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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 highly expressing human vascular endothelial growth factor receptor 2 extracellular domain (HEVEGFR-2) with eukaryotic protein structure in Pichia PASTORIS. Methods The recombinant plasmid pORF-HEVEGFR-2 was used to amplify full-length HEVEGFR-2 DNA. The recombinant Pichia pastoris X-33 was constructed by electroporation. Transformants (X-33.HEVEGFR-2) positive for recombinant HEVEGFR-2 protein were screened with drug-resistant phenotype and methanol induction. Results SDS-PAGE showed. A recombinant HEVEGFR-2 protein with a molecular weight of about 108 KDA was obtained. About 45% of the total amount of X-33-HEVEGFR-2 secreted protein. The recombinant protein in the supernatant of the mass concentration of 80 MG / L. Its HEVEGFR-2 part has a molecular weight of about 106 KDA. WESTERNBLOT confirmed that the protein specifically binds to rat anti-mouse VEGFR-2 monoclonal antibody. Conclusion Pichia pastoris can efficiently express the full-length of human vascular endothelial growth factor receptor 2 extracellular domain with eukaryotic protein structure.