人白细胞介素-15 cDNA的克隆及其在大肠杆菌中的表达

Cloning of Human Interleukin 15 cDNA and Its Expression in E.coli

从正常人外周血中分离粘附的单核细胞，加入ＬＰＳ刺激４ｈ后提取细胞ｍＲＮＡ反转录获得ｃＤＮＡ第一链，用两对特异引物进行巢式ＰＣＲ扩增出含ＢａｍＨＩ酶切位点的编码人１Ｌ－１５成熟蛋白的ｃＤＮＡ，其大小约３６０ｂｐ。用ＰＥＧ法回收其片段并将其连接到ｐＢＳＳＫ载体的ＳｍａⅠ位点上进行序列分析，结果表明其序列与文献报道一致。然后再将其片段亚克隆插入到ｐＧＥＸ－２Ｔ的ＢａｍＨＩ位点。筛选插入方向正确的克隆，转化大肠杆菌ＪＭ１０９，以１ｍｍｏｌ／ＬＩＰＴＧ诱导５ｈ收集菌体直接进行ＳＤＳ－ＰＡＧＥ，与阴性对照相比，ＭＷ４４ＫＤ处多出一条带，紫外扫描显示此带占菌体蛋白总量的８．８％。ＷｅｓｔｅｒｎＢｌｏｔ证实此带能够特异地与兔抗人ＩＬ－１５的抗体发生反应。证明ＲＴ－ＰＣＲ所得的人ＩＬ－１５ｃＤＮＡ在大肠杆菌中获得了表达，为进一步探讨人ＩＬ－１５的生物学作用打下基础。

mRNA was extracted from adherent PBMCs which were incubated with LPS(5μg/ml) in RPMI 1640 medium for 4h.The first strand cDNA Was synthesized by AMV reverse transcriptase.Human IL 15 cDNAwas amplified with nested PCR,its sequence is identical with the report.The PCR product was subcloned into pGEX 2T vector in BamH Ⅰ site ,then transformed into E.coli JM 109.Its expression was induced by 1mmol/L IPTG.The result of SDS PAGE analysis showed that there is a new protein band which is of 8.8% in total bacterial protein.Western Blot analysis proved that the induciable band could be specifically recognized by anti IL 15 antibody.These results show that human IL 15 cDNA amplified by RT PCR was expressed in E.coli.It is the basis for the study on biological roles of human IL 15 and for possible application in clinic in the future.