论文部分内容阅读
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
OBJECTIVE: To construct a scFv expression library of splenocytes from KGla immunized mice using phage display technology. Methods: The mouse myeloid leukemia cell line KGla was used to immunize mice. The spleen cells were obtained and the VH and Vκ genes were amplified by RT PCR and cloned into the phage display expression vector to construct the scFv library. The library volume and BstN1 digestion Clone analysis of library diversity. KGla as solid phase antigen, four rounds of adsorption and enrichment screening, screened by SDS PAGE screening scFv presentation. Results: The library had a capacity of 3 × 10 6 cfu, and the BstN1 Ⅰ digestion map of multiple clones showed diversity, suggesting that the capacity and diversity of the libraries could meet the needs of further screening and isolation of the target genes. The screened library could express exogenous scFv. CONCLUSION: The library construction provides the necessary and reliable basis for further isolation of molecular antibody genes on the surface of KGla cells