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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 (hIL) 10 cDNA from peripheral blood mononuclear cells cultured in vitro. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by lymphocyte separation. After cultured with RPMI1640 containing ConA for 24 h, hIL-10 cDNA was amplified by reverse transcription polymerase chain reaction (RTPCR) and cloned into pUCmT vector , Transformed into competent cells DH5α, blue-white screening, PCR and PvuI, NotI double digestion, recombinant DNA sequencing. Results The amplified product of RTPCR was 560bp, which was consistent with the expected fragment size. The white colonies were picked randomly. The plasmid extracted by small amount was identified by PCR and restriction endonuclease digestion. The size of the target band was consistent with that of the expected fragment. Genebank published hIL10 cDNA sequence exactly the same. Conclusion The hIL10-cDNA was amplified from PBMC and cloned into pUCmT vector. The method was simple and convenient, which provided a reliable precondition for further subcloning and expression studies.