TW201732041A - 經修飾之初乳蛋白質及其應用 - Google Patents
經修飾之初乳蛋白質及其應用 Download PDFInfo
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Abstract
一種經修飾之初乳蛋白質，其胺基酸序列係如SEQ ID NO：1所示之胺基酸序列，為自如SEQ ID NO：2所示之野生型初乳蛋白質之胺基酸序列之第33位置的異白胺酸被丙胺酸取代，第101位置的谷胺酸被半胱胺酸取代，以及第175位置的精胺酸被半胱胺酸取代而產生。
Description
本發明相關於一種初乳蛋白，特別是相關於一種經修飾之初乳蛋白質。
抗體，亦稱免疫球蛋白，係為一種主要由漿細胞分泌，並可被免疫系統用來識別以及中和外來物質，例如細菌或病毒等病原體的蛋白質。抗體包括IgA、IgD、IgE、IgG及IgM，其中IgA可在母乳、唾液、眼淚以及支氣管中的黏液被發現，對於作為身體抵禦外來病原體之第一道防線的黏膜免疫十分重要。詳細而言，許多病原體可通過接觸於呼吸道、腸道及生殖泌尿道的黏膜表面而感染宿主，而於黏膜的分泌性抗體IgA可透過與病原體的多個抗原決定位（epitope）結合，使得病原體無法與黏膜細胞結合而感染宿主。
畜產業於農業生產中扮演重要的地位，其中養豬育成不佳之主要因素與豬之高死亡率有關。一般而言，絕對性病原普遍被認為是豬隻疾病之主因，然而，根據中興大學獸醫病理學系之血清學調查結果顯示，高病原性的疾病，例如豬瘟或假性狂犬病並無明顯爆發之現象，但相對一些病原性較低之病原體，例如支原體屬（mycoplasma）、巴斯德氏菌屬（pasteurella）及沙門氏菌屬（salmonella），其於單獨感染時可能無明顯病害，但在豬隻的抗病能力低下加上複合多種病原之原發及繼發感染，會使病害加成以致死亡。也就是說，這樣的低病原性病原體對豬隻均數低下之影響甚大。
雌性哺乳動物於產後2-3天內所初分泌的乳汁稱之為初乳（colostrum），其後再分泌的乳汁則被稱為常乳。初乳中含有五種免疫球蛋白，分別是IgA、IgD、IgE、IgG及IgM，其中以IgG的含量最高，其他免疫球蛋白含量都較低。該些免疫球蛋白對於病毒、細菌、寄生蟲及酵母菌等病原體皆有良好的防禦作用。
然而，目前於初乳中，除了乳鐵蛋白已被純化、利用及培育基因轉殖動物外，其餘有益成分則鮮少被有效分離及使用。且，由於初乳的分泌時程過短，以及乳中的蛋白不穩定、收集不易及保存困難等因素，使得初乳效益雖多，但於實際應用層面上尚存在諸多困難。
因此，本發明的目的即在提供一種經修飾之初乳蛋白質，不僅可提高其於體外之穩定性，還具有預防或抵禦外來病原體之功效。
本發明為解決習知技術之問題所採用之技術手段係提供一種經修飾之初乳蛋白質，其胺基酸序列係如SEQ ID NO：1所示之序列，該經修飾之初乳蛋白質為自如SEQ ID NO：2所示之野生型初乳蛋白質之胺基酸序列之第33位置的異白胺酸被丙胺酸取代，第101位置的谷胺酸被半胱胺酸取代，以及第175位置的精胺酸被半胱胺酸取代而產生。
在本發明的一實施例中係提供一種編碼上述之經修飾之初乳蛋白質之胺基酸序列的DNA，具有SEQ ID NO：3所示之鹼基序列。
在本發明的一實施例中係提供一種口服劑型，包含上述之經修飾之初乳蛋白質。
在本發明的一實施例中係提供一種動物飼料組成物，包含上述之經修飾之初乳蛋白質。
在本發明的一實施例中係提供一種動物飼料組成物，該經修飾之初乳蛋白質係佔該動物飼料組成物0.01 wt%-0.02 wt%。
在本發明的一實施例中係提供一種醫藥組成物，包含一醫藥載劑、疫苗佐劑以及上述之經修飾之初乳蛋白質。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備增加一動物的免疫的飼料的應用。
在本發明的一實施例中係提供一種應用，應用中係透過增加該動物的免疫球蛋白IgA生成而增加該動物的免疫。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備投遞於一動物的飼料的應用。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備投遞於一動物的醫藥組成物的應用。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的飼料的應用。
