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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。
AIM To identify and identify a newly discovered mouse FR4 cleavage variant and examine its expression in mouse splenic lymphocyte subsets at the mRNA level. Methods A new cut variant was found in the FR4CDS region by PCR. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The expression of variants in spleen cells was detected by Western blot. The CD8 +, CD4 + CD25-, CD4 + CD25 + T lymphocytes. RT-PCR was used to detect the expression of new variants in various cell subsets. Results The gel electrophoresis showed that there was a band with more bases in the CDS region than that of the wild type FR4. Sequencing showed that this band contained an intron based on the wild type exon. Western blotting confirmed that the newly discovered Protein levels are also present in spleen cells in different T cell subsets express this new variant. Conclusions Both mRNA and protein levels confirm the existence of a novel FR4 cleavage variant. Expression of this variant in different T cell subsets suggests that it is indispensable for uptake of folic acid by lymphocytes.