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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
OBJECTIVE: To construct and express hIL 6 / GM CSF fusion protein molecule with dual biological activity of hIL 6 and hGM CSF. Methods: The genes of hIL 6 and hGM CSF were modified by PCR, and the sequence of GGSGS 3 was added between the two. The PCR products were cloned and cloned into pBV22 0 expression plasmid. The two fusion proteins are IL6 (1-184) GM CSF (IG12) and IL6 (2-4-4) GM CSF (9-127) ). The expression plasmids were introduced into E. Coli BL21 to induce expression respectively. The target protein was purified by Q Sepharose HP column and Sephacryls 2000 column. The biological activity of the fusion protein was determined by MTT colorimetry using hIL 6 dependent cell line B9 and hGM CSF dependent cell line TF1. Results: The sequencing results of the two fusion protein genes showed that their sequences were completely consistent with the theoretical design. The expression plasmids were all highly expressed in E.coli BL21. The expressed fusion proteins accounted for more than 25% of the total protein content. The expressed products existed as inclusion bodies and were separated by QSepharoseHP column and Sephacryls 2000 molecular sieve column After purification and renaturation of two-step column, two kinds of target proteins were obtained, their purity reached more than 95%. The results of the activity assay showed that both of the fusion proteins have high biological activity of hIL 6 and hGM CSF. Conclusion: The hIL 6 / GM CSF fusion protein with higher purity and dual biological activity was obtained