经改造的猪IGF-I基因在巴斯德毕赤酵母中的分泌表达

采用酵母偏爱密码子合成五条编码猪IGF-I基因的引物，利用重叠PCR技术拼接获得长度为210bp的猪IGF-I成熟蛋白基因。将该基因插入分泌表达载体pPIC9K中，转化巴斯德毕赤酵母菌。经表型鉴定、PCR分析及G418筛选得到Muts型多拷贝整合菌，以1.5%甲醇诱导培养后，经SDS-PAGE检测表达产物，在7.5 kDa处出现一特异蛋白条带，目的蛋白表达量达410mg/L。Dot blot检测表明，重组蛋白可与抗IGF-I多克隆抗体反应。

Expression of Reconstructed Porcine Insulin-like Growth Factor I in Pischia Pastoris

Five primers were synthesized using yeast biased codons, and a 210bp DNA fragment encoding mature peptide of porcine Insulin-like Growth Factor I was amplified by Over Lap Extension Polymerase Chain Reaction. The fragment was then inserted into pPIC9K vector, with the insert being downstream of AOX1 promoter and α-secreting signal peptide. The plasmid was digested with SalI and transformed into HIS4 mutant Pischia pastoris GS115 strain by LiCl. The phenotype of colonies were identified by comparison of the growth performance on MM and MD plates. Two colonies T1, T2 were selected from 4.0 mg/mL G418 plates, followed by induction of pIGF-I expression with 1.5% methanol. SDS-PAGE analysis of supernatant of the P. pastoris GS115 strain revealed an exogenous protein with molecular weight of approximately 7.5 kDa. Dot blot hybridization assay indicated a specific reaction with rabbit polyclonal antibody against the IGF-I protein. The optimal culture condition of T2 was achieved, and 410 mg IGF-I per liter of fluid culture was produced in P. pastoris strain.