人GCSF-白蛋白融合蛋白的构建及在毕赤酵母中的表达  

Construction and Expression of A Fusion Protein of Human Serum Albumin-Granulocyte Colony-Stimulating Factor in P.Pastoris

作　　者：徐钰[1] 孙钧铭 张莲芬[1] 窦文芳[1] 李洁[1] 朱威[1] 朱书峰[1] 金坚[1] 

机构地区：[1]江南大学生物工程学院生物制药系工业生物技术教育部重点实验室,江苏无锡214122 [2]无锡第三人民医院中心实验室,江苏无锡214041

出　　处：《药物生物技术》2008年第2期109-114,共6页Pharmaceutical Biotechnology

基　　金：国家"863"计划项目(2006AA02Z153);上海市科委生物医药重大科技攻关项目(06DZ19020);2006江苏省卫生厅科研项目(Z200611)

摘　　要：制备人粒细胞集落刺激因子(G-CSF)与人血清白蛋白(HSA)的融合蛋白。从新鲜人外周血中分离单核细胞,给予大肠杆菌脂多糖刺激后,RT-PCR制备G-CSF cDNA,以其为模板扩增得到人G-CSF的编码区序列(525 bp)。重叠PCR拼接HSA-GCSF融合基因(2277 bp),序列测定正确,中间未加入任何连接肽。插入表达载体pPIC9K中α交配因子的开放阅读框内,构建分泌型表达重组质粒pPIC9K-HSA-GCSF。电击转化毕赤酵母菌KM71,G418筛选得到高拷贝转化子。甲醇诱导表达融合蛋白,SDS-PAGE鉴定融合蛋白的相对分子质量约为84 k,Western blot分析融合蛋白为G-CSF与HSA的杂合分子,NFS-60细胞/MTT比色法测定融合蛋白具有G-CSF的生物学活性。结果:构建了可分泌表达HSA-GCSF融合蛋白的毕赤酵母工程菌,为进一步对其进行药学研究奠定了基础。To construct the fusion protein that is composed of granulocyte colony-stimulating factor（G- CSF） and human serum albumin（HSA）. Fresh human peripheral blood was separated to obtain mononuclear cells. Mononuclear cells were cultured with lipopolysaccharide, and used to prepare RNA. The coding region sequence of G-CSF（525 bp） was obtained by RT-PCR. Then the fusion gene of HSAGCSF（2 277 bp） was constructed by overlapping PCR without any connecting peptide. And the recombinant plasmid of pPIC9K- HSA-GCSF was constructed and transformed into P. pastoris KM71 by electroporation. The recombinant with high fusion gene copies was selected at the Geneticin culture dish, and was overexpressed with methanol in Pichia pastoris. SDS-PAGE and Western blot analysis indicated that the relative molecular mass of the fusion protein was about 84k, and the fusion protein was a heterozygote made of HSA and G-CSF. The specific activity of culture supernatant was about 1× 10^4 IU/mg assayed by the NFS-60/MTT colorimetric method, which provides a sound basis the for further research into its pharmaceutical field.

关 键 词：人粒细胞集落刺激因子 人血清白蛋白 融合蛋白 毕赤酵母 

分 类 号：S642.5[农业科学—蔬菜学] Q81[农业科学—园艺学]