TWI426128B - 一種提昇生物反應器生產抗病有用蛋白產量之方法 - Google Patents
一種提昇生物反應器生產抗病有用蛋白產量之方法 Download PDFInfo
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Description
本發明係關於一種提昇生物反應器生產抗病有用蛋白產量之方法及相關核酸建構物。詳言之,本發明係關於利用α肌動蛋白啟動子以及抗病有用蛋白的核酸序列建構質體,並搭配特定品系的斑馬魚作為生物反應器,大量提昇抗病有用蛋白產量的方法。本發明可運用於水產養殖業及其他養殖業,提供了一種改善養殖效率的方法,降低疾病所造成的損失。
乳鐵蛋白(lactoferrin)是一種人類及動物體內普遍存在的攜鐵醣蛋白,分子量約為8萬道爾頓,屬於運鐵蛋白家族(transferrin family)成員之一。哺乳動物的乳鐵蛋白素存在於乳汁、嗜中性白血球及各種外泌液如眼淚、唾液及臟器黏液中,在調節人及動物的生理功能上,扮演多項重要角色,如:維持體內鐵離子的恆定、抵抗許多種類的微生物感染、抗發炎反應、促進淋巴細胞的分化及調節巨嗜細胞(marcophage)與顆粒細胞(granulocyte)的增生、在腸胃道中可幫助鐵離子之運送及吸收等。在最近幾年的研究報告中,更指出其有抗癌細胞增生及腫瘤轉移的功能。
當胎牛乳鐵蛋白(bovine lactoferrin)被消化道中的胃蛋白酶(pepsin)水解,會形成兩性乳鐵蛋白素分子(amphipathic bovine lactoferricin,LFB),這段分子量為3142道爾頓的胺基酸序列是胎牛乳鐵蛋白的N端衍生物,帶有大量的正電荷,為一段抗菌多肽,可以將包含格蘭氏陽性菌及格蘭氏陰性菌在內的多種細菌或病源殺死或使之不活化。LFB的抗菌性來自一兩性β褶板的構造,在此構造中,疏
水性的胺基酸與帶正電的極性胺基酸分別排列於相異兩側,其中帶正電端可與格蘭氏陰性菌表面帶負電的脂多醣分子(lipopolysaccharide,LPS),藉此破壞其細胞膜的穩定性,在格蘭氏陽性菌方面則與細胞膜外的磷壁酸層(teichoic acid layer)結合。細菌與LFB結合後,會造成細胞內外之電化學梯度及酸鹼梯度被破壞,導致細胞死亡。
另一方面,研究指出LFB亦可經由干擾細胞內外氫離子梯度的方式抵抗真菌及寄生蟲的入侵,並可與硫酸乙醯肝素(heparan sulfate,HS)及葡糖胺聚糖(glycosaminoglycans,GAGs)交互作用,利用高度正電及雙硫鍵形成的二級結構,使病毒無法藉由與硫酸乙醯肝素或葡糖胺聚糖結合而感染細胞。由於LFB可對抗如此多樣的微生物感染,在水產養殖業的抗菌應用中也扮演了相當重要的角色。
由於具有許多操作上的優點,斑馬魚(zebrafish,Danio rerio)一直是種極佳的模式生物。真核生物的斑馬魚於製造蛋白質時有一套繁複的轉譯後修飾過程;斑馬魚相當地多產,出生三個月左右即性成熟,之後在適當的溫度環境下(27~29℃)每週可生產一到二次,一對成魚一次可產下200~300個卵。此外,建立一個可施用於斑馬魚的培養系統很容易也很便宜,且以斑馬魚進行基因轉殖的操作也很容易達成,轉殖完成的不同魚株容易篩選也容易繁殖。基於以上的優點,斑馬魚將會是個作為生物反應器的優良材料。
先前曾有運用巨細胞病毒(cytomegalovirus,CMV)啟動子驅動第七凝血因子(coagulation factor VII)基因,並以斑馬魚、非洲鯰魚(African catfish)、吳郭魚(tilapia)的受精卵作為生物反應器製造第七凝血因子的技術被發表,前一陣子也曾報導了利用CMV啟動子或mylz2啟動子,以斑馬魚為生物反應器生產石斑魚抗病蛋白epinecidin-1的技術。
