【摘要】 目的：探讨体外CD10~+干细胞与VSV病毒共培养分化后，能否从分化后细胞的基因组中扩增出成熟抗体IgG重链（VH）、轻链（VL）可变区基因片段。方法：37℃培养CD10~+干细胞至单层细胞，B细胞扩增培养基及添加的白介素因子、VSV病毒与CD10~+干细胞共培养15 d，留存每天细胞上清测定IgM含量，以15 d细胞为样本抽提总RNA，反转录为cDNA，以其为模板，用单链抗体（ScFv）噬菌体抗体库构建引物扩增VH、VL。结果：利用含VSV病毒、IL-2、IL-4、IL-7、IL-10、IL-21、CD40L的B细胞扩增培养基成功将CD10~+干细胞分化为CD38、CD138阳性浆细胞，IgM含量随时间递减，诱导15 d后成功扩增出成熟抗体IgG的370 bp VH、320 bp VL可变区基因片段。结论：成功完成了基于CD10~+干细胞的人源抗体基因获取，可用于各类人畜共患病病毒噬菌体抗体库构建。更多还原

【Abstract】 Objective:To investigate whether mature IgG heavy chain(VH) and light chain(VL) variable region gene fragments can be amplified from genome of CD10~+ stem cells co-cultured and differentiated with VSV virus in vitro. Methods:After CD10~+ stem cells were cultured to monolayer cells at 37 ℃, B cell amplification medium, interleukin factors, VSV and CD10~+ stem cells were co-cultured for 15 d. IgM content of cell supernatant was measured every day. Total RNA was extracted from 15 d cells and reversed into cDNA, which was used as template. Single chain antibody(ScFv) phage antibody library was used to construct primers to amplify VH and VL. Results:CD10~+ stem cells were successfully differentiated into CD38 and CD138-positive plasma cells by B cell amplification medium containing VSV, IL-2, IL-4, IL-7, IL-10, IL-21 and CD40L, and detected with decreasing IgM content over time. After 15 d of induction, 370 bp VH and 320 bp VL variable region gene fragments of mature IgG were successfully amplified.Conclusion:Human antibody genes based on CD10~+ stem cells have been successfully obtained, which can be used for construction of phage antibody libraries of various zoonotic viruses.更多还原