hJagged1^(ext)-Fc融合蛋白真核表达载体的构建和表达  

Construction and expression of a fusion protein containing extracellular domain of human Jagged1 and Fc fragment of human IgG1

作　　者：贾洪霞[1] 张萍[2] 周鹏[2] 李军锋[2] 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

出　　处：《细胞与分子免疫学杂志》2004年第3期278-281,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划 (973)资助项目 (No .0 0 1 CB50 990 6);国家自然科学基金资助项目(No.30 2 0 0 1 4 8)

摘　　要：目的 :构建含人Jagged1胞外段 IgFc融合基因片段的质粒 ,并在真核细胞中表达。方法 :从人骨髓细胞中用RT PCR扩增hJagged1基因的胞外段。经DNA测序后 ,将hJagged1基因的胞外段插入改造过的质粒pBluescriptskⅡ中 ,获得hJagged1ext Fc融合基因。将融合基因插入真核表达载体pEF BOSneo ,构建真核表达载体pEF BOSneo hJagged1ext Fc。以其瞬时转染COS7细胞 ,应用RT PCR、细胞免疫荧光技术 ,及夹心ELISA检测融合蛋白的表达。结果 :hJagged1基因的胞外段被有效地扩增。DNA序列分析表明 ,所构建的含hJagged1ext Fc融合基因的质粒与设计相同 ,hJagged1ext Fc融合蛋白得到正确表达。结论 :成功地构建了hJagged1ext Fc融合基因的真核表达载体 ,并在真核细胞中正确表达 ,为进一步研究Jagged1在造血干 /祖细胞的体外扩增奠定了基础。AIM: To construct a eukaryotic expression vector containing the extracellular domain of human Jagged1 and Fc fragment of human IgG1 fusion gene and to express the hJagged1 ext Fc fusion protein in mammalian cells. METHODS: The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. In order to obtain hJagged1 ext Fc fusion gene, the extracellular domain of Jagged1 gene was inserted into pBluescript skⅡ hcγ1, and then the fusion gene was inserted into pEF BOSneo to get the eukaryotic expression vector pEF BOSneo hJagged1 ext Fc. The recombination plasmid was transiently transfected into COS7 cells and the expression of the fusion protein was identified by RT PCR, immunofluorescent assay and sandwich ELISA. RESULTS: The extracellular domain of human Jagged1 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 mammalian cells. CONCLUSION: The hJagged1 ext gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, for example, the Jagged1 ext Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro .

关 键 词：造血干/祖细胞 NOTCH JAGGED1 融合蛋白 真核表达 

分 类 号：R331.2[医药卫生—人体生理学] Q78[医药卫生—基础医学]