Cloning And Expression Of Activation Macrophage Membrane Protein AMMP-2 Gene And Preparation Of Its Polyclonal Antibody

Macrophages play a unique role in the human immune system. We always think that it'sscavenger of the body. However, In recent years, it was found that macrophages is not only a cellgroup, but also is a complex group of different immune function. With the development of themolecular biology technology and computer technology, based on the level of DNA moleculespeculation and identified, more and more attention is putted on macrophage cell structure andfunction, It gradually become the research focus about macrophages conjungation-dependenttumoricidal mechanism.Our lab found that macrophage has tumoricidal active after fixed by polyphosphateformaldehyde during activation macrophages experiment. It destructs tumor in different ways. Itcertify that there are still other effects molecules on the macrophages surface.On this basis, we clone activation macrophage membrane protein AMMP-2 gene, expressionand preparation of polyclonal antibodies against recombinant AMMP-2 protein.First we query related proteins database, select protein Serial number is 00226142, and namedit AMMP-2 (membrane macrophage active protein-2).Obtain genetic sequences in the NCBI, andobtain related information of the structure in bioinformatics analysis about it, Determine protein isalkaline protein, it has a zone of 150bp bases spiral in membrane outer. It may be a signaltransduction structure, and predict protein 65 % exist within the nucleus. Then with BCG stimulatemice ,we obtain activation intra-abdominal macrophage , clone 902bp bases genetic fragments ofAMMP-2 membrane protein in RT-PCR, insert it in cloning vector and prokaryotic expression vector.two restructuring plasmid are pET28a-AMMP-2 and pGEX4T1-AMMP-2, Induce tworestructuring plasmid express in different temperature, time and IPTG concentrations inengineering bacterium Rosetta (DE3). Determining optimal expression conditions for 37 degrees,IPTG 0.8 mmol/L and 6 hours, two kinds of recombinant fusion protein existence form arecytoryctes, and pET28a-AMMP-2 restructuring plasmid expressing quantity is higher in two fusionprotein. Induce recombinant plasmid pET28a-AMMP-2, obtain purity of 0.52 ug/ml of fusionprotein through dilute renaturation, dialysis, and CM cellulose cation exchange purification column,preparation of polyclonal antibodies against recombinant AMMP-2 protein. Polyclonal antibody'titer reaches 1:12800, it shows specificity in organize in immunohistochemical technology. Protein AMMP-2 is in the liver, muscles and in the brain.To sum up, we first successfully obtain the AMMP-2 Gene segments, express and purify itsextra-membrane protein, prepare protein AMMP-2 polyclonal antibody and organization positioning.Obtain relevant function message of protein AMMP-2, it laid a preliminary basis for furtherresearch..