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目的将ｈＩＬ－７基因与ｈＧＭ－ＣＳＦ（７－１２７）基因通过一中间接头（Ｇｌｙ４Ｓｅｒ）３连接到一起，并在大肠杆菌中表达具有ｈＩＬ－７活性和极高ｈＧＭ－ＣＳＦ活性的ＩＬ－７－ＧＭ－ＣＳＦ（７－１２７）融合分子。方法通过引物设计和采用分级克隆的策略构建原核表达载体ｐＢＶ２２０－ＩＬ－７－ＧＭ－ＣＳＦ（７－１２７），并在大肠杆菌中表达ｈＩＬ－７－ＧＭ－ＣＳＦ（７－１２７）融合蛋白。连接ＩＬ－７与ＧＭ－ＣＳＦ（７－１２７）的中间接头为（Ｇｌｙ４Ｓｅｒ）３。通过选择密码子，在（Ｇｌｙ４Ｓｅｒ）３ｃＤＮＡ的中后部产生了一个ＢａｍＨⅠ位点，ＧＧＡ编码Ｇｌｙ，ＴＣＣ编码Ｓｅｒ。ＩＬ－７下游引物和ＧＭ－ＣＳＦ（７－１２７）上游引物的合成均起始于该位点。结果ｐＵＣ１８－ＩＬ－７－ＧＭ－ＣＳＦ（７－１２７）序列测定表明，ＩＬ－７、ＧＭ－ＣＳＦ（７－１２７）以及中间接头序列均与设计一致。活性测定结果表明，ｒｈＩＬ－７－ＧＭ－ＣＳＦ（７－１２７）的ｒｈＩＬ－７比活性约为１．６×１０６Ｕ／ｍｇ，ｈＧＭ－ＣＳＦ（７－１２７）比活性约为５×１０９～１×１０１０Ｕ／ｍｇ，比ｒｈＧＭ－ＣＳＦ标准品高５００～１０００倍。结论在ＩＬ－７－ＧＭ－Ｃ?
Objective To express the hIL-7 gene together with hGM-CSF (7-127) gene via an intermediate (Gly4Ser) 3 and express IL-7 with hIL-7 activity and high hGM-CSF activity in E. coli GM-CSF (7-127) fusion molecule. Methods The prokaryotic expression vector pBV220-IL-7-GM-CSF (7-127) was constructed by primer design and hierarchical cloning strategy. The fusion protein of hIL-7-GM-CSF (7-127) was expressed in E. coli. The middle linker that connects IL-7 to GM-CSF (7-127) is (Gly4Ser) 3. By selecting codons, a BamHI site was created in the middle and rear of the (Gly4Ser) 3 cDNA, with GGA encoding Gly and TCC encoding Ser. The synthesis of IL-7 downstream primer and GM-CSF (7-127) upstream primer all started at this site. Results Sequence analysis of pUC18-IL-7-GM-CSF (7-127) showed that IL-7, GM-CSF (7-127) and the intermediate linker sequence were consistent with the design. The results of bioassay showed that the specific activity of rhIL-7-GM-CSF (7-127) was about 1.6 × 106U / mg and the specific activity of hGM-CSF (7-127) was about 5 × 109-1 × 1010U / mg, 500 ~ 1000 times higher than rhGM-CSF standard. Conclusion In IL-7-GM-C?