臺灣博碩士論文加值系統

直至目前為止，X-/Y-精子分離技術對於應用於經濟動物產業仍無法滿足不傷胚胎、低花費、高效能及方便性的技術需求。本研究實際應用Ohno’s law，以哺乳動物之性特異性蛋白 (sex-specific proteins, SSPs) 較其他non-SSPs在演化上具高度保留性為基礎理論，來進行X、Y精子表面特異性蛋白 (SSPs) 篩選，以期做為X、Y精子免疫分離法之基礎。

實驗先以豬精子細胞膜表面蛋白作為抗原，進行雌雄兔性別差異免疫法以產生相對應抗體。再應用雌雄兔產生的抗體製備性別特異性親和管柱，通入豬精子細胞膜蛋白，進行 SSPs 之純化。實驗結果發現將經過一次純化的性特異性蛋白質利用二維電泳 (two-dimensional electrophoresis) 配合鍍銀染色法分析，分別在電泳膠片上可見13個 X-SSPs 點及9個 Y-SSPs 點。進行ㄧ次重覆性實驗後由於無法得到完整的蛋白質分佈情形，故以區域性做為劃分；X-SSPs分別為 pI值位於3，分子量位10-20 KDa間及 pI值4，分子量40-50二個區域；Y-SSPs區域為pI值4，分子量30-40、 pI值5，分子量50、 pI值6-7，分子量30-40及 pI值6-7，分子量20四個區域。進行二、三次重覆性實驗後分別在電泳膠片上可見5個 X-SSPs 點、9個 Y-SSPs 點以及1個X-SSPs點及14個Y-SSPs點。以MALDI-TOF質譜儀及MASCOT比對工具進行 SSPs 身份鑑定。其中由雄兔抗體管柱所辨識到的hypothetical蛋白質(X-SSP)證實來源為Hereford種母牛的第七對染色體所轉譯出的X-染色體性聯蛋白質。其他以本研究理論認定之X-SSPs 及Y-SSPs，則因蛋白質資料庫之目前資料不足暫時無法成功鑑定其身份。

綜言之， 本研究已證實Ohno''s law 之存在性，亦即精子細胞膜表面存有與X-/Y- 染色體性聯之特有蛋白質。本研究所認定之精子性特異性蛋白質 (hypothetical proteins及/或其他) 的存在，提供了未來進行預選子代精蟲免疫篩選法之研究基礎。

Current techniques of sexing sperm have not been widely available for agriculture breeding industry, due to its high costs, low efficacy and inconvenience. A non-invasive, immunological method for sexing sperm would be satisfactory to above-mentioned needs. This study was designed, that is based on the Ohno’s law that sex-specific proteins (SSPs) are evolutionary more highly conserved than non-SSPs, to isolate SSPs from X or Y sperm and create a foundation for further immunological sperm sexing. Opposite-sex immunizations (antigens: porcine sperm membrane crude proteins) of rabbits were applied to raise the corresponding antibodies for each sex. Sex-specific affinity columns (M-, F-columns) prepared from the antibodies of respective sex were used to purify putative SSPs. Sequential studies demonstrated that with application of high resolution two-dimensional electrophoresis (2-DE) and the silver stain after one-cycle affinity chromatography column (ACC), 13 X-SSPs and 9 Y-SSPs protein spots were detected on 2-DE display (experiment 1); multiple X-SSPs (pI=3, 10-20 KDa; pI=4, 40-50 KDa) and Y-SSPs spots (pI=4, 30-40 KDa; pI=5, 50 KDa, pI=6-7, 30-40 KDa; pI=6-7, 20 KDa) were detected (exp. 2); 5 X-SSPs and 9 Y-SSPs spots were detected (exp. 3); 1 X-SSPs and 14 Y-SSPs spots were detected (exp. 4). Selected spots were subject to MALDI-TOF mass spectrometry and database search. One of the hypothetical proteins from M-column (putative X-SSP) preparation was recognized to be the female protein that is encoded from Hereford cow sex chromosome 7. Identification of putative proteins was unsuccessful due to limited data base of porcine proteins. In conclusions, this study verifies the existing of Ohno’s law. The putative SSPs are present at a detectable form under these delicate preparations, and that are minor components in the membrane. These results indicate the feasibility of immunological sperm sexing techniques.

表次............................................................................ げ
圖次............................................................................ こ
中文摘要.................................................................... ご
英文摘要.................................................................... さ
第一章、前言...............................................................1
第二章、文獻探討.......................................................3
第一節 子代性別預選技術........................................3
一、 著床前胚之性別鑑定技術.................................4
二、 X-/Y-精子分離技術.......................8
第二節 膜蛋白的分離與純化....................................14
一、 膜的分離及膜蛋白的萃取.................................14
二、 膜蛋白的純化......................................................17
第三節 質譜儀於蛋白質分析上的應用.....................21
第三章、材料及方法....................................................22
第一節 性別差異免疫法............................................22
第二節 抗原抗體製備................................................22
第三節 親和性管柱製備............................................25
第四節 X及Y精子特異性膜蛋白之純化....................28
第五節 X及Y精子特異膜蛋白之偵測及分析............29

第四章、結果.................................................................33
第五章、討論.................................................................47
參考文獻.........................................................................51
附錄.................................................................................59

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