异种同源表皮生长因子受体EGFR胞外段在毕赤巴斯德酵母中的表达

目的　实现异种同源表皮生长因子受体 EGFR胞外段在毕赤巴斯德酵母中的分泌性表达。方法　将鼠和人的 EGFR胞外段目的基因 (mer,her)与分泌型毕赤酵母表达载体 p PICZa A重组 ,构建了相应的重组表达质粒 p PICZa A- mer和 p PICZa A- her,用 Sac 将重组质粒线性化后 ,电穿孔转化酵母菌株 GS115 ,利用 Zeocin筛选阳性克隆 ,挑出阳性克隆用甲醇小规模诱导表达后 ,进行 SDS- PAGE分析及 Western- blot检测 ,筛选高表达量的克隆作大规模诱导表达。结果　获得了鼠和人的 EGFR胞外段目的蛋白在毕赤巴斯德酵母中的分泌性表达 ,SDS-PAGE分析及 Western- blot检测得到预期条带 ,相对分子质量约为 95× 10 3。免疫印迹分析表明 ,表达蛋白具有良好的抗原性和特异性。结论　 p PICZa A- mer和 p PICZa A- her重组质粒在甲醇营养型酵母中可经甲醇诱导表达鼠和人的 EGFR胞外段目的蛋白。

Expression of Xenogeneic Homologous Epidermal Growth Factor Receptor Ectodomain in Pichia Pastoris

OBJECTIVE: To develop a secreted expression system of xenogeneic homologous epidermal growth factor receptor (EGFR) ectodomain using Pichia pastoris (P. pastoris), thus providing a basis for subsequent antitumor immunological researches on EGFR subunit protein vaccine. METHODS: In order to construct the secreted expression vector pPICZaA-mer and pPICZaA-her, we recombined the cDNA of xenogeneic (human) EGFR ectodomain (her) and the corresponding control mouse EGFR ectodomain (mer) with the empty plasmid pPICZaA, respectively. Then the recombinant plasmids were linearized by Sac I and transformed into P. pastoris strain GS115 by electroporation. The positive transformants were selected on YPDS plate including Zeocin and were induced to express by methanol. Consequently, the proteins in the culture supernatant were assayed with SDS-PAGE and Western-blot. So we could choose the high-level secreted expression recombinant strain to produce protein vaccine vastly. RESULTS: We got the secreted expression of mouse and human EGFR ectodomain in P. pastoris. The relative molecular mass of the two aimed proteins is about 95 x 10(3). The Western-blot analysis demonstrated that the expression proteins have much good antigenicity and specificity. CONCLUSION: The recombinant plasmids pPICZaA-mer and pPICZaA-her could be induced to express the EGFR aimed proteins by methanol in methanol-trophic yeast expression system.