Chen Hongyuan, Tan Yi, Guo Zhigang, Ma Weifeng, Cai Shaoxi, Du Jun, Huang Jun, Hu Houwen, Cai Shaohui
Department of Microbiology and Immunology, College of Guandong Pharmacy, Guang zhong 510006, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2008 Jun;25(3):647-51, 677.
To investigate the impact of phenotypic knockout of CXCR4 on Molt-4 cells via intrakine technology,the C-terminal alpha-helix gene SDF-1alpha/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-1alpha/54/ KDEL from the constructed plasmid SDF-WT-Gly x 4-Dec/PET-30a(+) at its C-terminal and subclone it into eukaryotic expression vectors pEGFP-C3 for generating recombinant vector cells by lipEGFP-C3/SDF-1alpha/54/KDEL, and then have it sequenced. After the transfection of recombinant plasmids into COS-7 posome, SDF-1alpha/54/KDEL protein is confirmed with Western blot. The recombinant plasmids pEGFP-C3/SDF-1alpha/54/KDEL are isolated and transiently transfected in Molt-4 cells by electroporation. Flow cytometric analysis shows a dramatic reduction of CXCR4 expression on Molt-4 cells. The conclusion is that SDF-1alpha/54/KDEL could assume a role in the phenotypic knockout of CXCR4, and the findings suggest that the inhibiting effect of SDF-1alpha/54 against CXCR4 is not influenced by the deletion of SDF-1alpha helix at the C terminal.
为了通过细胞内细胞因子技术研究CXCR4基因敲除对Molt-4细胞表型的影响，将人基质细胞衍生因子-1（SDF-1α）缺失的C末端α-螺旋基因SDF-1α/54/KDEL与保留信号四肽-KDEL融合，该信号可将新合成的受体保留在Molt-4细胞内质网中。随后，通过PCR从构建的质粒SDF-WT-Gly x 4-Dec/PET-30a(+)的C末端扩增靶基因SDF-1α/54/KDEL，并将其亚克隆到真核表达载体pEGFP-C3中，通过lipEGFP-C3/SDF-1α/54/KDEL生成重组载体细胞，然后进行测序。将重组质粒转染到COS-7细胞中后，通过蛋白质免疫印迹法确认SDF-1α/54/KDEL蛋白的表达。分离重组质粒pEGFP-C3/SDF-1α/54/KDEL，并通过电穿孔法将其瞬时转染到Molt-4细胞中。流式细胞术分析显示Molt-4细胞上CXCR4的表达显著降低。结论是SDF-1α/54/KDEL可能在CXCR4的表型敲除中发挥作用，研究结果表明SDF-1α/54对CXCR4的抑制作用不受C末端SDF-1α螺旋缺失的影响。
