Cloning Expression Of Selenoprotein W From Chicken And Characterization Of Monoclonal Antibodies Against Their Recombinant Proteins

Selenium is an important trace element which is essential for human and animals'life activities. Se covalent bonding in protein in the form of selenocysteine, we call it selenoprotein. Selenoprotein W (SelW) is a low molecular weight selenium protein in cells. In different organisms, SelW amino acid sequences are highly homologous. Previous studies showed that SelW were expressed in various tissues and organs, higher expression of skeletal muscle and brain as well. Rencently studies of mammals and rodents SelW's revealed that SelW regulated the metabolism of striated muscle, calcium metabolism and antioxidant and so on. However, the function of chicken's SelW has not been reported. In order to study the function of chicken's Sel W, we have designed primers according to the cDNA sequence of chicken Sel W, amplified cDNA sequences which containing complete ORF, using overlap extension PCR technique to the Sec codon of cDNA mutation, by sequence analysis, the results with the reference sequence homology 99%, and mutant products were cloned into pGEX-6P-1 expression vector, the recombinant expression plasmid pGEX-SelW, transformed into the Rosetta host bacteria by IPTG induction, the recombinant fusion protein were highly expressed, mainly in the form of inclusion bodies, purified by cut gel of recombinant fusion protein, and then cut off the GST tag using thrombin, the second's cut gel purified protein. Polyclonal antibody by Western blotting showed that the corresponding fusion protein GST-SelW reactions to produce specific bands, but not with empty vector proteins after induction of the GST tag.Purified protein immuned BABL / c mice, using cell fusion techniques, screening by indirect ELISA and four vices cell subclone, obtained two hybridoma, named 1B8 and 4H5. Identified by the monoclonal antibody subclass identification kit, both IgM class of antibody titers of the supernatant were 1:6400. Preparation of the monoclonal antibody 1B8 and 4H5 ascites, ascites titer are 1:12800 and 1:6400. Two hybridoma chromosome number were 103±5, significantly more than SP2 / 0 myeloma cells from 65 to 75. Two hybridoma continuous passage in vitro and cryopreservation of three months after the recovery did not affect the stability of antibody secretion. no cross reaction with chicken GST tag and selenoprotein N. Affinity analysis showed that the two monoclonal antibody on the affinity of the target protein higher than chicken SelW recombinant protein and synthetic peptide, and high affinity for the antibody-sensitive, stable and efficient inspection applications provide necessary guarantees. In short, successful preparation of the two monoclonal antibodies for the chicken SelW rapid, specific detection provid a detection reagent, and provide the material basis for further research for chicken SelW's functions.