摘要
目的 构建及表达具有hGM CSF和hIL 6双重生物学活性的hGM CSF IL 6融合蛋白分子。方法 应用PCR技术对hGM CSF和hIL 6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 ,并构建成克隆有 4种融合蛋白基因的pBV2 2 0表达质粒 ,4种融合蛋白分别是GM CSF(9～ 12 7) IL 6 (2 9～ 184 ) (简称GI1)、GM CSF(9～ 12 7) IL 6 (1～ 184 ) (简称GI2 )、GM CSF(1～ 12 7) IL 6 (1～ 184 ) (简称GI3)、GM CSF(9～ 12 7) IL 6 (2 4～ 184 ) (简称GI4 ) ,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以获取目的蛋白。使用hGM CSF依赖细胞株TF1和hIL 6依赖细胞株B9通过MTT法测量融合蛋白的生物学活性。结果 对 4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到总蛋白含量的 30 %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 4种目的蛋白 ,其纯度均达到 95 %以上。活性测定结果表明 4种融合蛋白均具有较高的hGM CSF和hIL 6的双重生物学活性。结论 获得了具有较高纯度和?
Objective To construct and express hGM-CSF/IL-6 fusion protein with high purity and both hGM-CSF and hIL-6 biologic activities. Methods The novel gene coding for the fusion protein of four hGM-CSF/IL-6 was constructed by step cloning in pBV220 expression vector. The mutant hGM-CSF and hIL-6 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 BL-21. To obtain the fusion protein, Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration were performed. The biological activities were examined by MTT method. Results Four fusion proteins were all expressed in E.coli host strain BL-21 as inclusion body. The expression levels were more than 30% of the total cell lysate. Through Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration, four kinds of hGM-CSF/IL-6 fusion protein with high purity were obtained. These proteins all showed both hGM-CSF and hIL-6 biologic activities. Conclusion hGM-CSF/IL-6 fusion proteins with high purity and both hGM-CSF and hIL-6 biological activities were demonstrated.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第2期128-133,共6页
Chinese Journal of Microbiology and Immunology
基金
云南省自然科学基金 (青年基金 )资助项目( 2 0 0 1C0 0 3 1Q)