本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的飼料的應用，該疾病包括豬繁殖和呼吸障礙綜合症（porcine reproductive and respiratory syndrome, PRRS）、口蹄疫、豬流行性下痢病毒（porcine epidemic diarrhea, PED）及禽流感。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的醫藥組成物的應用。
在本發明的一實施例中係提供一種如上述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的醫藥組成物的應用，該疾病包括豬繁殖和呼吸障礙綜合症（porcine reproductive and respiratory syndrome, PRRS）、口蹄疫、豬流行性下痢病毒（porcine epidemic diarrhea, PED）、禽流感及人類之流行性感冒。
經由本發明所採用之技術手段，本發明之經修飾之初乳蛋白質之三級結構相較於野生型之初乳蛋白更為穩定。且，經該經修飾之初乳蛋白質可透過提高免疫球蛋白IgA的生成而增強黏膜免疫，進而預防或治療非特定病原之疾病之感染。
以下根據第1圖至第8圖，而說明本發明的實施方式。該說明並非為限制本發明的實施方式，而為本發明之實施例的一種。
依據本發明的一實施例的一經修飾之初乳蛋白質，其胺基酸序列係如SEQ ID NO：1所示之胺基酸序列，該經修飾之初乳蛋白質為自如SEQ ID NO：2所示之野生型初乳蛋白質之胺基酸序列之第33位置的異白胺酸被丙胺酸取代，第101位置的谷胺酸被半胱胺酸取代，以及第175位置的精胺酸被半胱胺酸取代而產生。而該經修飾之初乳蛋白質之胺基酸序列係由具有SEQ ID NO：3所示之鹼基序列的DNA予以編碼。
詳細而言，本發明之經修飾之初乳蛋白質係為純化結合於細菌之細胞壁上的胜肽聚醣的蛋白質，且該蛋白質經修飾其序列而得，並依其特性命名為病原辨識蛋白（Pathological Recognition Protein, PRP）。
進一步而言，本發明的經修飾之初乳蛋白質可製成一口服劑型，例如固體、半固體、或液體的口服劑型。詳細而言，該固體的口服劑型包括一藥片、一多顆粒、一粉末、或一膠囊。
進一步而言，本發明的經修飾之初乳蛋白質可經與飼料混合後而製成一動物飼料組成物。且，該經修飾之初乳蛋白質係佔該動物飼料組成物0.01wt%-0.02wt%。當然，本發明不以此為限。在其它實施例中，該經修飾之初乳蛋白質佔該動物飼料組成物之比例可根據情況而有所不同。
進一步而言，本發明的經修飾之初乳蛋白質可用於製備增加一動物的免疫的飼料的應用，其係透過增加該動物的免疫球蛋白IgA生成而達到。詳細而言，該動物可為哺乳動物，例如豬或牛。
進一步而言，本發明的經修飾之初乳蛋白質可用於製備投遞於一動物的醫藥組成物的應用，並包括醫藥載劑及疫苗佐劑。
進一步而言，本發明的經修飾之初乳蛋白質可用於製備預防或治療引發黏膜免疫之疾病的飼料或醫藥組成物，其中該疾病包括豬繁殖和呼吸障礙綜合症（porcine reproductive and respiratory syndrome, PRRS）、口蹄疫、豬流行性下痢病毒（porcine epidemic diarrhea, PED）及禽流感。
概括而言，在本實施例中，係以豬（在其它實施例中，亦可採用其他哺乳動物，例如：牛）的初乳為材料，透過鈉十二烷基的硫酸鹽聚丙烯酰胺凝膠電泳法（SDS-PAGE）分析，以純化可能會結合於細菌之細胞壁上的胜肽聚醣的初乳蛋白質純化，並經液相層析串聯式質譜儀（LC/MS/MS）鑑定分析及基因庫比對後，而初步了解該胜肽聚醣結合蛋白之身分及特性，而將該胜肽聚醣結合蛋白命名為病原辨識蛋白（Pathological Recognition Protein, PRP）。待取得該病原辨識蛋白之胺基酸序列後，係選殖出豬的病原辨識蛋白基因，並且構築於酵母菌表現系統。並將該體外表現的病原辨識蛋白陸續完成表現量評估、發酵環境測試、小鼠管餵後腸道菌相檢驗、活體大腸桿菌及沙門氏菌抑制試驗等確定其蛋白質活性，如下所示。
