huIL-6-GM-CSF(9-127)融合蛋白的表达及活性测定

Expression of huIL-6-GM-CSF(9-127) fusion protein and its biologic activities

目的 构建及表达具有huIL 6和huGM CSF双重生物学活性的huIL 6 GM CSF(9- 12 7)融合蛋白分子。方法 应用PCR技术对huIL 6和huGM CSF的基因分别加以改造 ,同时在二者之间加上连接肽序列 (G G S G S) 3 ,克隆PCR产物 ,并构建成pBV2 2 0 IL 6 GM CSF(9- 12 7)表达质粒 ,将表达质粒导入E .coliDH5α中 ,诱导表达融合蛋白。通过QSepharoseH .P .离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化以获取目的蛋白。使用MTT法测量其huIL 6和huGM CSF生物学活性。结果 pUC18 IL 6 GM CSF(9- 12 7)的序列与理论设计完全一致 ,表达质粒在E .coliDH5α中得到高效表达 ,表达的融合蛋白占总蛋白含量的 30 %以上 ,表达产物以包涵体的形式存在。通过QSepharoseH .P .离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化及复性后获得目的蛋白 ,其纯度达到 96 %以上。融合蛋白具有huIL 6和huGM CSF的双重生物学活性 ,其促进huIL 6依赖细胞株B9和huGM CSF依赖细胞株TF 1增殖的比活性分别为 2 .86× 10 7U/mg和 3.33× 10 8U/mg。结论 获得了具有较高纯度和双重生物学活性的huIL 6 GM CSF(9- 12 7)

Objective To construct and express hIL 6 GM CSF(9-127) fusion protein with high purity and dual biologicatl activities of huIL 6 and huGM CSF. Methods The novel gene coding for the fusion protein of hu IL 6 GM CSF(9-127) was constructed using strategy of step by step cloning in pBV220 expression vector. The amino acids 1～8 of huGM CSF were deleted by PCR technique. The huIL 6 and mutant huGM CSF(9-127) cDNAs were linked via a linker sequence coding 15 amino acid residues (G G S G S) 3. Fusion protein was expressed in E.coli host strain DH5α.To obtain the fusion protein, Q Sepharose H.P. ion exchange chromatography and Sephacryl S 200 gel filtration were performed. The biologic activities were detected by MTT method. Results Fusion protein was expressed in E.coli host strain DH5α in the form of inclusion body. The expression level was more than 30% of the total cell lysate. Through Q Sepharose H.P. ion exchange chromatography and Sephacryl S 200 gel filtration, hu IL 6 GM CSF(9-127) fusion protein with high purity was obtained. The protein showed dual biological activities of huIL 6 and huGM CSF. The specific activity of huIL 6 was 2.86×10 7 U/mg, and for huGM CSF, it reached 3.33× 10 8 U/mg .Conclusion huIL 6 GM CSF(9-127) fusion protein with high purity and dual biological activities of huIL 6 and huGM CSF was obtained.