人白细胞介素15 cDNA克隆及其在大肠杆菌中表达

MOLECULAR COLONING OF HUMAN IL15 cDNA AND ITS EXPRESSION IN E. coli

从ＬＰＳ活化的人外周血单核细胞总ＲＮＡ中，用ＲＴＰＣＲ方法扩增出编码成熟人白细胞介素１５（ｈＩＬ１５）的ｃＤＮＡ，并将其克隆于ｐＢｌｕｅＳｋｍ质粒中，序列测定结果与预期一致。再将ｈＩＬ１５ｃＤＮＡ克隆于表达载体ｐＢＶ２２０，构建了高效表达克隆ｐＢＶＩＬ１５。热诱导４ｈ，ｈＩＬ１５蛋白表达即达高峰。表达的重组蛋白经ＳＤＳＰＡＧＥ及凝胶密度扫描分析，分子量约１３ｋＤ，占菌体总蛋白的３３％，经包涵体提取及ＳｅｐｈａｃｒｙｌＳ１００凝胶过滤纯化，重组蛋白纯度达９５％以上。

おuman IL15 cDNA fragment was cloned from LPSstimulated human monocytes by RTPCR and inserted into pBlueSkm plasmid, identified by endonuclease and DNA sequencing. The recombinant plasmid referred to as pBVIL15 was constructed by inserting the cloned hIL15 cDNA into prokaryotic expression vector pBV220 and transformed into E. coli HB101. The highest level expression of rhIL15 was achieved at 4 h after heat induction. The expressed rhIL15 protein, with molecular weight of about 13 kD, is about 33% of the total bacterial protein as detected by SDSPAGE and densitometry analysis and was purified to over 95% purity by extracting the inclusion bodies and gel filtration chromatography on Sephacryl S100. The purified rhIL15 protein had marked effect on proliferation of ConAactivated murine T lymphoblasts.