發明人於2009年11月26號發表了以斑馬魚的肌肉及魚卵為生物反應器,利用β肌動蛋白啟動子驅動外源抗病蛋白基因,生產LFB
物反應器,利用β肌動蛋白啟動子驅動外源抗病蛋白基因,生產LFB蛋白的技術(Cheng-Yung Lin,Ping-His Yang,Chia-Lin Kao,Han-I Huang and Huai-Jen Tsai.Transgenic zebrafish eggs containing bactericidal peptide is a novel food supplement enhancing resistance to pathogenic infection of fish.Fish & Shellfish Immunology 2010;28:419-427.)。透過此技術,外源抗病蛋白可被表現於全身肌肉細胞及魚卵中,且此基因可有效遺傳至其子代,而不僅限於當代。更進一步,此技術生產的魚隻及其魚卵可直接作為添加物加工製成養殖飼料,並能夠保有抗菌活性而不需要再作進一步的純化。然而,本技術仍存在有抗菌蛋白有效產量上的缺陷,有待提昇其產量以增進利用效率。
本發明之一目的在於提供一種提昇生物反應器生產抗病有用蛋白產量之方法,其係包含下列步驟:(a)構築一質體,其至少包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列、一蛋白酶切割位之核酸序列以及一α肌動蛋白啟動子;(b)將構築之質體轉殖入斑馬魚胚胎中;(c)馴養魚隻,以及(d)收集魚卵、魚胚或成魚,得到抗病蛋白。
本發明之另一目的在於提供一種核酸建構物,其至少包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列及一α肌動蛋白啟動子。
本發明之又一目的在於提供一種表現載體,其包含有一前述之核酸建構物,該核酸建構物上至少包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列及一α肌動蛋白啟動子,並可另包含一
蛋白酶切割位之核酸序列。
本文所述之LFB係指兩性乳鐵蛋白素分子(amphipathic bovine lectoferricin);本文所述之GFP係指綠色螢光蛋白(green fluorescent protein)。
本發明係關於一種提昇生物反應器生產抗病有用蛋白產量之方法,其包含下列步驟:(a)構築一質體,其包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列、一蛋白酶切割位之核酸序列以及一α肌動蛋白啟動子;(b)將構築之質體線性化後轉殖入選定品系之斑馬魚胚胎中;(c)馴養魚隻,使之成長、交配及產卵;以及(d)收集魚卵、魚胚或成魚,即可得到富含抗病蛋白之生物體。
本發明之提昇生物反應器生產抗病有用蛋白產量之方法中,該外源抗病蛋白之核酸序列較佳係為乳鐵蛋白素(lactoferricin)核酸序列,而該α肌動蛋白啟動子可使外源抗病蛋白基因表現於全身之肌肉細胞中。在一最佳實施例中,該α肌動蛋白啟動子係為α肌動蛋白1a啟動子,能驅動該外源抗病蛋白專一且大量表現於骨骼肌中,其表現量先前技術所使用的β肌動蛋白啟動子。
本發明之方法中,該螢光蛋白核酸序列係用以標示抗病蛋白的表現,以其轉譯產物之螢光強度作為抗病蛋白表現位置定位及表現量之定量依據。該螢光蛋白核酸序列可為各種顏色螢光蛋白核酸序列,在一較佳實施例中,該螢光蛋白核酸序列係為綠色、紅色、黃色、藍色、靛色螢光蛋白核酸序列;在一最佳實施例中,該螢光蛋白核酸序列係為綠色螢光蛋白核酸序列。
本發明以斑馬魚作為生物反應器表現功能性外源抗病蛋白,選定
