摘要
目的克隆人IL-10cDNA的全长序列,构建原核表达载体并诱导其表达。方法无菌条件下静脉采取正常人外周血10ml。加入等体积的生理盐水稀释后,加入淋巴细胞分离液,离心收集细胞,提取细胞总RNA。以细胞总RNA为模板,进行RT-PCR,克隆出IL-10cDNA,经双酶切后插入表达载体pMon中,用pMon-IL10重组体转染大肠杆菌DH5α。用萘啶酮酸诱导使重组人IL-10在大肠杆菌中表达,对表达产物进行SDS-PAGE电泳及Western-blot分析。结果人外周血单核细胞经ConA刺激后,IL-10转录水平增加,有利于IL-10cDNA的克隆;克隆的IL-10cDNA经测序证实与基因库报告的序列完全一致,插入pMon载体后经萘啶酮酸诱导能够在大肠杆菌DH5α表达;表达的蛋白质能与兔抗人IL-10特异性结合。结论人IL-10cDNA被成功克隆并能够在大肠杆菌中表达。
Objective To clone the full length of human interleukin- 10 cDNA,construct prime expression vector pMon - IL10 and induce its expression in E, coll. Methods Total RNA was extracted from ConA - stimulated human peripheral blood mononuclear cells （PBMC）. The cDNA of human IL- 10 was cloned from RNA by RT- PCR and subeloned into pMon vectors. The E. coli DH5α cells were transfected with the constructed pMon - IL10 recombinant, and the conbinant human IL- 10 expression in E. coli was induced by nalidixic acid. Finally, the expressed IL- 10 protein was assayed by SDS- PAGE and Westem blot. Results The transcription of Ⅱ - 10 in PBMC was stimulated by ConA, making the cloning of IL- 10 cDNA easier. Gene sequencing confirmed that the sequence of human IL- 10 cDNA cloned was identical to that reported in Genbank. Under nalidixic acid induction, the recombinant human IL- 10 was effectively expressed in E. coli, and the expressed protein could specifically combine with the rabbit anti - human IL- 10 antibody, Conclusion The full - length of IL- 10 cDNA has been successfully cloned and expressed in E. coli DH5α.
出处
《徐州医学院学报》
CAS
2005年第4期305-308,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省教育厅资助课题(00KJD320001)
