【摘要】 [目的]将抗乙酰胆碱受体单链抗体(ScFv)A 7基因转化至毕赤酵母GS 115,筛选阳性转化子并诱导蛋白表达,制备单链抗体A 7蛋白.[方法]将构建的含有抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的重组真核表达载体pPIC 9 K-ScFv A 7经SalⅠ酶切线性化后,利用电转化方法转化受体毕赤酵母菌GS 115,以甲醇诱导蛋白表达,应用鼠抗c-myc单克隆抗体进行斑点杂交试验,检查所表达的单链抗体A 7蛋白.[结果]在酵母菌的培养上清液中发现单链抗体A 7蛋白的表达,而阴性对照菌中未见表达产物.[结论]抗乙酰胆碱受体主要免疫原区单链抗体A 7蛋白已成功地在真核表达系统中表达.更多还原

【Abstract】 OBJECTIVE To transform single chain variable fragment(ScFv) A7 against acetylcholine receptor in myasthenia gravis into Pichia yeast and to express the protein in the eukaryotic expression system.METHODSThe recombinant vector,which was isolated and purified from E coli DH5α was linerized by endonuclease Sal Ⅰ and was transformed into Pichia pastoris GS115 by electroporation.The genome of GS115 was isolated to conduct the insertion condition of the target gene.The target gene was induced to express by methanol.The expressed products were detected by dot blotting assay using a mouse anti-c-myc monoclonal antibody.RESULTSScFvA7 gene was inserted into the chromosome of GS115 successfully and ScFv A7 proteins were found in the supernatant after 120 hours methanol induction.CONCLUSIONThe ScFvA7 protein has been expressed successfully in the eukaryotic expression system.These lay a foundation for the activity and specificity identification of this protein.更多还原