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水蛭素基因轉殖與非基因轉殖母豬乳腺與乳汁之蛋白質體比較
Comparison of protein expression in the mammary gland and milk from hirudin transgenic and non-transgenic sows using a proteomic approach
摘要
水蛭素（hirudin）為凝血素（thrombin）之抑制物，可抑制止血作用（hemostasis）與血栓（thrombosis）形成。水蛭素基因轉殖豬將可用於生產重組水蛭素（recombinant Hirudin; rHirudin）供醫療應用。本研究之目的為評估水蛭素轉殖基因是否影響水蛭素基因轉殖母豬乳腺與乳汁蛋白質表現。乳腺與乳汁樣品採自分娩當天之水蛭素基因轉殖與非基因轉殖母豬，取均質後乳腺及脫脂後乳汁之可溶性蛋白質供二維電泳分析，電泳膠片以colloidal Coomassie blue染色後掃描供影像分析或挖取蛋白質點供身份鑑定用。基因轉殖與非基因轉殖母豬乳腺蛋白質點以Melanie 3影像分析軟體進行定量，在所有定量分析的147個乳腺蛋白質點中有9個蛋白質點之表現量在基因轉殖與非基因轉殖母豬間具顯著差異（P < 0.05），其中在基因轉殖母豬乳腺表現量下降之蛋白質點為凝溶膠蛋白（gelsolin）及4個血紅素（hemoglobin）異型體（isoforms），表現增加之蛋白質點為異檸檬酸去氫酶1（isocitrate dehydrogenase 1）、絲胺酸胜肽酶抑制物（serpin peptidase inhibitor）及2個未知身份蛋白質。在所有定量的85個乳汁蛋白質點中有4個蛋白質點在基因轉殖與非基因轉殖母豬間具顯著差異（P < 0.05），其中在基因轉殖母豬乳汁表現下降的蛋白質點為血紅素結合蛋白（haptoglobin）及2個未知蛋白質，另1個在基因轉殖母豬乳汁表現量上升蛋白質點則為未知身份蛋白質。綜合以上，本研究之定量分析發現在水蛭素基因轉殖與非基因轉殖母豬間乳腺與乳汁分別有9個與4個蛋白質點之表現差異顯著，此等差異表現蛋白質之確切生理意義及未知身份蛋白質點之身份仍有待進一步研究。
並列摘要
Hirudin is a potent inhibitor of thrombin which plays a key role in hemostasis and thrombosis. Transgenic pigs carrying hirudin gene have been generated for producing recombinant hirudin in milk. The recombinant hirudin will be applicable therapeutically. The purpose of this study was to evaluate whether hirudin transgene affects the expression levels of mammary gland and milk proteins in hirudin transgenic sows. Mammary gland and milk samples were collected from two hirudin transgenic and two non-transgenic sows on the day of farrowing. Tissue samples were homogenized and soluble proteins were isolated for analysis. Milk proteins were collected after removing milk fat. Soluble proteins from mammary gland and milk samples were assayed by 2-DE and stained with colloidal Coomassie blue. Gels were scanned for further image analysis or subjected to excising protein spots for identification. A total of 147 protein spots on all mammary gland 2-DE gels were quantified using Melanie 3 software. The levels of nine protein spots were significantly different between the transgenic and non-transgenic sows (P < 0.05). The down-regulated proteins in the mammary gland of transgenic sows were gelsolin and four isoforms of hemoglobin. Isocitrate dehydrogenase, serpin peptidase inhibitor and the other two unidentified protein spots were up-regulated in the mammary gland of transgenic sows. A total of 85 milk protein spots on all milk 2-DE gels were quantified. The levels of four protein spots (P < 0.05). Haptoglobin and two other unidentified proteins were down-regulated in milk from transgenic sows. The identity of the up-regulated protein in milk from transgenic sows remains unidentified. In summary, this study found that there were nine mammary gland protein spots and four milk protein spots differed between hirudin transgenic and non-transgenic sows, respectively. The exact physiological relevance of the differentially expressed proteins and the identities of those unidentified protein spots in transgenic pigs require further elucidation.