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

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FSHβ-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-PAGE和Western blot分析表明:重组FSHβ-CTP-α的转化子可以正确有效地表达目的蛋白,分子量约为29kD。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91.849mIU/mL,显著高于低拷贝转化子的平均37.419mIU/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。

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 FSHa-suburfit and β-subunit genes, then it was inserted into pPIC9K vector.The recombinant plasmid pPIC9K FSHβ-CTP-α was wansformed into Pichia pastoris GS 115 by electroporation. The multi-copy inserts His^＋Mut^＋ were gained by the screening of phenotype and hyper-resistance to G418.After methanol induction, the supernatant was analysised by SDS-Polyacrylamide Gen Electrophoresis and Western blot. The results show that the transformants of FSHβ-CTP-α 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 mlU/mL and the low-inserts is 37.419 mlU/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.