两种hIL-6/GM-CSF融合蛋白基因的构建、表达及活性比较

目的 :构建及表达具有hIL 6和hGM CSF双重生物学活性的hIL 6 /GM CSF融合蛋白分子。方法 :应用PCR技术对hIL 6和hGM CSF的基因分别加以改造 ,同时在两者之间加上连接肽序列 (G G S G S) 3 ,克隆PCR产物 ,并构建成克隆有两种融合蛋白基因的pBV2 2 0表达质粒 ,两种融合蛋白分别是 :IL 6 (1～ 184 ) GM CSF(9～ 12 7) (简称IG1)和IL 6 (2 4～ 184 ) GM CSF(9～ 12 7) (简称IG2 )。将表达质粒分别导入E .coliBL 2 1中诱导表达。通过QSepharoseHP离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化目的蛋白。使用hIL 6依赖细胞株B9和hGM CSF依赖细胞株TF1,通过MTT比色法测定融合蛋白的生物学活性。结果 :对两种融合蛋白基因的测序结果表明 ,其序列与理论设计完全一致。表达质粒在E .coliBL 2 1中均得到高效表达 ,表达的融合蛋白均占总蛋白含量的 2 5 %以上 ,表达产物以包涵体的形式存在 ,通过QSepharoseHP离子交换柱和SephacrylS 2 0 0分子筛柱二步柱纯化及复性后 ,获得两种目的蛋白 ,其纯度均达到 95 %以上。活性测定结果表明 ,两种融合蛋白均具有较高的hIL 6和hGM CSF的双重生物学活性。结论 :获得了具有较高纯度和双重生物学活性的hIL 6 /GM CSF融合蛋白

Construction,expression and biologic activities of two rhIL-6/GM-CSF fusion proteins

AIM: To construct and express the gene encoding hIL-6/GM-CSF fusion protein. METHODS: The genes encoding the two hIL-6/GM-CSF fusion proteins were constructed in pBV220 expression vector. Fusion proteins were expressed in E.coli BL-21 and purified through Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration columns. The biologic activities of the fusion proteins were detected by proliferation of hIL-6 dependent cell line B9 and hGM-CSF dependent cell line TF1 with MTT assay. RESULTS: Both fusion proteins were expressed in E.coli BL-21in the form of inclusion body. The expression levels were more than 25% of the total cell lysates. Both fusion proteins were obtained with high purity which had both hIL-6 and hGM-CSF biologic activities. CONCLUSION: Two hIL-6/GM-CSF fusion protein with high purity and bilolgic activities have been acquired successfully.