title: [Construction of intrakine mutant SDF-1alpha/54/KDEL and its inhibiting effects upon CXCR4 expression on cell membrane].
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.
authors.affiliation: Department of Microbiology and Immunology, College of Guandong Pharmacy, Guang zhong 510006, China.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Tan
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Guo
authors.forename: Weifeng
authors.initials: W
authors.lastname: Ma
authors.forename: Shaoxi
authors.initials: S
authors.lastname: Cai
authors.forename: Jun
authors.initials: J
authors.lastname: Du
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Houwen
authors.initials: H
authors.lastname: Hu
authors.forename: Shaohui
authors.initials: S
authors.lastname: Cai
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000235:genetics*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018274:Electroporation; D055786:Gene Knockout Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009154:Mutation; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D017154:Stromal Cells / Q000378:metabolism; D014162:Transfection
chemical_list: D054377:Chemokine CXCL12; D019718:Receptors, CXCR4; D011994:Recombinant Proteins