人白细胞介素-3基因在酿酒酵母中的分泌表达

Secretor Expression of Human Interleukin 3 in Yeast

利用ＰＣＲ突变方法，改造了人白细胞介素－３（ｈＩＬ－３）ｃＤＮＡ，在成熟蛋白编码顺序５＇端前突变入了Ｘｂａ Ｉ酶切位点和酵母类胰蛋白酶加工位点Ｌｙｓ－Ａｒｇ的密码子。改造后的ｈＩＬ－３ ｃＤＮＡ插入大肠杆菌－酵母穿梭质粒ｐＶＴ１０２ｕ／α，使其准确融合于α－交配因子前导序列之后，并置于启动子ＡＤＨ１调控之下。转化入酿酒酵母宿主菌Ｓ－７８筛选后，３０℃培养４８ｈ。

Using the PCR technique,mutated human interleukin 3(hIL-3) gene was'obtained, in which the restriction site Xba I and site of trpsin-like endoprotease were made in front of the 5' terminal encoding sequence. This gene was fused to the leader sequence of a-mating factor behind and cloned into pVT10 2u/a, a shuttle plasmid between yeast and E. coli, the fused gene can be transcripted under the coNtrol of ADH1 promoter in yeast. After fransformation of the plasmid containing the hIL-3 gene into saccharomyces cerevisiae S-78 and fermentation at 30癈 for 48 hours, it was observed that the culture supernatant was able to obviously stimulate preliferation of the hIL-3 dependent TF-1 cell line. The expressed rhIL-3 in the supernatant was more than 1. 0mg/L identified and quantified by ELISA. It was also found that the biological activity of rhIL-3 in this supernatant was about 1. 8x103u/ml after being analysised by 3H-thymidine incorproation methed using TF-1 cells.