Porcine progesterone detection through phage display technology

• Main Authors: Min-chen Li, 李旻宸
• Other Authors: Wen-Jen Yang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/11796410341338900113

碩士 === 國立高雄大學 === 生物科技研究所 === 100 === Raising the efficiency of pig-breeding is a major point to increase the economy of pig farm. Several studies showed that the concentration of progesterone in blood was a crucial factor used to determine the pregnancy or not in sow. After embryo implantation, the concentration of this hormone stably rises and keeps in a high level to maintain a suitable environment for embryo development until delivery. It is significantly different with the non-pregnant sow that influenced by estrogen and returned to estrous leading to the decrease of progesterone concentration. Therefore, progesterone was used as a diagnostic marker, and analyzed it with phage display technology to evaluate its potential of replacing the antibody against progesterone and facilitating the development of diagnostic kit. Western blot analysis showed that the molecular weight of progesterone-3-O-CMO-BSA conjugate was 68.4 kDa. It indicated that progesterone was conjugated onto BSA successfully. After 2 rounds of biopanning with ph.D.-C7C and ph.D.-7 phage display peptide libraries and removing the phages bind to BSA, the residual numbers of phage were 315 pfu and 175 pfu, respectively. Several phages were randomly selected, amplified, purified and then assayed by enzyme linked immunosorbent assay (ELISA) and dot blot analysis to evaluate their binding affinity to progesterone. Several phages with high binding affinity were selected for DNA sequences analyses and deduced into amino acid sequences. Three consensus sequences were found after sequences alignment. Moreover, several selected sequences were found corresponding to the progesterone receptor (PGR) of porcine (NP_001159960) and progesterone receptor membrane component 1 (PGRMC1) of porcine (NP_999076) deposited in NCBI database. These phages were assayed with pure progesterone, it showed that phage C7C-74 and D7-19 have higher affinity and specificity to progesterone and their sequences were corresponding to the ligand biding site of porcine PGR. In conclusion, the selected phages have the potential for the development of diagnostic kit in porcine pregnancy detection.