Preparation and characterization of monoclonal antibodies against nucleostemin, a protein that controls cell proliferation in stem cells and cancer cells.
Nucleostemin (NS) is a novel protein that promotes the proliferation of stem cells and cancer cells. The aim of this study is to prepare specific monoclonal antibodies against nucleostemin as a tool in nucleostemin research. To generate the antibodies, the cDNA coding nucleostemin was cloned from human gastric cancer cell line NCI-N87 with reverse transcriptase-polymerase chain reaction (RT-PCR). The partial cDNA fragment was inserted into prokaryotic expression vector pGEX-4T1 to make glutathione S-transferase (GST) fusion protein in Escherichia coli, and the whole-length cDNA fragment was cloned into vector pcDNA3 for expression in mammalian cells. The spleen cells, from the BALB/c mouse immunized with the GST-nucleostemin fusion protein, were used to prepare the monoclonal antibody with hybridoma technique. After screening with enzyme-linked immunosorbent assay (ELISA) and subcloning, six specific monoclonal antibodies against nucleostemin were obtained. Moreover, they also worked well in Western blotting, immunoprecipitation, and immunohistochemistry. These antibodies will be helpful in elucidating biological functions and the molecular mechanism of nucleostemin.
Yang
Haixia
H
Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research, Peking University School of Oncology, Beijing, P.R. China.
Zhang
Jianzhi
J
Wu
Jian
J
Shou
Chengchao
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D019204:GTP-Binding Proteins; D005822:Genetic Vectors; D005982:Glutathione Transferase; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C469949:GNL3 protein, human; D009687:Nuclear Proteins; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins