因在毕赤酵母中的克隆和表达  

Cloning and Expression of Recombinant Human Cytomegalovirus Peptide Gene in Pichia pastoris

作　　者：谢琪璇[1] 余巍[1] 潘善培[1] 肖銮娟[1] 彭雅林[1] 张春雪[1] 

机构地区：[1]暨南大学生殖免疫研究所,广州510632

出　　处：《中国生物工程杂志》2005年第3期29-34,共6页China Biotechnology

基　　金：深圳市科技计划重点资助项目

摘　　要：为了在毕赤酵母中表达带有组氨酸纯化标记的重组人巨细胞病毒嵌合肽 (rHCMVp) ,根据其基因序列 ,设计引物从pPIC9K rHCMVp上扩增得到目的基因片段 ,并导入毕赤酵母诱导型表达载体pPICZαA中。通过电击将线性化的重组质粒转化到毕赤酵母X33细胞中 ,筛选获得表达量较高的重组菌株 ,研究了该菌株生长的培养条件 ,包括不同诱导时间、甲醇浓度、pH值对人巨细胞病毒嵌合肽表达的影响。 2L发酵罐进行了高密度发酵 ,经 1 %的甲醇、pH6 0的条件下诱导48h ,最终菌体密度OD60. 0 达到 1 80 ,每升发酵液中含目的蛋白 78. 7mg ,产量比摇瓶提高了 4 8倍。rHCMVp可通过高密度发酵大量获得。In order to express the recombinant human cytomegalovirus peptide(rHCMVp) with a hexahistidine tag in Pichia pastoris allowing rapid purification by IMAC, the gene sequence was amplified from plasmid pPIC9K-rHCMVp,and then transmited into expression vector pPIZαA.The recombinant plasmid was linearized and transformed into Pichia pastoris X33 strain.Then a high level expression strain was screened by fermantarion in flasks.The growth conditions of the transformant strain were optimized in shake flasks including methanol concentration,pH and inducing time. Then the conditions were applied to the production of rHCMVp in high-density fermentation.The highest expression of rHCMVp was obtained when the Pichia pastoris were induced with 1%(v/v) methanol in pH6.0 culture medium for 72h in shake flasks. After the genetically engineered yeast were induced in the 2-liter fermentor for 48h, the OD 600 of cells was 180.The concentration of rHCMVp in the supernatant was 78.7mg/L that was 4.8 times more than that induced in the shake-flask for 72h.The rHCMVp could be obtained largely by high-density fermentation.

关 键 词：人巨细胞病毒 HCMV 克隆和表达 诱导型 目的基因 毕赤酵母 重组质粒 嵌合肽 表达载体 表达量 

分 类 号：Q786[生物学—分子生物学] R373[医药卫生—病原生物学]