从体外培养的外周血单个核细胞克隆人白细胞介素-10cDNA

Cloning of human interleukin-10 cDNA from in vitro-cultured peripheral blood mononuclear cells

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)10cDNA。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RPMI1640培养24h,提取总RNA,逆转录聚合酶链反应(RTPCR)扩增hIL-10cDNA,克隆入pUCmT载体,转化感受态细胞DH5α,蓝白筛选,PCR及PvuI、NotI双酶切鉴定,重组DNA测序。结果RTPCR扩增产物560bp,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hIL10cDNA序列完全一致。结论从PBMC中扩增hIL10-cDNA,克隆至pUCmT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。

Objective To clone human interleukin-10 cDNA from in vitro cultured peripheral blood mononuclear cells.Methods Peripheral blood mononuclear cells were separated from human peripheral venous blood with lymphocyte separation fluid and in vitro cultured for 24 h with RPMI 1640 containing concanavalin A.RNA was extracted from PBMCs,human interleukin-10 cDNA was amplified by RT-PCR and cloned into pUCm-T vector,and recombinant was transformed into competent cells DH5α.Miniprep DNA was prepared after blue-white color screening.PCR amplifying,restriction mapping and recombinant sequencing were performed.Results The 560 bp fragment amplified by RT-PCR was the same as the anticipated one in size.Miniprep DNA was prepared from randomly isolated white colony,the fragment obtained from PCR amplification,restriction mapping was the same as the anticipated one in size and sequencing showed its complete homology to hIL-10cDNA published in Genebank.Conclusion The cloning of human interleukin-10 cDNA from PBMCs and recombination with pUCm-T vector is feasible and relatively simple,which provides dependable prerequisite for further subcloning and gene expression study.