摘要
目的:在巴斯德毕赤酵母中表达人巨细胞病毒gp52C末端和pp150C末端串联片段的嵌合肽.方法:用SacⅠ和BglⅡ分别酶切CMVp-pPIC9K重组质粒,电打孔法导入毕赤酵母GS115后,在缺组氨酸的MD板上筛选出转化子,然后根据甲醇利用快速型(Mut+)和甲醇利用缓慢型(Muts)菌株的不同生长特点,筛选出Mut+和Muts型转化子,用PCR法进一步鉴定阳性克隆.分别用甲醇诱导Mut+和Muts型转化子表达目的蛋白4d,取培养产物冻干浓缩,进行SDS-PAGE和Westernblotting,筛选出能特异表达目的蛋白的菌株,分析蛋白的含量及纯度.结果:重组人巨细胞病毒可在甲醇利用快速型毕赤酵母中有效表达,其表达量约占培养上清分泌蛋白的76 5%.结论:重组人巨细胞病毒(HCMV)嵌合肽可在真核细胞毕赤酵母中成功表达.
Aim:To express the recombinant peptide from gp52 and pp150 C-terminal peptides of human cytomegalovirus (HCMV) in the Pichia pastoris GS115. Methods: After linearised by Sac Ⅰ or Bgl Ⅱ, respectively, the recombinant plasmids were transformed into Pichia pastoris GS115 by electroporation. The Mut+ and Muts transformants were screened according to their different growth characteristic in MD plates without histidine. Some of positive transformants were confirmed by PCR. All Mut+ and Muts clones were induced with methanol. After 4 days of methanol induction, the dialyzed and lyophilizated products were analyzed by SDS-PAGE and Western blot. Results: Recombinant peptied of HCMV was expressed effectively in Mut+ Pichia pastoris and camp up to 76.5% of total proteins in supernatant. Conclusion: High-level expression of secreted recombinant peptide of HCMV has been successfully archived in Pichia pastoris expression system.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
2003年第1期73-78,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
深圳市科委科技计划项目