之斑馬魚品系可穩定遺傳所轉殖之基因而非僅當代表現,且透過選定之啟動子大量提昇了外源抗病蛋白的表現量,魚隻抗病蛋白表現量穩定,並可大量繁殖。在一最佳實施例中,本發明之方法所選定之斑馬魚品系係為ZαL-3。
本發明之方法中,該蛋白酶切割位之核酸序列的設計緊連外源抗病蛋白核酸序列,在轉譯後,該轉譯產物經由蛋白酶切割,自動釋出有抗病功用之蛋白質進行作用,不需再作純化。在一最佳實施例中,該蛋白酶切割位之核酸序列係為胃蛋白酶切割位之核酸序列。
本發明另係關於一種核酸建構物,其包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列及一α肌動蛋白啟動子。
本發明之核酸建構物中,該外源抗病蛋白之核酸序列較佳係為乳鐵蛋白素(lactoferricin)核酸序列,該α肌動蛋白啟動子最佳係為α肌動蛋白1a啟動子,能驅動該外源抗病蛋白專一且大量表現於骨骼肌中。
本發明之核酸建構物中,該螢光蛋白核酸序列可為各種顏色螢光蛋白核酸序列,在一較佳實施例中,該螢光蛋白核酸序列係為綠色、紅色、黃色、藍色、靛色螢光蛋白核酸序列;在一最佳實施例中,該螢光蛋白核酸序列係為綠色螢光蛋白核酸序列。
本發明之核酸建構物另可包含一蛋白酶切割位核酸序列,該序列緊連外源抗病蛋白核酸序列,在轉譯後,該轉譯產物經由蛋白酶切割,自動釋出有抗病功用之蛋白質進行作用,不需再作純化。在一較佳實施例中,該蛋白酶切割位之核酸序列係為消化道相關蛋白酶切割位之核酸序列;在一最佳實施例中,該蛋白酶切割位之核酸序列係為胃蛋白酶切割位之核酸序列。
本發明又係關於一種表現載體,其包含有一前述之核酸建構物,該核酸建構物上至少包含一外源抗病蛋白之核酸序列、一與其連接之
螢光蛋白核酸序列及一α肌動蛋白啟動子,並可另包含一蛋白酶切割位之核酸序列。
以下將以具體實施例說明本發明之提昇生物反應器生產抗病有用蛋白產量之方法、核酸建構物及表現載體,然這些實施例僅用以作為本發明代表性的不同面向及特徵,並不得被推論為本發明僅侷限於此等實施例所揭示之內容。
以具有哥薩克(Kozak)序列之綠色螢光蛋白(green fluorescent protein,GFP)的cDNA片段(SEQ ID NO:1)作為模板,使用LFBTG-F1引子(SEQ ID NO:2)與LFBTG-R引子(SEQ ID NO:3)進行聚合酶鏈鎖反應,再將所得之729個鹼基對片段當作模板,使用LFBTG-F2引子(SEQ ID NO:4)與LFBTG-R引子進行聚合酶鏈鎖反應,得到760個鹼基對的產物。最後將760個鹼基對片段當作模板,使用LFBTG-F3引子(SEQ ID NO:5)與LFBTG-R引子進行聚合酶鏈鎖反應,得到789個鹼基對的最終產物。
上述核酸建構物經限制酵素BamHI作用後補齊(fill in),再以限制酵素XhoI作用後結合入pZαGFP質體(SEQ ID NO:6),經限制酵素AgeI作用後補齊,再以XhoI作用之切位形成pZαLFBG質體(SEQ ID NO:7),建構成的表現載體(pZαLFBG質體)如圖一所示,而此表現載體包含之核酸建構物如圖二所示。
pZαLFBG質體啟動子選用α肌動蛋白1a啟動子(α-actin 1a promoter),其基因能於肌肉細胞中表現抗病蛋白,並且終生肌肉皆能表現。
A. 斑馬魚之飼養:斑馬魚(zebrafish,Danio rerio)野生種個體成長至兩個月時,即馴養於28.5℃、光/暗週期為14/10小時的恆溫培養箱中,雄、雌成魚比例為2:3,約40尾,飼養於60公分×20公分×30公分的玻璃水族箱中,以適量的人工乾燥飼料與豐年蝦餵之。
B. 受精卵的收集:顯微注射前一天晚間11點,在恆溫箱即將進入暗週期前,將魚集中於寵物箱中,以隔離網隔離,於隔日早上光週期開始後,每隔15至20分鐘收集一次魚卵。每次約可注射30至40顆魚卵,一次實驗可注射250至300顆左右的卵。
