PARENT SESSION
Pregnancy and Parturition

PRODUCTION AND CHARACTERIZATION OF RECOMBINANT EQUINE PRORELAXIN.

Neumann, Jennifer¹, Huang, Yue-Jin², Lazaris, Anthoula², Karatzas, Costas², Ryan, Peter³, Bagnell, Carol¹, ^{1 2 3}

ABSTRACT- Late-term pregnancy loss due to placental insufficiency is a clinical problem of economic importance to the equine industry world-wide. Equine relaxin (eRLX) is a placental product, and reports of low circulating RLX in late-term aborting mares (1992 Biol Reprod 46:648) led us to hypothesize that eRLX may be a valuable marker of placental function and fetal well-being. Since eRLX is not commercially available and tissue extraction and purification are labor-intensive, the objective of this study was to produce recombinant equine prorelaxin (reRLX). The full-length, 593-bp, equine preprorelaxin gene was modified by the addition of a histine tag and subcloned into a pSecTag expression vector. The eRLX-his/pSecTag expression vector was transfected into immortalized bovine mammary epithelial (MAC-T) cells grown in Dulbecco's minimum essential medium, supplemented with fetal bovine serum (10%), insulin (5 ug/ml) and antibiotics. Transfected cells were selected using hygromycin B (200 ug/ml). Proteins from cell extracts and 5x-concentrated media were separated on a 12.0 % acrylamide gel under non-reducing conditions and transferred to nitrocellulose membranes. A 20.7 kDa band corresponding to the expected size of equine proRLX (19 kDa) with a histine tag was detected in the media samples using a rabbit anti-porcine RLX serum and visualized using an enhanced chemiluminescence detection system. Immunological activity of the reRLX was confirmed by homologous equine RLX radioimmunoassay (RIA). Media from transfected MAC-T cells contained 5.72 ± 0.31 ng/ml of reRLX, while only 0.37 ± 0.11 ng/ml of the recombinant hormone was detected in cell lysates, indicating most of the reRLX is secreted from the cells. In mock-transfected MAC-T cells, there was no evidence for immunoreactive reRLX in cells or media. These data show an immunologically active reRLX can be produced in a mammalian cell line. Large-scale production of reRLX will be important for the creation of an enzyme-linked immunoassay to measure plasma eRLX in at-risk mares, as well as for use in clinics and in basic research. This will provide clinicians with a means of monitoring equine placental function and fetal well-being and thus provide a valuable addition to equine veterinary medicine. (Supported by 2000 NJAES Equine Initiative Fund)

KEY WORDS: equine, relaxin, recombinant protein, pregnancy