基于CD10^(+)干细胞的诱导分化及人源抗体基因制备

Induction of differentiation based on CD10^(+) stem cells and preparation of human antibody genes

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。

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.