小白鼠貯精囊分泌液胰蛋白?抑制因數的生化研究

Abstract

從小白鼠貯精囊分泌液中可利用硫酸銨沉澱法，膠體過濾層析以及逆相高效液相層析，純化到一種胰蛋白?抑制因數（mouse seminal vesicle secretion trysin inhibltor , MSVS TI ) ，屬於 Kazal - type 的胰蛋白?抑制因數，分子量約 7kDa ，本身不含醣。由胺基酸定序，確定 MSVSTI 是由小白鼠前列腺所得到一種 MP12 cDNA 在貯精囊內所表現的蛋白質產物。 我們實驗室利用遺傳工程技術，將 MSVS TI 的 cDNA 鑲入表現質體 pGEX-2 ，於大腸菌中表現，以大量純化融合蛋白質 GST – TI ( glutathione－S－transferase－trypsin inhibitor) 。並以融合蛋白質 GST- TI 製備抗 MSVS TI 之抗血清，此抗血清可以專一性地辨認貯精囊分泌液中的 MSVS TI。 利用抗 MSVS TI之抗血清，對小白鼠的生殖器官及肝臟、胰臟、脾臟作西方墨跡分析（Western blotting) , 發現 MSVS TI 僅分佈在雄性小白鼠的貯精囊及前列腺，其含量會隨著小白鼠的成熟而累積，且其表現依存於雄性素的存在；利用 MSVS TI cDNA 所作探針，對雄性小白鼠生殖器官作北方墨跡分析（Northern blotting ) ，可得到相同的結果。 利用抗 MSVS TI 抗血清，我們使用閒接免疫螢光檢視法來觀察 MSVS TI 結合在精子上的位置，可以發現 MSVS TI 會結合在精子頭端的acrosome 位置，而產生勾月狀螢光。

A Kazal-type trypsin inhibitor from mouse seminal vesicle secretion was purified to homogeneity via a series of purification steps including ammonium sulfate fractionation, gel filtration, and HPLC on a reverse phase C4 column. The molecular weight of the protein was determined to be 7kDa by both gel chromatography and sodium dodecylsulfate polvacrylamide gel electrophoresis. The protein contained no carbohydrate. Results of direct amino acid sequence determinations indicated that this protein was the product of MP12 cDNA identified from mouse prostate (Mills et al., 1987a) By recombinant DNA manipulation, E.coli cells were transformed with an expression vector constructed by inserting the DNA sequence coding for the inhibitor into pGEX-2. The cloned cells were able to produce high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione-S-transferase. The chimeric polypeptide could be partially purified through an affinity column of glutathione agarose beads. Antiserum induced from the chimeric polypeptide could recognize native MSVS TI specifically. The antiserum was used for the Western blot procedure to examine tissue distribution of MSVS TI among reproductive tracts. MSVS TI was found only in prostate glands and seminal vesicles. The results were confirmed by Northern blot analysis.