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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。
Objective: To construct a plasmid containing human Jagged1 extracellular IgF fusion gene fragment and express it in eukaryotic cells. Methods: The extracellular domain of hJagged1 gene was amplified by RT PCR from human bone marrow cells. After DNA sequencing, the extracellular domain of hJagged1 gene was inserted into the modified plasmid pBluescriptsk Ⅱ to obtain hJagged1ext Fc fusion gene. The fusion gene was inserted into eukaryotic expression vector pEF BOSneo to construct eukaryotic expression vector pEF BOSneo hJagged1ext Fc. Transiently transfected COS7 cells, RT-PCR, cell-mediated immunofluorescence and sandwich ELISA were used to detect the expression of fusion protein. Results: The extracellular domain of hJagged1 gene was efficiently amplified. DNA sequence analysis showed that the constructed plasmid containing the hJagged1ext Fc fusion gene was identical to the designed one and the hJagged1ext Fc fusion protein was correctly expressed. CONCLUSION: The eukaryotic expression vector of hJagged1ext Fc fusion gene was successfully constructed and correctly expressed in eukaryotic cells, which laid the foundation for further study on the in vitro expansion of Jagged1 in hematopoietic stem / progenitor cells.