从体外培养的外周血单个核细胞克隆人白细胞介素-10 cDNA
Chinese journal of experimental surgery(2005)
Abstract
目的从体外培养的外周血单个核细胞克隆人白细胞介素(hIL)-10 cDNA.方法淋巴细胞分离液自静脉血中分离出外周血单个核细胞(PBMCs),含ConA的RPMI 1640培养24 h,提取总RNA,逆转录-聚合酶链反应(RT-PCR)扩增hIL-10cDNA,克隆入pUCm-T载体,转化感受态细胞DH5α,蓝白筛选,PCR及Pvu I、Not I双酶切鉴定,重组DNA测序.结果 RT-PCR扩增产物560 bp,与预期片段大小一致,随机挑取白色菌落,小量提取的质粒经PCR、限制性内切酶酶切鉴定,目的条带与预期片段的大小均一致,测序结果与Genebank公布的hIL-10cDNA序列完全一致.结论从PBMC中扩增hIL-10cDNA,克隆至pUCm-T载体,方法较为简便,为进一步进行亚克隆及表达研究提供了可靠的前提条件.
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Key words
peripheral blood mononuclear cells,cdna,vitro-cultured
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