【摘要】 目的确认并鉴定一个新发现的小鼠FR4剪切变异体,并在mRNA水平检测其在小鼠脾淋巴细胞亚群中的表达。方法PCR克隆FR4CDS区时发现存在一个新的剪切变异体,酶切和测序予以鉴定,Western blot检测脾细胞中变异体的表达,流式分选脾细胞中CD8+、CD4+CD25-、CD4+CD25+T淋巴细胞,RT-PCR检测新的变异体在各细胞亚群中的表达。结果凝胶电泳显示存在一个CDS区碱基数大于野生型FR4的条带,测序表明此条带在野生型外显子基础上包含一个内含子成分,Western blotting证实新发现的剪切体在蛋白水平也存在,脾细胞中不同T细胞亚群均表达此新型变异体。结论mRNA和蛋白水平均证实存在一种新型FR4剪切变异体,不同T细胞亚群均表达此变异体暗示其在淋巴细胞摄取叶酸功能上不可或缺。更多还原

【Abstract】 Objective To identify and characterize a newly found splice variant of murine FR4,and analyze its mRNA expression in different splenic T lymphocyte subpopulations. Methods A novel splice variant was found through murine FR4 CDS cloning,and then confirmed by digestion and sequencing. Furthermore,the monoclonal antibody 12A5 and TH6 of wild-type FR4 were used to detect the novel variant in splenocytes at protein level by Western blot. Based on these findings,flow cytometric sorting was used to isolate splenic CD8+,CD4+CD25-,CD4+CD25+ T lymphocyte subtypes and the mRNA expression of the T lymphocyte subtypes was analyzed. Results Gel electrophoresis revealed the existence of a new band whose basepair is larger than wild-type FR4; sequencing results suggested this new band containing an additional intron compared with wild-type FR4; western blotting confirmed the existence of the novel splice variant at protein level,and moreover,it was expressed in different splenic T cell subtypes equally. Conclusion A novel FR4 splice variant is confirmed at both mRNA and protein levels,and the expression in all T cell subtypes reveals the variant is indispensable for folate uptake of lymphocytes.更多还原