Preparation And Identification Of Monoclonal Antibodies Against Bovine NRAMP1 N-Terminal Fragment

Natural resistance-associated macrophage protein 1(Nramp1),is an iron transporter expressed exclusively within phagocytosis cells (such as macrophages and neutrophils). Nramp1 was found to localize to late endosomes, where it mediates the export of iron and manganese into the cytosol. It has been proposed that Nramp1 promotes host resistance by depleting the phagolysosome of divalent metals(such as Fe²⁺and Mn²⁺), which are essential for pathogen growth. It enhances the organism resists ability of intracellular pathogenic microorganisms. In order to detect transgenic cells, embryos and transgenic cloned cattle of carrying Nramp1 and breed transgenic cattle that resist intracellular pathogens (such as Mycobacterium tuberculosis, Brucellosis, etc). We prepared bovine Nramp1-N monoclonal antibody (McAb).The experimental results are as follows:1 We have sieved the best way of expressing Nramp1-N Fusion Protein by induction of IPTG, Shaked cultivation at 35℃, Final concentration of IPTG: 0.1 mmol/L, the time: 10 h. We obtain Nramp1-N Fusion Protein that was higher purity by AKTA protein purified systerm.2 Using indirect ELISA to detect the titer of antibody in serum. The titer of antibody in mouse and rabbit immunized by Nramp1-N Fusion Protein reach up to 1:1×10⁵ and 1:1×10⁶.3 After fusion myeloma cells and immune spleen cells, using indirect ELISA to detect masc-clone, Cloning efficiency: 29.2 %, Masc-Cloning efficiency: 46.4 %. Subclone the positive cells by limiting dilution for several times to get identical cells, we got two positive hybridoma cells named D3 and F6. Using ELISA and Western Blot to detect specificity of two McAbs. The results showed two McAbs is aim at Nramp1-N. Using Colchcine to karyotype analysis of the two hybridoma cells, The average number of chromosome of the hybridoma cell lines was 53±2.4 The ELISA titer for bovine Nramp1-N antibodies in culture supernant was 1:500 and 1:200; The ELISA titer in ascites was 1:1×10⁵ and 1:1×10⁴. The antibody-serecting ability of the two hybridoma cells keep stable after serial subcultivation and resuscitation several times. The antibody-serecting ability of them keep stable after frozening and subcloning several times. Affinity constant of monoclonal Antibodys from D3 and F6 are 2×10⁸ and 2×10⁷. The Ig subtypes of D3 and F6 antibodies were IgG1/κand IgM/κ.