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目的:构建含有myc-RBPJ(R218H)(recombination binding protein-J)、myc-KyoT2融合基因片段的质粒,并在真核细胞中表达、鉴定。方法:由本科室保存质粒酶切分别得到RBPJ(R218H)和myc-KyoT2基因片段,将RBPJ(R218H)基因插入载体PCMV-myc中,获得融合基因myc-RBP-J(R218H),将myc-RBPJ(R218H)和myc-KyoT2插入真核表达载体pIRES2-EGFP,构建真核表达载体myc-RBPJ(R218H)-IRES2-EGFP、myc-KyoT2-IRES2-EGFP。以其瞬时转染HEK293细胞,应用免疫印迹与流式细胞术检测融合蛋白与绿色荧光蛋白的表达。结果:正确获得myc-RBPJ(R218H)融合基因,DNA序列分析表明所构建的含myc-RBPJ(R218H)、myc-KyoT2融合基因的质粒与设计相同,myc-RBPJ(R218H)、myc-KyoT2融合蛋白与绿色荧光蛋白均正确表达。结论:成功构建了含有myc-RBPJ(R218H)、myc-KyoT2融合基因的真核表达载体,并在真核细胞中正确表达,为进一步研究慢性粒细胞白血病与Notch信号转导通路之间的关系奠定了基础。
OBJECTIVE: To construct a plasmid containing myc-RBPJ (R218H) (recombination binding protein-J) and myc-KyoT2 fusion gene and express it in eukaryotic cells. Methods: The RBPJ (R218H) and myc-KyoT2 gene fragments were obtained by the undergraduate department’s conserved plasmids. The RBPJ (R218H) gene was inserted into the vector PCMV-myc to obtain the fusion gene myc-RBP- RBPJ (R218H) and myc-KyoT2 were inserted into eukaryotic expression vector pIRES2-EGFP to construct eukaryotic expression vector myc-RBPJ (R218H) -IRES2-EGFP and myc-KyoT2-IRES2-EGFP. HEK293 cells were transiently transfected into the HEK293 cells. The expression of fusion protein and green fluorescent protein (GFP) were detected by Western blotting and flow cytometry. Results: The myc-RBPJ (R218H) fusion gene was correctly obtained. The DNA sequence analysis showed that the constructed myc-RBPJ (R218H) and myc-KyoT2 fusion gene plasmids had the same design as the designed myc-RBPJ Protein and green fluorescent protein are correctly expressed. CONCLUSION: The eukaryotic expression vector containing myc-RBPJ (R218H) and myc-KyoT2 fusion gene was successfully constructed and correctly expressed in eukaryotic cells. To further study the relationship between chronic myeloid leukemia and Notch signal transduction pathway Foundation.