摘要
以PCR法从携带有鹌鹑血管内皮生长因子受体quekl（qVEGFR）的质粒中扩增获得qVEFR胞外段第2～4区cDNA片段，并将其定向克隆到毕赤酵母表达载体pPiCZα中，获得重组表达质粒pPICZaA-qVEGFR2。然后将用SacⅠ酶线性化的pPICZαA-qVEGFR2质粒电转化到毕赤酵母菌GS115中，经Zeocin抗性和PCR筛选得到阳性重组菌株。重组菌株用甲醇进行诱导表达后，通过SDS-PAGE和Western blot分析鉴定蛋白表达产物，结果证实qVEGFR2胞外段第2～4区cDNA在毕赤酵母中获得了分泌性表达，为进一步研究和制备肿瘤蛋白疫苗打下了基础。
The extraceUular domain 2-4 loop cDNA of quail vascular endothelial growth factor receptor quekl （qVEGFR2）was obtained from plasmid carrying qVEGFR2 by PCR. Then it was cloned into expression vector pPICZαA of Pichia pastoris. To oonstruct recombinant expression plasmid pPICZaA-qVEGFR2, linearized pPICZaA-qVEGFR2 with SacI was transformed to electropomted Pichia pastoris GS115, Subsequently, positive clone was selected by PCR and its phenotype was determined. SDS-PAGE and Western blot assays of culture medium from a methanol-induced expression strain demonstrated that recombinant qVEGFR2 proteins were expressed and secreted into the culture medium. These resuits could provide a basis for further researches on tumor protein vaccine as well as for the preparation of tumor protein vaccine with Pichia pastoris.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2008年第1期157-160,167,共5页
Journal of Biomedical Engineering
