Cloning And Expression Of BCG Activated Macrophage Membrane Protein NP-2 Gene And Preparation Of Its Polyclonal Antibody

Macrophages play a unique role in the human immune system. We always think that it's scavenger of the body. However, In recent years, it was found that macrophages is not only a cell group, but also is a complex group of different immune function. With the development of the molecular biology technology and computer technology, based on the level of DNA molecule speculation and identified, more and more attention is putted on macrophage cell structure and function, It gradually become the research focus about macrophages conjungation-dependent tumoricidal mechanism.Our lab found that macrophage has tumoricidal active after fixed by polyphosphate formaldehyde during activation macrophages experiment. It destructs tumor in different ways.It certify that there are still other effects molecules on the macrophages surface. On this basis, we clone activation macrophage membrane protein NP-2 gene, expression and preparation of polyclonal antibodies against recombinant NP-2 protein.First we query related proteins database, select Serial number IPI00221418 protein, and named it NP-2 (new protein-2).Obtain genetic sequences in the NCBI, and obtain related information of the structure in bioinformatics analysis about it, Determine protein is alkaline protein. It may be a signal transduction structure,and predict protein 34.8 % exist within the Cytoplasm.Then with BCG stimulate mice ,we obtain activation intra-abdominal macrophage , bases genetic fragments of NP-2 membrane protein in RT-PCR, insert it in cloning vector and prokaryotic expression vector. restructuring plasmid pGEX4T1-NP-2, Induce restructuring plasmid express in different time and IPTG concentrations in engineering bacterium Rosetta (DE3). Determining optimal expression conditions for 37 degrees, IPTG 1.0 mmol/L and 6 hours, the kind of recombinant fusion protein existence form are cytoryctes,.Induce recombinant plasmid pGEX4T1-NP-2, obtain purity of 0.50 ug/ml of fusion protein through dilute renaturation, dialysis, and GS4B purification column, preparation of polyclonal antibodies against recombinant NP-2 protein. Polyclonal antibody 'titer reaches 1:12800, it shows specificity in organize in immunohistochemical technology.To sum up, we first successfully obtain the NP-2 Gene segments, express and purify its extra-membrane protein, prepare protein NP-2 polyclonal antibody . Obtain relevant function message of protein NP-2, it laid a preliminary basis for further research .