Cloning and Detection of Equine Histidine-Rich Glycoprotein

Abstract Histidine-rich glycoprotein (HRG) is a plasma glycoprotein that is presumed to be involved in immune response, coagulation, and fibrinolysis. Recent studies have revealed that the blood HRG levels are markedly decreased in the murine model for sepsis, and that recombinant HRG injections significantly improved their survival rate. Although sepsis is a common secondary condition in horses, changes in the blood HRG levels during critical diseases in horses have been unexplored. To elucidate the association of HRG with certain diseases in horses, the establishment of a procedure for the detection of HRG is necessary. In the present study, we investigated HRG messenger RNA (mRNA) expression by real-time polymerase chain reaction using complementary DNA (cDNA) generated from various organ samples of Thoroughbred horses and determined that HRG mRNA was expressed in the liver. Consequently, HRG was cloned using cDNA from the liver samples, and the whole sequence of equine HRG and the amino acid sequence of HRG were identified. Since HRG comprises a significant number of histidine residues, we purified equine HRG in serum using a nickel resin. In addition, the purified equine HRG was detected by Western blot analysis. We also generated recombinant equine HRG, and cross-reactivity of the antibody was confirmed. We demonstrated the complete sequence of the equine HRG and detected serum HRG in healthy horses for the first time. This information may prove valuable for future investigations of serious inflammatory diseases in horses, such as the systemic inflammatory response syndrome.