儲精囊分泌液Kazal-type 胰蛋白?抑制子之重組蛋白質的生化特性研究

Abstract

自老鼠（mouse）儲精囊分泌液純化的胰蛋白?抑制子（Trypsin Inhibitor ，簡稱TI）屬Kazal-type protease inhibitor,分子量約6.5kDa ，本身不含醣。利用遺傳工程方法，將PCR放大的成熟TI DNA片段鑲入pGEX－2T載體中，於大腸桿菌（E.coli）表現，產生融合蛋白質（GST-TI）。利用親合性管柱色層分析法純化此融合蛋白質，經凝血酵素水解GST-TI再利用逆相高壓液態色層分析得到重組的TI。並以融合蛋白質當抗原製備抗體血清。藉上述抗體血清作西方點墨法分析（Western blotting) ，結果顯示TI分佈在雄性老鼠之前列腺及儲精囊；另外作免疫組織化學標定（Immunocytochemistry) ，探討TI在前列腺及儲精囊的組織分佈，結果TI出現在前列腺之管泡狀腺體 (tubuloalveolar gland）的上皮組織，而儲精囊管壁上皮組織偵測不到TI；若施以老鼠睪丸切除（castration) ，則前列腺之管泡狀腺體的上皮組織的TI消失，注射testosterone,TI又出現。

A Kazal-type trypsin inhibitor (TI) has been previously purified from seminal vesicle secretion in our laboratory. The protein contain no carbohydrate and its molecular weight was estimated to be 6.5kDa by both gel filtration chromatagraphy and electrophoresis. By genetic engineering manupulation, the DNA fragment of the whole TI was amplified through polymerase chain reaction, and ligated into pGEX-2T vector .The constructed vector was transfected into E.coli cells.The cloned cells were able to produce high yield of a 34kDa fusion protein consisting of glutathione-S-transferrase (GST) and TI. The GST carrier can be removed from the fusion protein by cleavage with thrombin and the recombinant TI can be isolated through reverse-phase C4 column HPLC. Antiserums induced from the fusion protein showed high affinity and specificity for native TI. With the antiserum, tissue distribution of the TI in the mouse was examined. Western blots indicated that the TI was expressed in the prostate gland and in the seminal vesicles. The results of immunocytochemistry showed that the TI was present in the secretory epithelium of ventral prostate gland but was absent in that of seminal vesicles. Immunodetectable TI declined in the secretory epithelium of ventral prostate gland after castration and returned upon testosterone administration.