EGF在毕赤酵母中的表达及活性测定

目的高效表达和纯化有活性的EGF蛋白。方法将含有表皮生长因子(EGF)基因的质粒pAO815通过LiAc法转入毕赤酵母细胞中,筛选在MM平板上生长缓慢的Muts型酵母工程菌,甲醇诱导基因表达产生EGF,柱纯化后利用SDS-PAGE检测到EGF的生成,MTT法及新生小鼠皮下法分别检测活性。结果 SDS-PAGE电泳检测EGF成功表达和纯化,MTT法测定EGF生物活性约为2.8×106IU/mL。结论本研究构建的工程菌株能高效表达EGF,且纯化的EGF具有与天然EGF同样的体外和体内活性。

Expression of EGF in Pichia Pastois and activity determination

Objective To efficiently express and purify the active EGF protein.Methods Transfer the plasmid pAO815 carrying epidermal growth factor(EGF) gene into Pichia pastoris cells by LiAc method;screen out the Muts yeast engineered bacteria,growing slowly on the MM plate;- methanol-induced gene expression produced EGF;purified using column EGF generation was detected by the SDS-PAGE analysis;MTT assay and the skin of newborn mice were used to detect activity.Results SDS-PAGE electrophoresis detected the successful expression and purification of EGF.The biological activity of EGF determined by MTT method was approximately 2.8×106 IU/mL.Conclusion The constructed engineered strain in this study can efficiently express EGF and the purified EGF has the same activity in vitro and in vivo as the natural EGF.