用醋酸锂法转化巴氏毕赤酵母表达人核心蛋白聚糖  

Expression of DCN in the eukaryotic by LiAC

作　　者：郝小夏[1] 

机构地区：[1]山西医科大学汾阳学院,032200

出　　处：《山西医药杂志（上半月）》2010年第5期401-403,共3页Shanxi Medical Journal

基　　金：山西省自然科学基金(20011061)

摘　　要：目的用醋酸锂法转化巴氏毕赤酵母表达人核心蛋白聚糖(DCN)。方法将重组体pPic9k-DCN经BglⅡ酶切线性化,通过醋酸锂法转入酵母宿主HIS-/GS115细胞中,然后在含不同浓度G418的YPD平板上筛选阳性克隆;用含1%甲醇的BMMY培养基诱导表达该蛋白;并观察表达产物对HepG2细胞生长的影响。结果①通过醋酸锂法将酶切线性化的重组载体成功转入酵母菌HIS-/GS115,并用聚合酶链反应(PCR)法进行了鉴定;②用含1%甲醇的BMMY培养基诱导表达出目的蛋白;③用表达产物与HepG2细胞共同孵育,发现其对HepG2细胞增生有抑制作用。结论本实验成功地用真核表达系统表达了人核心蛋白聚糖,并观察到其对HepG2细胞生长有抑制作用。Objective The eukaryotic expression system was used for expressing DCN.Methods The recombinant plasmid was linearized by cutting of restriction enzyme.The linear DCN gene was introduced into HIS-/GS115 cells by LiAC.The transformants were transferred onto YPD plates that contained different concentrations of G418 for selecting the positive clones.Selected transformants were cultured in BMMY medium with 1% methanol for inducing the expression of DCN protein.Effects of expression product on the growth of HepG2 cells were observed.Results ①The linear recombinant vector was introduced into HIS-/GS115 cells by LiAC and confirmed by PCR.②The expression of DCN protein was induced in 1% methanol BMMY medium.③While HepG2 liver cancer cell line were treated with the expression of DCN,HepG2 liver cancer cell line was inhibited by the expression of DCN.Conclusion To express DCN core protein in Pichia Pastoris is possible.We have observed that HepG2 liver cancer cell line was inhibited by the expression of DCN.

关 键 词：蛋白聚糖类 毕赤酵母 醋酸锂 

分 类 号：Q78[生物学—分子生物学]