Anti-kg1a Cell Surface Molecules Genetically Engineered Antibodies And Antigen Molecules

Hematopoiesis is a highly regulated process during which the interactions of cell-cell and cell-matrix regulate the future fate of stem cells and committed progenitors along specific lineages. Stem/progenitor cellular surface molecules play essential roles in this process, such as stem cell proliferation and differentiation and directing migration and homing of hematopoietic progenitor cells to distinct sites of the organism. To identify and characterize novel hematopoietic stem/progenitor cell-surface molecules and their antibodies is an important and exciting issue in fundamental and clinical research. Our goal of this research is to identify novel and functional engineering antibodies of hematopoitic progenitor cell surface molecules, and search for novel hematopoitic progenitor cell surface molecules and their genes with the identified antibodies.Firstly, three female BALB/C mice were given three times of i.p. injections of 1c10~7KGla cells, an undifferentiated subline of human acute myelogenous leukemia cell line KG1. Spleens of donor mice were removed and the cells were harvested. Total splenocyte RNA was extracted with the guanidinium isothiocyanate single-step procedure. The primers used for amplification of mouse heavy chain variable region(VH) and K light chain variable region(V K ) were designed and synthesized. The amplificated gene repertoires of VH and V K by RT-PCR were subsequently ligated into phagmid display vector pSEX81, which expresses scFv-pIII fusion proteins, using HindIII/pvuII and BamHI/MluI restriction sites and electroporated into E. Coli. XL1-Blue cells to construct the phage display library. The size and the diversity of the library were evaluated by titer and BstN I restriction enzyme digestion pattern analysis. Then the library was selected by using the KG1a cells as panning antigen by 4 rounds of binding-elution-enrichment procedure, and the expression of the library were examined by SDS-PAGE. The results showed that a scFv library of 3X10~6 individual...