C. 顯微注射實驗:欲注射之質體線性化後,利用二次水調整其濃度,再加入5倍PBS之酚紅(phenol red)稀釋混合至所需注射濃度25奈克/微升。將DNA溶液利用斑馬魚顯微注射器(zebrafish microinjector,Drummond代理)吸入毛細管中,注射之毛細管開口直徑為10到15微米,以毛細管插入一細胞時期之動物極中,並將DNA注入,所注入之DNA量約為0.0023微升。將注射過的受精卵以滅菌水清洗,之後放在28.5℃恆溫箱培養,之後依斑馬魚胚胎生長發育進行連續觀察。
A. 檢測與篩選:
(a)基因體DNA的抽取:將兩天大的胚胎約100尾加入500微升的裂解液(lysis buffer,包含10毫莫耳Ph值為8.0的TrisCl、10毫莫耳的EDTA、200毫莫耳的氯化鈉、0.5%的SDS)與200微克/毫升的蛋白酶K,置於55℃反應2小時,以酚/氯仿混合液萃取DNA後,加入2倍體積的無水酒精,在4℃下以每分鐘14,000轉的速度離心5分鐘,再以1毫升之70%酒精清洗沉澱的DNA,再在4℃下以每分鐘14,000轉的速度離心5分鐘,風乾沉澱物,溶於500微升的TE緩衝液,於-20℃下保存。
(b)聚合酶鏈鎖反應(PCR)檢驗:在0.5毫升微量離心管中依序加入1微升10毫莫耳的dNTPs、2.5微莫耳的10倍PCR緩衝液(含1.5毫莫耳的氯化鎂)、1微升5微莫耳的順向引子、1微升5微莫耳的反向引子、1微升的DNA模板(以1奈克之環狀質體作為對照組),以滅菌水補至25微升。混合均勻後加入1滴礦物油,置於PCR機器(DNA Thermal Cycle model 480,Perkin Elmer代理)。反應條件為:94℃ 10分鐘,加入1微升商業用蛋白酶Prozyme(2活性單位/微升)後,以94℃ 1分鐘的DNA解離、60℃ 1分鐘的DNA黏合、72℃ 2分鐘的DNA聚合的循環進行,共進行30個循環,最後以72℃ 10分鐘的條件完成反應,並取出10微升,以0.8%之膠體電泳檢視結果。
B. 遺傳:將顯微注射後的胚胎置於盛有少量水的圓形凹槽載玻片中,在顯微鏡(Leica MZ12)下觀察心臟發育的情形,5天後將存活下來的胚胎移至水族箱中飼養,以粉狀人工飼料(Artificial rotifer,OSI)1天餵食多次,約1週後換成豐年蝦1天餵食2次,約10週後可達性成熟。將親魚和野生種以1:1配對,保留可產生PCR檢驗陽性之第一子代F1的親魚,
並將形成品系的第一子代育成以供後續分析。
C. 產量標準化或產品規格化:將帶有外源基因轉殖之斑馬魚品系進行大型魚缸繁殖。以先前得到的轉殖品系量化後,再把異型合子品系(heterozygotic strain)繁育成同型合子品系(homozygotic strain),這樣生下來的子代就會百分之百地帶有轉殖基因。另外,要常與野生種互相交配,以免造成個體愈來愈不健康。
以含有α肌動蛋白a1啟動子、外源抗病蛋白LFB基因及綠色螢光蛋白GFP基因的線性化重組質體pZαLFBG注射斑馬魚胚胎,獲得穩定轉殖品系ZαL3。於發育72小時(受精後)在螢光顯微鏡下觀察斑馬魚體之綠螢光表現情形,其結果如圖三。由圖三可發現綠螢光標定於全身骨之骼肌(圖三A),如頭部肌肉(圖三B)、胸鰭與體壁肌肉(圖三C)、軀幹部肌肉(圖三D),且由綠螢光之高亮度可知螢光蛋白表現量高,顯示外源抗病蛋白可專一且高度表現於轉殖魚體的全身骨骼肌中。
取以α肌動蛋白a1啟動子作為驅動的斑馬魚穩定轉殖品系,於發育72小時(受精後)以不同的曝光時間拍攝帶有綠螢光之斑馬魚體,並在相同的培養及曝光條件下,拍攝以β肌動蛋白1啟動子作為驅動的斑馬魚穩定轉殖品系魚體,兩相比較之結果如圖四。結果顯示,無論在2.5秒(圖四B1及四B2)、4秒(圖四C1及四C2)或是8秒(圖四D1及四D2)的曝光時間下,以α肌動蛋白a1啟動子作為驅動的斑馬魚其螢光強度皆比以β肌動蛋白1啟動子作為驅動的斑馬魚高出許多,顯示以α肌動蛋白啟動子取代β肌動蛋白啟動子,確實可改良斑馬魚生物反應器之效率,使外源抗病蛋白的產量大幅提升。
