Investigation of the hepsin-interacting molecules by RAP in situ and recombinant Flag-hepsin fusion protein

• Main Authors: Ying-Hui Su, 蘇英卉
• Other Authors: Shu-Wha Lin
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/07221125414714473818

碩士 === 國立臺灣大學 === 醫事技術學研究所 === 90 === Hepsin is a gene discovered by screening a human liver cDNA library with reverse-transcribed probes synthesized based on the highly conserved region of the catalytic site of serine protease. Hepsin is a type II transmembrane protein over-expressed at cancer metastasis and initiation of blood coagulation driven by activated factor VII. However, in vivo studies demonstrated that hepsin KO mouse had neither shown embryonic lethality, bleeding disorder nor growth anomaly in adulthood, and was physically normal compared with its normal counterpart based on biochemical analysis. To date, the physiological function of hepsin is yet to be determined. This thesis aims at searching for hepsin-interacting molecules. The strategy adopted comprised two parts: Part I, searching for hepsin-interacting molecules using RAP in situ ( RAP：Receptor Alkaline Phosphatase or Receptor Affinity Probe) technique; Part II, confirmation of the relationship between hepsin and blood coagulation factor. First, recombinant alkaline phosphatase (AP) and alkaline phosphatase-soluble hepsin (AP-sHep) fusion protein established in our laboratory were used in RAP in situ, searching for hepsin-interacting molecules directly. Cell culture medium containing AP-sHep and mouse embryos at different developmental stages were added together and we found that the hindbrain of these embryos (at embryonic day 10~12) showed obvious staining results. In addition, histological sections of the same tissue showed positive results, indicating that certain substance, which is under empirical identification, reacts with hepsin. Besides, 4 cell lines treated with PMA (Phorbol 12-myristate 13-acetate) and RA (retinoic acid) were tested if they would express hepsin-interacting molecules but no positive results were obtained. In the second part, electrophoresis was used to purify flag-hepsin fusion protein abundantly expressed in E coli. expression system. Anti-hepsin antibody used in immunohistochemical experiments was then prepared. The antigen applied in rabbit vaccination was a synthetic peptide comprising 18 amino acids at the carboxyl terminus of hepsin (residues 400-417). The antibody was purified by purification column coupled with the same synthetic peptide and was used in subsequent immunohistochemical experiments. In revealing the relationship between hepsin and blood coagulation, the ability of FXIa and FVIIa to catalyze flag-hepsin was tested by cleavage of Flag-hepsin with FXIa and probing with antibodies. The results showed no hepsin activation catalyzed by the above factors, either in the presence of phospholipids, or poly-lysine as a mediator, or not.