EGF在毕赤酵母中的表达及活性测定

Expression of EGF in Pichia Pastois and activity determination

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。

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.