Construction of intrakine mutant SDF-1alpha/54/KDEL and its inhibiting effects upon CXCR4 expression on cell membrane.
- Author:
Hongyuan CHEN
1
;
Yi TAN
;
Zhigang GUO
;
Weifeng MA
;
Shaoxi CAI
;
Jun DU
;
Jun HUANG
;
Houwen HU
;
Shaohui CAI
Author Information
1. Department of Microbiology and Immunology, College of Guandong Pharmacy, Guang zhong 510006, China.
- Publication Type:Journal Article
- MeSH:
Animals;
COS Cells;
Cell Membrane;
metabolism;
Cercopithecus aethiops;
Chemokine CXCL12;
genetics;
Cloning, Molecular;
Electroporation;
Gene Knockout Techniques;
Genetic Vectors;
genetics;
Humans;
Mutation;
Receptors, CXCR4;
genetics;
metabolism;
Recombinant Proteins;
genetics;
Stromal Cells;
metabolism;
Transfection
- From:
Journal of Biomedical Engineering
2008;25(3):647-677
- CountryChina
- Language:Chinese
-
Abstract:
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.
