Full-length CDNA Clone, Expression And ELISA Detection Establishment Of Peroxiredoxin6from Ovis Aries

Peroxiredoxin6(Prdx6) is belong to a family of Se-independ-end peroxidasesand is widely distributed in almost all of the vivos. Prdx6from different species havedifferent component of amino acid residues, but a Cys locating in the caralyticalyactive center is conserved. Prdx6is a bifunctional protein showing both GSHperoxidase and PLA2of activities within different environment of optimum pH.Several studies have shown that Prdx6has multiple biological functions and itsdifferential expression pattern is relatvie with a variety of disease. As a result, Prdx6is expected to become certain disease-related biological marker. Gene sequence ofPrdx6in several species are successfully cloned and expressed. Moreover, themonoclonal or polyclonal antibodies against Prdx6from human and rat arecommercialized. However, the study of Prdx6from Ovis aries is limited nowadays,and it is evident that lack of the specific antibody and detection methods of Prdx6from Ovis aries.Based on previous studies of constructing suppression subtractive hybridization(SSH) cDNA library of buffy coat from Brucella infected sheep and screening thepartial cDNA sequence of the differential expressed gene Prdx6from Ovis aries inour lab, the full-length cDNA sequence of Ovis aries Prdx6was cloned using5/-RACE technique in this study. The full-length cDNA of the sheep Prdx6was of1753bp with an open reading frame (ORF) of597bp encoding Prdx6protein of224amino acid residues with the predicted molecular weight of25.06kDa. For expressingthe recombinant Prdx6of sheep in E. coli, the recombinant plasmid pET-30a-Prdx6wwas constructed by means of gene engineering. The recombinant proteins of sheepPrdx6were successfully expressed with appropriate inducing conditions of1.5mmol/L at37℃for5.5h. The quantity of expressed Prdx6in E. coli was10.7%ofthe whole bacterial proteins by SDS-PAGE analysis. To prepare the monoclonal antibody (McAb) against sheep Prdx6, the femaleBALB/c mice aged8weeks old were immunized with the purified recombinant Prdx6protein (rPrdx6). Spleen cells and lymphonodi poplitei cells of the rprdx6-immunizedmice were fused with SP2/0cells and via three times of subcloning, five hybridomascell lines named D41G4, F1F1, B11B9,3RD5and3RB3which secreted monoclonalantibodies respectively belonging to the subclass of IgG1, IgG1, IgG1, IgG2a andIgG2b were selected. By western-blotting two hybridomas cell lines named F1F1andB11B9were confirmed to secrete the monoclonal antibodies with the activities ofbinding to the native Prdx6protein of sheep. The Prdx6-McAbs secreted by F1F1andB11B9were manufactured from BALB/c mice intraperitoneally injected withhybridomas. The ascites antibodies were purified by CA-AS method.An indirect competitive inhibition enzyme-linked immunosorbent assay(ic-ELISA) was developed for the quantitative detection of Prdx6with therecombinant protein and monoclonal antibody of sheep Prdx6. The basic protocol wasas following. The rPrdx6was added into microtiter plates with a concentration of1.0μg/mL, and then the plates were incubated at37℃for2h. One hundred microliterof0.1M NH4Cl was added into each well, and then the plates was incubated for1h at37℃. The McAb diluted by1120000times was used to carry out the competitionreaction at37℃for1h. One hundred microliter of goat anti-mouse IgG-HRPdiluted with4000times was added into wells and then the plates were incubated for1h at37℃. After incubated10mins with OPD in the dark at37℃, the reaction wasterminated by adding stop solution, and then determine the absorbance values at490nm was determined. The regression equation of the standard curve was y=-38.948x﹢177.46with correlation coefficient R2=0.9931. The lower limit detection was173.78ng/mL.In this study, the full-length cDNA of Prdx6from Ovis arise was cloned, and thesheep rPrdx6was induced to express. Moreover, the monoclonal antibody andicELISA detection method of sheep Prdx6were established. This study results willfacilitate future study of the Prdx6and its relationship with certain diease.