表現人類與小鼠Hepsin基因重組蛋白及抗體製備

Abstract

Hepsin屬第二型穿膜絲胺酸蛋白酶，普遍表現於各組織細胞，其中又以肝臟表現量最高。Hepsin能在缺乏組織因子下活化血液凝血第七因子，啟動外源性凝血反應；近幾年研究發現，hepsin在前列腺癌細胞株常是過量表現，而被推測與癌細胞的生長及轉移相關；。文獻指出hepsin可活化pro-HGF以及pro-uPA，然此功能仍缺乏活體內實驗證明。hepsin基因剔除小鼠不僅胚胎發育正常，成鼠也未出現凝血反應異常，所以目前對hepsin詳細生理功能及作用機轉仍不清楚。 為了研究hepsin的功能，本篇論文目標在製備抗hepsin抗體。首先，藉由大腸桿菌表現系統與Pichia pastoris酵母菌表現系統表現hepsin重組蛋白，成功使用大腸桿菌表現系統表現出人類hepsin基因重組蛋白以及小鼠hepsin基因重組蛋白，並將人類hepsin基因重組蛋白免疫小鼠。此外，為了分析將來作為抗體功能評估的方法，本論文也利用SK-Hep1的hepsin穩定表現細胞株建立活體動物實驗模式；於活體實驗中發現，表現突變型hepsin的肝癌細胞株其腫瘤生成能力較低。

Hepsin is a type II transmembrane serine protease, which is present in most tissues, with the highest expression level in liver. It is shown to interact with coagulation Factor VII, and convert zymogen factor VII to factor VIIa. Previous studies suggest that hepsin is involved in cell growth and migration. Hepsin was also shown to activate proHGF and pro-uPA by in vitro study. These findings are promising yet lacking of in vivo functional significance. Moreover, because embryonic developmental defects and hemorrhage abnormality were not shown in hepsin knockout mice. The biological role of hepsin remains unclear. To study hepsin function, this study aims at generating anti-hepsin antibodies. E. coli and Pichia pastoris expression systems were used to produce the hepsin recombinant protein. In this study, I have expressed the human and mouse hepsin and used the human recombinant protein to generate the specific anti-human hepsin polyclonal antibody. Besides, to set up the in vivo tumorigenesis assay by inoculating the SK-Hep1 /hepsin stable clone into nude mice could be as a kind of functional assay of the generated antibodies. In the nude mice tumorigenesis assay, I discovered that the tumorigenesis ability of SK-Hep1 stable clone which express the mutant hepsin was lower than those express wild type hepsin or none.