Prokaryotic Expression And Localization Of Chicken XOD Subunit And Its Protein Expression Analysis After NIBV Infection

Xanthine oxidase(XOD) plays a vital role in the body’s purine catabolism, and study found that XOD also plays an important role in the body’s pathological process. In this study, a pair of primers were designed according to the reported gene sequence of G. gallus XOD(D13221.1) in NCBI, and used to amplify a XOD subunit sequence(987 bp) through RT-PCR technique. The aim gene fragment was inserted into the PMD-18-T vector by genetic cloning technique. The cloning XOD gene was ligated into the pET-32a(+) to construct the recombinant pET-XOD vector after determining the correct insertion. The pET-XOD vector was transformed into host cells Rosetta(DE3) to produce the recombinant protein. The recombinant proteins were purified, to produce the antiserum, by Ni NTA affinity chromatography method; using indirect ELISA to determine the titer of the antiserum; using Western blot to determine the specificity of the antisera. These antisera were used to locate the distribution of XOD in chicken liver and kidney through immunohistochemistry and immunofluorescence. The relative protein expression of XOD and Bcl-2 in the kidney of chicken with NIBV infection was determined by Western blot.The results showed that the prokaryotic expression system of chicken XOD was successfully constructed and the recombinant XOD proteins(about 54 KDa) with high concentration were purified. Western blot and ELISA analyses showed that the antisera raised by recombinant XOD protein could specifically recognize the chicken liver XOD and the titer of antiserum was about 1:102400. Immunohistochemistry and immunofluorescence showed that the XOD mainly presented in the cytoplasm of chicken hepatocytes and proximal tubular epithelial cells. The relative protein expression of XOD and Bcl-2 in the kidney of chickens with NIBV infection were significantly increase(P<0.05).In this experiment, the high concentration of recombinant chicken XOD protein was obtained by prokaryotic expression. And the antiserum raised by this recombinant protein could be used for localization of the distribution of XOD and determination of the relative protein expression of XOD in the chickens, providing a theoretical basis for further pathological mechanism of chickens with NIBV infection.