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构建人基质细胞衍生因子(SDF-1)突变体SDF-1α/54/KDEL重组真核表达质粒,并考察其对T细胞性白血病细胞株Molt-4细胞表面受体CXCR4表达的影响,为尝试表型敲除肿瘤细胞CXCR4以抑制肿瘤的转移提供理论和实验依据。以SDF-WT-Gly×4-Dec/PET30a(+)质粒为模板,用PCR法扩增出SDF-1α/54并将其亚克隆至真核表达质粒pEGFP-C3构建成真核表达载体pEGFP-C3/SDF-1α/54/KDEL。经酶切及测序验证后,脂质体介导转染入COS-7细胞,通过Western blot检测SDF-1α/54/KDEL的表达。电穿孔法将重组载体瞬时转染CXCR4高表达的Molt-4,并用流式细胞仪检测CXCR4含量的变化。DNA测序证明:读码框完全正确,重组真核表达载体含有SDF-1α/54基因和编码4肽KDEL的基因,Western blot证明融合蛋白能在COS-7细胞表达。电穿孔转染Molt-4细胞后发现,SDF-1α/54/KDEL可以显著降低胞膜表面CXCR4表达量。由此提示:KDEL介导的SDF-1α/54对Molt-4细胞CXCR4具有表型敲除作用,且此效应不受SDF-1αC端α螺旋缺失的影响。
To construct SDF-1α / 54 / KDEL recombinant eukaryotic expression plasmid of human stromal cell-derived factor (SDF-1) and investigate its effect on the expression of CXCR4 on T cell leukemia cell line Molt-4, Phenotypic knockout of tumor cells CXCR4 to provide a theoretical and experimental basis for inhibiting tumor metastasis. SDF-1α / 54 was amplified by PCR from SDF-WT-Gly × 4-Dec / PET30a (+) plasmid and subcloned into eukaryotic expression plasmid pEGFP-C3 to construct eukaryotic expression vector pEGFP -C3 / SDF-1α / 54 / KDEL. After digestion and sequencing, the liposomes were transfected into COS-7 cells and the expression of SDF-1α / 54 / KDEL was detected by Western blot. The recombinant vector was transiently transfected into Molt-4 with high expression of CXCR4 by electroporation, and the change of CXCR4 content was detected by flow cytometry. DNA sequencing proved that the reading frame was completely correct. The recombinant eukaryotic expression vector contained the gene SDF-1α / 54 and the gene encoding 4-peptide KDEL. Western blot showed that the fusion protein was expressed in COS-7 cells. Transfection of Molt-4 cells by electroporation showed that SDF-1α / 54 / KDEL could significantly reduce the expression of CXCR4 on the membrane surface. Thus, KDEL-mediated SDF-1α / 54 phenotype knockdown effect on CXCR4 Molt-4 cells, and this effect is not affected by the C-terminal α helix deletion of SDF-1α.