The Preliminary Study On The Expression, Purification And Function Of Prdx6-11R Fusion Protein

Objective To construct a prokaryotic expression vector of Prdx6-11R fusion protein,induce its expression and purify the expression products and study the Prdx6-11R fusionprotein in human corneal epithelial cells in vitro and the role of the transmembrane functionand the protein through the liver and kidney organization by entering into the cell membraneto inhibit the role of H2O2. This makes the foundation for the function of Prdx6protein andthe carrying biologically active substance of large molecules reach the tissue site.Methods The epididymis cDNA library was used to amplify a part of the Prdx6gene (Asequence) for the connection with the B sequence which was made by us. The amplified geneswere connected to pET32b vector to construct a recombinant pET32b/Prdx6-11R expressionvector, and then inducted the expression of Prdx6-11R fusion protein by IPTG. The inducedcells was broken by super sound way to release the fuion protein．The collected supernatantliquid was purified by Ni affinity chromatography Purify System and was removed theendotoxin．Add the fusion protein Prdx6-11R into the culture medium，and incubate togetherwith the human corneal epithelial cells in vitro and was injected into the mice intraperitoneally,then observe the protein with a fluorescence microscopy to detect the fluorescence of cornealepithelial cells, liver and kidney. Deal with the human corneal epithelial cells with HydrogenPeroxide (H2O2) and Prdx6-11R fusion protein，and observe cell viability conditions toexplore if the Prdx6-11R fusion protein is helpful for inhibiting the cytotoxicity of H2O2forcorneal epithelial cells.Results The equencing results showed that the sequence of amplified fragment wasconsistent with the purpose of sequence，and the pET32b/Prdx6-11R expression plasmids were sucBsfully constructed. The purity of the Prdx6-11R proteins is above95%with theconcentration above2mg/mL. After dealing with corneal epithelial cells, liver and kidney byPrdx6-11R fusion protein, the concentration of Prdx6increased in cells and tissues, and showsa dose-and time-dependent effect. Prdx6-11R inhibits the role of H2O2on corneal epithelialcells, and the oxidative damage of corneal epithelium cells has been inhibited.Conclusions The recombinant expression plasmid pET32b/Prdx6-11R was constructedand expressed in E.coli successfully. The activity of Prdx6was included in the fusion protein.The fusion protein purified has a significant role that it can enter into the corneal epitheliumcells, liver and kidney. Prdx6-11R fusion protein can promote Prdx6into human cornealepithelial cells and significantly improve the antioxidant effect of Prdx6in the cornealepithelium cells.