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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。
Adherent mononuclear cells were isolated from normal human peripheral blood and cells were extracted by reverse transcription of LPS for 4h and reverse transcribed to obtain the first strand cDNA. Two pairs of specific primers were used for nested PCR to amplify BamHI CDNA encoding human 1L-15 mature protein, approximately 360 bp in size. The fragment was recovered by PEG method and ligated into the SmaI site of pBSSK vector for sequence analysis. The results showed that the sequence was consistent with that reported in the literature. The fragment was then subcloned into the BamHI site of pGEX-2T. The clones inserted in the correct orientation were screened and transformed into E. coli JM109. After the cells were induced by 1mmol / L IPTG for 5h, SDS-PAGE was carried out to collect the cells. Compared with the negative control, MW44KD showed a more band and UV- 8.8% of the amount. WesternBlot confirmed that the band was able to specifically react with anti-human IL-15 antibody. It is proved that the human IL-15 cDNA obtained by RT-PCR is expressed in Escherichia coli, which lays the foundation for further exploring the biological effect of human IL-15.