Prokaryotic Expression Of Activation Macrophage Membrance Protein ENPP-4 Gene And Preparation Of Polyclonal Antibodies

Preliminary studies in macrophage activation experiment, we found that macrophages fixed by polyphosphate formaldehyde still have strong tumor cell killing activity, and this is different from the known antitumor molecules in the last decade, indicating the surface of macrophages might have other effect elements. Cell surface molecules of activated macrophages through a variety stimulus have been compared, and up-regulated proteins of BCG-activated macrophages were identified. We selected one of the specific proteins, an unknown protein, named ENPP-4 to carry out in-depth understanding of the protein and thus indirectly related to mechanisms of anti-contact support.ENPP-4 protein is a member of ENPP protein family, which has a familial relationship. ENPP family has a variety of biological functions, including nucleotide recycling, modulation of purinergic receptor signalling, regulation of extracellular pyrophosphate levels, stimulating cell activity and the regulation of insulin receptors and so on. Abnormal ENPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type II diabetes. In this study we cloned the ectodomain gene of ENPP-4 for prokaryotic expression and generation of polyclonal antibodies against the recombinant protein, in order to specifically study the functions of ENPP-4 in the further research.In this experiment, The plasmid pET28a was used to construct the recombinant prokaryotic expression plasmid pET28a-ENPP-4. After DNA sequencing identification, the E.coil was induced under the conditions of bacteria initial concentration OD600=0.6,IPTG concentration was 0.6 mmol/L at 37℃, then bacteria were haversted 6h later. We obtained highly purity of the fusion protein through dilute renaturation and dialysis renaturation. After quantification, the prokaryotic expressed protein was injected into rabbits to generate anti-ENPP-4 antibodies.The control serum was collected from ear vein before rabbits immunized, after six boost immunization, valence of antibody were detected by indirect Elisa, the results showed that Polyclonal antibody titer reaches 1:6400. Western-blotting showed ENPP-4 polyclonal antibody has a high specificity against the total protein of transformed E. coli. Using polyclonal antibodies for the analysising of tissue distributions of ENPP-4 protein, the results showed ENPP-4 protein were highly expressed in the spleen, muscle, stomach and ovarian.In conclusion, we successfully obtained the ENPP-4 gene segments, expressed and purified its extra-membrane protein, prepared polyclonal antibodies against ENPP-4 and detected the tissue distribution of ENPP-4. Obtain relevant function message of protein ENPP-4, it laid a preliminary basis for further research.