摘要
目的探讨在毕赤氏巴斯德酵母(PICHIA PASTORIS)中高效表达有真核蛋白结构的人血管内皮生长因子受体2胞外段(HEVEGFR-2)的可行性。方法从重组质粒PORF-HEVEGFR-2经PCR获全长HEVEGFR-2 DNA,构建重组毕赤氏巴斯德酵母分泌性表达载体,电转化PICHIA PASTORIS X-33。用抗药性表型和甲醇诱导筛选出重组HEVEGFR-2蛋白表达阳性的转化子(X-33.HEVEGFR-2)。结果SDS-PAGE显示。获分子量约108 KDA的重组HEVEGFR-2蛋白。约占X-33-HEVEGFR-2分泌性表达蛋白总量的45%。该重组蛋白在表达上清中的质量浓度达80 MG/L。其HEVEGFR-2部分分子量约106 KDA。WESTERNBLOT证实,该蛋白能特异地与大鼠抗小鼠VEGFR-2单克隆抗体结合。结论毕赤氏巴斯德酵母能高效表达有真核蛋白结构的人血管内皮生长因子受体2胞外段蛋白全段。
Objective To investigate the feasibility of high expressing extracellular domain of human vascular endothelial growth factor receptor-2 （heVEGFR-2） with eukaryotic protein structure in Pichia pastoris. Methods We used PCR to amplify the DNA fragment encoding heVEGFR-2 from pORF-heVEGFR-2. The recombinant Pichia pastoris secretory expression vector（pPICZαA-heVEGFR-2）was constructed and transferred into Pichia pastoris X-33 by electroporation. The high expression transformnants were identified through its drug-resistant phenotype and methanol induction. Results As indicated by SDS-PAGE, the recombinant heVEGFR-2 protein with a molecular weight （MW） approximately 108 kDa, which reached 80 mg/L in the mass concentration, comprised 45 % of the total expressed secreted proteins from Pichia pastoris X-33, The section of heVEGFR-2 had a MW ap- proximately 106 kDa. The results of western blot analysis demonstrated that this protein could be specifically recognized by the rat monoclonal antibody against mouse VEGFR-2 （rat McAb against mVEGFR-2）. Conclusion The heVEGFR-2 with eukaryotic protein structure can get a high expression in Pichia pastoris.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第1期1-4,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家973计划(NO.2001CB510001)资助~~