純化蛋白質：
革蘭氏陽性介質（GEM）顆粒製備：無菌沾取Lactoccous lactis菌液，接種於MRS（DifcoTM Lactobacilli MRS Broth）培養基上並篩選出單一菌落，挑選單一菌株至250 ml的MRS培養液，於厭氣環境37℃培養18小時。將培養完畢的菌液分裝於50 ml的離心管中，以13000 xg離心10分鐘，去除上清液，以原體積1/2的ddH2
O懸浮菌塊，以13000 xg離心10分鐘後，去除上清液，加入原體積1/5的acid solution（0.6M TCA, pH=1），鬆蓋，置於沸水隔水加熱30分鐘，再以13000 xg 離心10分鐘，去除上清液，並以原體積1/2的PBS懸浮菌塊，上述步驟重複3次，再以13000 xg 離心10分鐘後，去除上清液。最後分別以原體積1/10的PBS回溶菌塊，使用細胞計數器計算評估每ml中所含之GEM顆粒數並保存於-80℃備用。
乳清製備：將所取得初乳分裝於50 ml 之離心管，以10000 xg、4℃條件，離心30分鐘後留取下層乳汁，並移至另一50 ml 之離心管，再依體積加入100%的醋酸並使最終醋酸濃度為1%，將其置於37℃恆溫箱10分鐘，進行酸化作用。其後以其1/10體積的1M醋酸鹽中和乳汁，最後以10000 xg、4℃條件，離心10分鐘後吸取上清液，此即為乳清。將製備好的乳清採Bradford法進行蛋白質定量，保存於-20℃備用。
GEM顆粒與乳清之結合試驗：以100μl的GEM顆粒（約5.6×108
GEM）與乳清（約7mg）混和後，置於震盪器上在室溫下震盪30分鐘，再以13000 xg離心10分鐘，去除上清液。而沉澱物則以1 ml的PBS buffer懸浮，並以13000 xg離心10分鐘。上述步驟重複3次後，再以1 ml的elution buffer（含1 M NaCl）懸浮，最後以13000 xg離心10分鐘，去除上清液後用20 μl的PBS buffer回溶，再加入等體積的2倍sample buffer混合均勻後，置於乾浴槽以95℃加熱10分鐘，再以13000 xg離心10分鐘，吸取上層溶液，進行蛋白質膠體電泳分析。
免疫小鼠：將2.2×109
GEM顆粒與初乳乳清（約25 mg）混和震盪均勻後，以13000 xg離心10分鐘去除上清液，沉澱物則以1 ml的PBS buffer懸浮，並以13000 xg離心10分鐘，上述步驟重複3次，再以1 ml的1 M NaCl 懸浮，最後以13000 xg離心10分鐘，去除上清液後用50 μl的PBS buffer回溶，加入等體積的2倍蛋白質Sample buffer混合均勻，置於乾浴槽以95℃加熱10分鐘，再以13000 xg離心10分鐘，吸取上層部份，以PBS buffer定量至100 μl，加入等量的完全佐劑（Freund’s Adjuvant, Complete），於4℃環境中震盪混和12 hr，作為首次免疫小鼠之針劑。之後佐劑則改混和以不完全者（Freund’s Adjuvant, Incomplete）乳化抗原蛋白。免疫試驗中抗原注射以三週為一免疫週期，第一週以混和完全佐劑之抗原蛋白進行小鼠腹腔免疫針劑注射，每週以穿刺片採集小鼠臉頰血，製備血清及保存於-20℃備用；至第三次免疫，取得小鼠血清作為西方墨點法之一次抗體，偵測初乳乳清中胜肽聚醣親合蛋白，若比較於前二次免疫血清之抗體濃度有明顯上升，則再給予第四劑混和不完全佐劑之抗原蛋白針劑注射，並於隔週進行小鼠之全血採集。
