山羊重组促卵泡素长效类似物基因在毕赤酵母中表达.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2008, March 25; 24(3): 409-414 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 杨欢利, 赵西彪, 宋晓娟, 丁家桐 , 225009 : 为了获得长效FSH 制剂, 利用重叠PCR 技术将山羊FSH 的α, β亚单位, 通过hCGβ亚单位羧基末端延长肽(CTP) 基因序列的连接, 构建成单链长效类似物基因FSH β-CTP-α 。将其克隆至表达载体pPIC9K, 重组载体线性化处理后电转 化至毕赤酵母 GS115 中, 经判型和 G418 筛选后获得高拷贝菌株 His+Mut+ 。经甲醇诱导表达后, 进行 SDS和 Western blot 分析表明: 重组FSH β-CTP-α的转化子可以正确有效地表达目的蛋白, 分子量约为29 kD 。放射免疫分析法 (RIA)测定表达上清, 高拷贝转化子的平均表达量为91.849 mIU/mL, 显著高于低拷贝转化子的平均37.419 mIU/mL 的表 达量, 为FSH 的结构研究和长效FSH 制剂的生产奠定了基础。 : 山羊促卵泡素, 长效类似物基因, 毕赤酵母 Expression of Goat Follicle-stimulating Hormone Analogous Gene in Pichia pastoris Huanli Yang, Xibiao Zhao, Xiaojuan Song, and Jiatong Ding College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China Abstract: In order to obtain the long-acting FSH preparation, the single strand long-acting analogous gene FSHβ-CTP-α was successfully constructed by the C-terminal peptide(CTP) of carboxyl-terminal region of human chorionic gonadotropin with the goat FSHα-subunit and β-subunit genes, then it was inserted into pPIC9K vector.The recombinant plasmid pPIC9K FSHβ-CTP-α was + + transformed into Pichia pastoris GS115 by electroporation. The multi-copy inserts His Mut were gained by the screening of pheno- type and hyper-resistance to G418.After methanol