山羊重组促卵泡素长效类似物基因在毕赤酵母中表达

Expression of Goat Follicle-stimulating Hormone Analogous Gene in Pichia pastoris

摘要:

为了获得长效FSH制剂, 利用重叠PCR技术将山羊FSH的a, b亚单位, 通过hCGb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FSHb-CTP-a。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-PAGE和Western blot分析表明: 重组FSHb-CTP-a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。

Abstract:

In order to obtain the long-acting FSH preparation, the single strand long-acting analogous gene FSHb-CTP-a was successfully constructed by the C-terminal peptide(CTP) of carboxyl-terminal region of human chorionic gonadotropin with the goat FSHα-subunit and b-subunit genes, then it was inserted into pPIC9K vector.The recombinant plasmid pPIC9K FSHb-CTP-a 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 induction, the supernatant was analysised by SDS-Polyacrylamide Gen Electro-phoresis and Western blot. The results show that the transformants of FSHb-CTP-a could express the objective protein successfully and the molecular weight is about 29 kD. The concentration of supernatant was detected by Radio-immunoassay and the average expression of multi-inserts is 91.849 mIU/mL and the low-inserts is 37.419 mIU/mL. The expression of multi-inserts is higher than the low-inserts significantly. This research lay the foundation for studying the structure of FSH and the production of long-acting FSH preparation.