Cloning And Expression Of The Porcine TrxR2 And Selenoprotein H Genes And The Selenoprotein Antibody Preparation

Abstract:Selenium is an essential trace element for many forms of life including humans, playing a key role in many physiological functions. Selenium functions mainly as the form of selenoprotein. Until now, there are 25 kinds of selenoprotein in swine but most of which have unidentified functions. There is, so far, no report about the full gene sequences of the TrxR2 and SelH. In this study, the full-length cDNAs of TrxR2 and SelH genes (GenBank No.GU181287, HM018602) including the complete signal peptide sequence and the selenocysteine were cloned from fresh lung tissue by RT-PCR. Sequence analysis showed that the cDNA of TrxR2 shares over 85% homologies with human, cattle, dogs, monkeys and apes, and the cDNA of selenoprotein H shares homologies ranging from 93% to 95% with horses, human, monkeys, apes and dogs. The obtained cDNAs were ligated into prokaryotic expression vector pET-30a(+), and the the recombinant plasmids pET-30a (+)-TrxR2 and pET-30a(+)-T-SelH were delivered into E. coli BL21 (DE3) pLysS cell. After 3 hours of IPTG induction, the recombinant selenoproteins with a histidine tag appended to the C terminus were purified by Ni-Sepharose affinity chromatography, The purified recombinant TrxR2 and SelH proteins exhibited molecular masses of approximately 67 KD and 23KD, respectively, as determined by SDS-PAGE analysis. The purified proteins were used as immunogen to immunize rabbits to produce selenoprotein antibodies. The specificity of rabbit antibodys were analyzed by ELISA and Western blotting analyses. The obtained results showed that the raised antibodies dispalyed good specificities. As determined by ELISA analysis, TrxR2 was found to be highest in the hypothalamus and the lung followed. The highest concentration of porcine SelH was found in the thyroid, followed by skeletal muscle and lung. There were no significant differences in content among the remaining tissues., The success of the high quality selenoprotein antibody preparation lays the foundation for further research on the function and distribution of selenoproteins, as well as developing a simple method for detecting selenoproteins.