【摘要】 目的 :用噬菌体展示技术构建KGla免疫小鼠的脾细胞scFv表达文库。方法 :用人髓系白血病细胞系KGla细胞免疫小鼠 ,取其脾细胞 ,RT PCR扩增VH和Vκ基因并克隆入噬菌体展示表达载体 ,构建scFv文库 ,并测定文库的库容量和BstN1酶切单个克隆分析文库的多样性。用KGla作为固相抗原 ,进行四轮吸附 洗脱 富集的筛选 ,SDS PAGE检验筛选后文库scFv的呈示表达。结果 :文库的库容为 3× 10 6cfu ,单个克隆的BstN1Ⅰ酶切图谱显示多样 ,提示文库的容量和多样性均能满足进一步筛选分离目的基因的需要 ,筛选后的文库能很好地表达外源scFv。结论 :文库的构建为进一步地分离KGla细胞表面分子抗体基因提供了必要而可靠的基础更多还原

【Abstract】 Objective:To construct a scFv library by phage display technique from the spleen cells of mice immunized with KGla cells. Methods: Three mice were immunized with KGla cells, and their spleen cells were harvested. The genes of VH and Vκ were amplified by RTPCR and a scFvphage display antibody library was constructed with the amplified V gene. The library was selected by using the KGla cells as panning antigen by 4 rounds of bindingelutionenrichment procedure, and the expression of the library were examined by SDSPAGE. Results: We were able to produce a scFv library containing 3×106individual clones which showed different patterns after digested with restriction endonuclease BstN I. It indicated that the capacity and diversity of the library is sufficient for screening specific scFv.The surface display expression of the library was also verified. Conclusion: A scFv library of antiKGla cell surface molecules has been constructed. It will be useful for isolating specific scFv against the cell surface molecules of hematopoietic progenitors.更多还原