圖一為本發明表現載體(以pZαLFBG質體為例)之建構流程成示意圖。建構完成之質體上包含哥薩克(Kozak)序列、α肌動蛋白啟動子、外源抗病蛋白序列、螢光蛋白序列及各種限制酵素切位。
圖二為本發明之核酸建構物,其上包含哥薩克(Kozak)序列、α肌動蛋白啟動子、外源抗病蛋白序列及螢光蛋白序列,亦可再包含一轉譯後酵素可切割位置(以胃蛋白酶切位為例)。
圖三為發育72小時後斑馬魚體之綠螢光表現情形。圖三A顯示螢光標定於全身之骨骼肌,如頭部肌肉(圖三B)、胸鰭與體壁肌肉(圖三C)及軀幹部肌肉(圖三D)。
圖四為不同曝光時間下,以α肌動蛋白a1啟動子驅動與以β肌動蛋白1啟動子驅動下,螢光亮度之比較。圖四A1、四B1、四C1與四D1係為以α肌動蛋白a1啟動子驅動,圖四A2、四B2、四C2與四D2係為以β肌動蛋白1啟動子驅動,圖四A1與四A2為明視野。結果顯示,無論是曝光2.5秒(圖四B1及四B2)、4秒(圖四C1及四C2)或是8秒(圖四D1及四D2),以α肌動蛋白a1啟動子驅動者螢光強度皆高出以β肌動蛋白1啟動子驅動者許多。
<110> 國立台灣大學
<120> 一種提昇生物反應器生產抗病有用蛋白產量之方法/METHOD FOR ENHANCING PRODUCTION OF DISEASE-RESISTANT USAGE PROTEINS USING BIOREACTORS
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Claims (11)
- 一種提昇生物反應器生產抗病蛋白產量之方法,其包含下列步驟:(a)構築一質體,其包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列、一蛋白酶切割位之核酸序列以及一α肌動蛋白啟動子;(b)將構築之質體線性化後轉殖入選定品系之斑馬魚胚胎中;(c)馴養魚隻,使之成長、交配及產卵;以及(d)收集魚卵、魚胚或成魚,即可得到富含抗病蛋白之生物體;其中該外源抗病蛋白之核酸序列係為乳鐵蛋白(lactoferrin)核酸序列。
- 根據申請專利範圍第1項之方法,其中該乳鐵蛋白之核酸序列可為其片段,即乳鐵蛋白素之(lactoferricin)核酸序列。
- 根據申請專利範圍第1項之方法,其中該蛋白酶切割位係為胃蛋白酶切割位。
- 根據申請專利範圍第1項之方法,其中該螢光蛋白核酸序列係為綠色、紅色、黃色、藍色、靛色或其他顏色螢光蛋白核酸序列。
- 根據申請專利範圍第1項之方法,其中該外源抗病蛋白係專一表現於骨骼肌中。
- 一種核酸建構物,其包含一外源抗病蛋白之核酸序列、一與其連接之螢光蛋白核酸序列及一α肌動蛋白啟動子,其由5’端至3’端之先後排列順序依序為α肌動蛋白啟動子、外源抗病蛋白核酸序列、螢光蛋白核酸序列。
- 根據申請專利範圍第6項之核酸建構物,其中該外源抗病蛋白核酸序列係為乳鐵蛋白素(lactoferricin)核酸序列。
- 根據申請專利範圍第7項之核酸建構物,其中該螢光蛋白核酸序列係為綠色、紅色、黃色、藍色、靛色或其他顏色螢光蛋白核酸序列。
- 根據申請專利範圍第7項之核酸建構物,其中該核酸建構物另可包含一蛋白酶切割位之核酸序列。
- 根據申請專利範圍第9項之核酸建構物,其中該蛋白酶切割位係為胃蛋白酶切割位。
- 一種表現載體,其包含有一如申請專利範圍第7至11項中任何一項的核酸建構物。
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