利用乳蛋白基因啟動子於豬乳腺上皮細胞中表現並分泌外源蛋白質

Abstract

利用 DNA 重組技術生產重組蛋白質已發展超過 30 年之久。至今，超過百種醫藥用重組蛋白質已被美國食品與藥物管理局(Food and Drug Administration, FDA)核准使用於醫療上。早期用來大量生產重組蛋白質的生物宿主以微生物為主，例如細菌或酵母菌，但使用微生物系統生產蛋白質常有蛋白質轉譯後修飾(post-modification)功能不完全的問題，此可能影響蛋白質產物的穩定性與生物功能性。因此，目前用來生產重組蛋白質之生物宿主，仍以哺乳類動物細胞為主。猪乳腺上皮細胞 SI-PMEC 是一自泌乳猪乳腺組織所分離並篩選出來的細胞株，此細胞株已持續培養超過 8 個月(約 70 代)。SI-PMEC細胞生長在 Matrigel 上，並給予泌乳激素(prolactin)，細胞會分化形成三維的類乳腺網狀結構，並高度表現乳蛋白基因。將帶有綠螢光蛋白與螢光酶表現載體轉殖至 SI-PMEC細胞中，可測得顯著地這兩個報導基因的表現；更進一步，SI-PMEC 細胞已成功地被用 來產生重組抗凝血水蛭素(hirudin)。 由於α-乳白蛋白和β-酪蛋白是哺乳動物乳中主要的蛋白質。因此，在我們的研究中，假設此兩種蛋白質基因的啟動子(promoter)在 SI-PMEC 細胞中有較強的表現能力，因此利用這兩個蛋白質基因的啟動子與其信號胜肽(signal peptide)序列來建構表現載體，製造出能分泌至細胞外的重組蛋白質，即報導螢光酶。本實驗結果顯示：(1) 轉殖有表現基因載體的 SI-PMEC 細胞，培養於第一型膠原蛋白與 Matrigel 基質上，細胞呈分化外貌狀，且可高度表現報導蛋白質；(2) firefly 螢光酶一旦被分泌至 SI-PMEC 細胞外，其活性很快地衰減，因此不適用於細胞分泌性質的報導蛋白質，反之 Gaussia 螢光酶被分泌至細胞外時，其活性穩定；(3) 沒有構築乳蛋白基因的信號胜肽序列時，螢光酶在細胞內的表現量較有信號胜肽序列時為高；(4) 以 α-乳白蛋白和 β-酪蛋白的信號胜肽序列置換Gaussia 螢光酶本身所帶有的信號胜肽序列，其分泌螢光酶的效率降低；(5) α-乳白蛋白基因啟動子在 SI-PMEC 細胞中有顯著較高的表現與分泌螢光酶的效率；(6) 在更換培養基處理下，轉殖有表現載體的分化 SI-PMEC 細胞可持續的表現與分泌螢光酶。 本研究提供一個可生產與分泌重組蛋白質的乳腺細胞培養系統，儘管本系統完備之前還有很多條件須探討和最佳化，但此一系統用於生產醫用蛋白質可能具有產物純化程序簡單與因可連續操作而產能高的優勢，值得後人參考利用之。

Using recombinant DNA technology to produce recombinant proteins have developed over 30 years. Until today, over hundred therapeutic recombinant proteins have been approved by Food and Drug Administration (FDA) and used in medical purposes. In the early days, the microorganisms, e.g., bacteria and yeast, were the major host cells to produce recombinant proteins. However, the major limitation of the microorganisms is possibly lack of post-translation modification that may affect the stability and activity of produced proteins. For the reason, the therapeutic recombinant proteins are mainly produced by mammalian cells so far. Spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) was isolated from mammary gland of a lactating Landrace pig and maintained for more than 8 months (70 passages). When SI-PMEC cells grew on Matrigel and stimulated by prolactin, the cells could differentiate and form three-dimensional mammary gland-like structure and strongly express the gene of milk proteins. The SI-PMEC cells transfected with the express vector encoding the enhanced green fluorescence protein (EGFP) and luciferase were approved they could highly express both reporter genes. Furthermore, the cells have been used to produce recombinant anticoagulant hirudin successfully. Based on the knowledge of α-lactalbumin and β-casein is the major proteins in mammal milk, we suppose that the gene promoter of α-lactalbumin and β-casein can strongly express in mammary epithelial cells. Therefore, the sequences of promoter and signal peptide of both genes were constructed into expression vectors that were used to produce secreted recombinant proteins, firefly and Gaussia luciferase, in this study. Our results showed that: (1) the expression vectors transfected SI-PMEC cells cultured on the dish with type-I collagen or Matrigel as substratum could differentiate and strongly express the exogenous proteins; (2) while firefly luciferase was secreted to the culture medium, the luciferase activity decreased quickly; on the contrary, the activity of Gaussia luciferase secreted to the culture medium was stable; (3) the expressed luciferase of SI-PMEC cells transfected with a express vector without signal peptide sequence is higher than that with the signal peptide sequence of milk protein genes; (4) changing the native signal peptide of reporter Gaussia luciferase by the signal peptide sequences of α-lactalbumin and □-casein genes showed a reduced efficiency of produced luciferase secretion; (5) using the α-lactalbumin promoter to drive Gaussia luciferase expression, the culturing SI-PMEC cells had the highest expression of luciferase in the cultured medium; (6) the express vector transfected and differentiated SI-PMECs could continuously express and secrete the luciferase when treating the cultures with medium change. Our studies provide a culture system of mammary epithelial cells to produce and secrete recombinant proteins. Even though there are many conditions that have to be discussed and optimized in this system but also many advantages that are worthy to utilize, such as collecting the secreted recombinant proteins easily, simplifying the procedure of product purification, and possibly a high productivity in a continuous culture system.