融合蛋白hJagged1^(ext)-Fc毕赤酵母表达载体的构建及表达  

Construction and Expression of A Fusion Protein Containing Extracellular Domain of Human Jagged1 and Fc Fragment of Human IgG1 in Pichia Pastoris

作　　者：李国辉[1] 康志杰[1] 黄斯勇[1] 何飞[2] 徐恒[1] 张丽[1] 伍艳兰[1] 牛晓丽[1] 马长升[3] 韩骅[2] 梁英民[1] 

机构地区：[1]第四军医大学唐都医院血液科,陕西西安710038 [2]第四军医大学基础部医学遗传学与发育生物学教研室,陕西西安710032 [3]第四军医大学生物医学工程系,陕西西安710032

出　　处：《中国实验血液学杂志》2008年第4期910-914,共5页Journal of Experimental Hematology

基　　金："十一五"国家"863"重大项目;"十一五"国家"863"重点项目基金资助;编号2006AA02A111

摘　　要：本研究旨在构建人IgG1 Fc蛋白及融合蛋白hJagged1ext-Fc,并在毕赤酵母GS115中表达。用RT-PCR法从人骨髓细胞中扩增hJagged1基因的胞外段。在测序正确后,将hJagged1基因的胞外段插入构建好的PIC-Fc载体,得到PIC-hJagged1ext-Fc。用MD平板筛选重组子,G418法筛选高拷贝转化子,经甲醇诱导表达后,采用SDS-PAGE分析表达蛋白。结果表明:hJagged1基因的胞外段被有效地扩增。序列分析显示所构建的含phJagged1ext-Fc融合基因的质粒与设计相同,人IgG1 Fc蛋白及融合蛋白hJagged1ext-Fc得到正确表达。结论:成功地构建了hJagged1ext-Fc融合基因的毕赤酵母表达载体,并在毕赤酵母中正确表达,为进一步研究Jagged1在造血干/祖细胞的体外扩增奠定了基础。In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jaggedl and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jaggedl gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jaggedl gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jaggedl gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1^ext-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1^ext gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1^ext-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.

关 键 词：JAGGED1 hJagged1^ext-Fc 造血干/祖细胞 NOTCH 融合蛋白 毕赤酵母 

分 类 号：Q789[生物学—分子生物学] Q782