ABSTRACT
Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional cytokine implicated in normal hematopoiesis. We previously reported that SDF-1alpha enhanced the survival of hematopoietic stem and progenitor cells in synergy with other cytokine such as GM-CSF, steel factor, or thrombopoietin. As adult stem cells are very rare, many investigators are trying to expand hematopoietic stem/progenitor cells in vitro. In this study, we constructed an adenoviral vector and produced high titer of recombinant adenoviruses directing robust expression of SDF-1alpha determined by ELISA. We also produced control empty adenoviruses and recombinant LacZ adenoviruses. In order to check the feasibility of SDF-1alpha in ex vivo expansion system, we compared HUVEC cells tranduced by a SDF-1alpha recombinant virus with HUVEC cells transduced by a LacZ recombinant virus in supporting activity of hematopoietic cells, and found that expression of SDF-1alpha in HUVEC cells increased viable blood cell population obtained from the same number of CD34+ cells. The SDF-1alpha recombinant adenovirus seems to be useful for future application in hematopoiesis studies.
Subject(s)
Humans , Adenoviridae , Adult Stem Cells , Blood Cells , Chemokine CXCL12 , Enzyme-Linked Immunosorbent Assay , Granulocyte-Macrophage Colony-Stimulating Factor , Hematopoiesis , Hematopoietic Stem Cells , Human Umbilical Vein Endothelial Cells , Research Personnel , Stem Cell Factor , Stem Cells , ThrombopoietinABSTRACT
BACKGROUND: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah- 1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating beta-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. METHODS: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. RESULTS: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. CONCLUSION: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , beta Catenin , Carcinogenesis , Cell Proliferation , Clinical Coding , Clone Cells , Colon , Colorectal Neoplasms , Diagnosis , DNA, Complementary , Ecthyma, Contagious , Enzyme-Linked Immunosorbent Assay , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.
Subject(s)
Animals , Humans , Amino Acid Sequence , Antibodies, Monoclonal , Cell Division , Cell Line , Cell Line, Tumor , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Melanoma , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.