Stathmin基因毕赤酵母表达体系的构建及鉴定  

Construction and identification of Stathmin gene Pichia pastoris expression system

作　　者：杨铭[1] 林芳[1] 和婷[1] 董轲[1] 张惠中[1] 

机构地区：[1]第四军医大学唐都医院中心实验室,陕西西安710038

出　　处：《国际检验医学杂志》2016年第9期1161-1163,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81372836)

摘　　要：目的构建Stathmin基因毕赤酵母表达体系,并对表达产物进行纯化和鉴定,为Stathmin相互作用蛋白的进一步研究提供实验基础。方法通过PCR方法从SKBR3乳腺癌细胞中扩增出人Stathmin基因片段,克隆入毕赤酵母表达载体pPIC3.5K,构建重组载体pPIC3.5K-Stathmin,转化GS115工程菌,经含基因素(G418)的酵母膏胨葡萄糖琼脂(YPD)培养基筛选出阳性克隆。用0.5%甲醇诱导表达,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western Blotting鉴定表达产物。结果 DNA测序结果表明,所获基因片段与Stathmin基因序列一致。在含有G418 600μg/mL的YPD培养基上筛选出的pPIC3.5K-Stathmin转化子,经PCR鉴定为阳性克隆。SDS-PAGE可见约37×103处有目的条带表达,经Western Blotting鉴定,此条带为Stathmin蛋白。结论成功构建了Stathmin酵母表达载体,并在毕赤酵母中表达成功,为Stathmin相互作用蛋白研究以及生物治疗药物的制备奠定了基础。Objective To provide the experimental basis for the further research of the interacting proteins with Stathmin,the Stathmin gene Pichia pastoris expression system was constructed,the expressed Stathmin product was purified and identified.Methods Stathmin gene was amplified from tumor cell line of SKBR3 by PCR method and cloned into the yeast expression vector pPIC3.5K.The recombinant vector pPIC3.5K-Stathmin was constructed and transformed into Pichia pastoris GS115.The positive clones were screened by YPD medium containing Geneticin 600μg/mL.Expression was induced with 0.5% methanol and expression products were identified by SDS-PAGE and Western Blotting.Results DNA sequencing result showed that the gene fragment was consistent with Stathmin gene sequence.pPIC3.5K-Stathmin was selected from YPD culture medium containing Geneticin,and the positive clones were identified by PCR.SDS-PAGE showed that a 37×10^3 protein band could be seen on the PAGE gel after Coomassie Blue staining,which was further confirmed and identified as Stathmin protein by Western Blotting.Conclusion Stathmin yeast expression vector is successfully constructed and expressed in Pichia pastoris,which laid the foundation for the study of interacting proteins with Stathmin,and for the preparation of the biological treatment drugs of Stahtmin target.

关 键 词：基因 毕赤酵母表达体系 鉴定 

分 类 号：R730.5[医药卫生—肿瘤]