The Preparation Of Monoclonal Antibody Against Wild And Mutated Nucleophosmin

Leukaemia is a hematopoietic stem cell clonal disease with critical heterology.Medical expert suggested that AML(Acute myeloid leukemia) should be discover, diagnose and treatment early. Up to now,WHO(world health organization) classified AML by MIC classification according to cell morphology, immunology and cytogenetic,but about 50% de novo AML divided into classified acataleptic group because normal karyotpe, this influenced the diagnose and prognosis directly. It is very serious to find more detection target.NPM(nucleophosmin) is a ubiquitously expressed multifunctional protein which main located in nucleoli granular zone, act as a chaperone for many protein such as histone, p53, ARF(ADP ribosylation factor), Bax, effect the cell's proliferation and Apoptosis. NPM is over-expression in solid tumor usually, but mutation in hematologic malignancies. The location of mutated NPM protein is changed from nucleus to kytoplasm, and it's biological function changed too. Many research revealed mutated NPM is directly implicated in the pathogenesis of Leukaemia.AML have many type of NPM mutation,NPM-mA is the most common one.Recently many scholar suggested mutated NPM should be include into the detection index to leukemia of WHO. Up to now the detection method of mutated NPM included immunohistochemistry, PCR, DHPLC. Immunohistochemistry was used in our country usually, but this method was limited because the monoclonal antibody was being import.1975 K?hler and Milstein invented the hybridoma technique, it brought the preparation of monoclonal antibody into reality. They fused mouse myeloma cell and mouse spleen cell which sensitized by sheep erythrocyte together, and get a group of cells, which could be cultured in vitro and secreted monoclonal antibody against sheep erythrocyte, this hybrid cell line named hybridoma, using this method people can get antibody which just discriminate single antigenic determinat.Our topic use molecular cloning technique and hybridoma cell technique, construction fusion expression vector of NPM or NPM-mA with Trx (thioredoxin) respectively, induced expression and purified fusion protein, use the fusion protein immunizated mouse and preparation monoclonal antibody.Main result and conclusion:1. Epitope are Confirmed to be exist in the same and different amino acid sequence between NPM and NPM-mA by software, high-fidelity enzyme amplification the ORF(open reading frame) sequence of NPM and NPM-mA, clone them into prokaryotic expression vector pET-32a(+), IPTG induce expression bacteria with recombination vector, secretory expression two fusion protein, purify the fusion protein by His Binding Collumn.2. Use the two fusion protein as antigen to immunizated mouse, preparation monoclonal antibody according to the classic hybridoma cell technique, get a NPM monoclonal antibody by indirect ELISA screening, this antibody can cross-reaction with NPM-mA; specific NPM-mA monoclonal antibody wasn't obtained yet.