细胞内趋化因子重组突变体SDF-1α/54/KDEL的构建及其对CXCR4受体在细胞膜表面表达的抑制作用

Construction of Intrakine Mutant SDF-1α/54/KDEL and Its Inhibiting Effects upon CXCR4 Expression on Cell Membrane

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 investigate the impact of phenotypic knockout of CXCR4 on Molt-4 cells via intrakine technology,the C-terminal α-helix gene SDF-1α/54/ KDEL of human stromal cell-derived Faceor-1 deletion is fused to a retention signal 4-peptide -KDEL that retains the newly synthesized receptor within the Molt-4 cells endoplasimc reticulum. Subsequently, PCR is used to amplify the target gene SDF-1α/54/ KDEL from the constructed plasmid SDF-WT-Gly×4-Dec/PET-30a(+) at its C-terminal and subclone it into eukaryotic express...