肝細胞生長因子 (HGF) 是一種分泌型、在生理上屬多功能性的蛋白質。HGF通過與Met相結達到信號傳達的目的，可以誘導目標細胞進行有絲分裂或形態發生，提高細胞的活動力和侵入性等作用影響細胞的遷移，如此可促使癌細胞的轉移。HGF的α亞基包含了N端區域及四個kringle區域，其與受體結合的關鍵區域位於α亞基上。為了闡明α亞基的多個區域對受體結合及信號傳達的影響，本研究以kringle為單位設計了四個重組基因區域，分別為NK1、NK2、NK3和NK4，並以大腸桿菌為宿主，分別進行四個重組蛋白的表現並利用GST親和性層析管柱來進行重組蛋白的純化。所獲得的重組蛋白皆與GST形成融合蛋白，將重組蛋白進行細胞學測試。研究證明，四個缺失型重組蛋白對人類乳癌細胞的增生機轉有一定的影響，其中GST-NK1與GST-NK2的抑制效果比GST-NK3與GST-NK4明顯。這些結果說明由大腸桿菌所生產的GST-NKs融合蛋白對乳癌細胞具有抑制其生長的作用。本研究也為未來實測細胞增生與細胞轉移等提供了基因表現、純化的基本技術及研究路徑。

Hepatocyte growth factor (HGF) is a multifunctional protein, which secrets via Golgi complex after synthesized, and is hydrolyzed into an active heterodimer containing an α and a β chain by extracellular protease. It is known that HGF functions through surface domain of Met, and thus induces mitosis and metastasis. The interaction domain of HGF is believed to be located in the α-chain. In order to study these findings structurally and functionally, we designed and constructed four different recombinant coding regions of the gene (NK1, NK2, NK3, and NK4) which was then successfully expressed in E. coli. Purification of these four different recombinant proteins with glutathione-agarose column showed that all of the four constructs had been successfully expressed with some degradations. Cell proliferation assay showed that the recombinant proteins inhibited the growth of breast cancer cells to some extent. The assay also showed that GST-NK1 and GST-NK2 were better inhibitors than GST-NK3 and GST-NK4 to the cancer cells. It is concluded that E. coli expression is an appropriate system for achieving functional HGF.

中文摘要……………………………………………………………… 5
英文摘要……………………………………………………………… 6
第一章：概論………………………………………………………… 7
第二章：材料與方法…………………………………………………18
第三章：生長因子基因的重新改造…………………………………39
第四章：重組生長因子的表達及純化………………………………54
第五章：重組生長因子的細胞學鑑定………………………………62
第六章：結論…………………………………………………………71
參考文獻………………………………………………………………72

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