蛋白質西方墨點試驗：聚偏二氟乙烯（PVDF membrane）以無水甲醇潤濕15分鐘，再浸於transfer buffer中備用。將轉漬槽轉印夾依序將吸水棉、濾紙、欲轉印之蛋白質電泳膠片、PVDF membrane、濾紙及吸水棉疊置，中間避免氣泡產生。槽內以transfer buffer注滿，外以冰浴降溫，轉印電壓為100伏特時間1小時，轉印完成後將PVDF membrane浸泡於含有5%（w/v）脫脂奶粉的TBS buffer中，於4℃搖晃至少二小時，再以TBS buffer浸洗，每次五分鐘，共六次，以上述免疫試驗所得的小鼠免疫初乳乳清中胜肽聚醣親合蛋白之抗體為探針，使用時以TBS buffer稀釋1000倍作為一次抗體，與PVDF membrane於室溫下搖晃反應一小時，同樣再以TBS buffer浸洗，每次五分鐘，共六次，將帶有Alkaline Phosphatase之抗體做為二次抗體，以TBS buffer稀釋1500倍作為二次抗體，與PVDF membrane於室溫下搖晃反應一小時，再以TBS buffer浸洗，每次五分鐘，共六次，最後加入BCIP/NBT液態受質呈色，當呈色效果適當時，儘快以二次清洗終止呈色反應。
基因取得：
液相層析串連質譜儀：LC-MS/MS為液相層析儀串連兩組質譜儀進行樣本分析。原理為利用液相層析儀之高分析能力，將含有許多胜肽片段之混和物加以分離，再利用質譜儀中離子原將樣本氣化為離子態，產生分子大小及電荷不同之胜肽，即進入第一階段質量分析器（mass analyzer），利用電子或碰撞氣體等外力撞擊欲分析之離子，以產生更小的離子碎片，再以第二階段質量分析器進行樣本碎片離子之質荷比（mass to charge ratio；m/z）測量，便可利用其所帶電荷推算該分析物之質量。藉由液相層析儀分離出之胜肽，經過兩次離子化及裂解，經過比對分析，便可獲得胜肽之胺基酸序列，再利用如Mascot Analysis（Matrix Science, London, UK）等生物資訊搜尋服務平台軟體，進行DNA序列資料庫比對分析以獲得相對應之基因。
豬病原辨識蛋白的選殖與活性分析：由LC-MS/MS所獲的部分胺基酸序列將用為設計degenerate primers的依據，此引子既與Olgo-d（T）作為掉取豬乳腺cDNA中病原辨識蛋白基因的工具，待RT-PCR後顯示於凝膠電泳上所有條帶將逐一選殖於TA-vector，經核酸定序及生物資訊比對後既可獲得豬病原辨識蛋白完整基因，此基因序列如SEQ ID NO：3所示。選殖及修改序列後的豬病原辨識蛋白先轉構築於大腸桿菌表現系統，誘發菌體表現後並加以純化，純化後的病原辨識蛋白將測試原與GEM顆粒結合的能力是否存在及穩定，結合後以Western blotting進行分析。
酵母菌表現載體構築：為避免原使用的大腸桿菌表現系統汙染飼養環境，因此改用飼料中既有使用的酵母菌為載體表現病原辨識蛋白，選殖、修改及定序後的豬病原辨識蛋白基因既構築於 pYES2.
1V5-His TOPO vector（Invitrogen），此載體為酵母菌表現型者，因此構築後轉染於酵母菌INVSc1株中，惟此系統原有誘發表現的機轉以被修改為對溫度及營養性者，在特定溫度及營養物質存在時方啟動表現病原辨識蛋白，但在尚未申請專利前對於培養條件的資訊束無法詳細告知。
小鼠腸道微生物試驗：腸道菌相的觀察：小鼠以管餵方式給予體重的1/100病原辨識蛋白，兩天管餵一次歷時六天，對照組將僅餵與同體積之二次滅菌水，實驗結束時犧牲動物取小腸（胃下1.5~2.5 cm）進行分析，小鼠腸道內容物將以滅菌過的PBS稀釋成適當倍數，再以培養基培養，24hr後計算其菌落數，菌相以菌落之對數値（log cfu）表示之。另以細菌快速鑑定方法（API 20E）鑑定腸道桿菌科細菌及其他革蘭氏陰性菌，其結果將以下列表1判定之。
【表1】
發酵槽發酵條件測試：經構築染後的酵母菌係以連續式發酵槽（Winpact Bioreactor and Fermentor）培養，酵母菌先行活菌及懸浮後，按OD值稀釋及更換培養液後在誘發環境下培養八小時，期間監控溫度、轉速、pH值及溶氧量等變因使維持在恆定範圍。而每批培養後的酵母菌將取樣係以western blotting分析病原辨識蛋白的表現。將發酵八小時之發酵液進行酵母菌分離，約一公升發酵液可得約九~十克之重組酵母菌，並將該重組酵母菌存放於乾燥環境備用。
初乳中結合菌膜蛋白的純化與蛋白的定性：異性蛋白結合至微生物細胞膜或細胞壁，在固著方式大致上可分為五種：(1)透過transmembrane protein上疏水性的transmembrane domain而固著於微生物細胞膜上，(2)藉脂蛋白N端上的半胱氨酸胺經乙醯化共價結合於細胞膜的長鏈脂肪酸，(3)透過LPXTG motif anchor提供蛋白質暫時停留於細胞膜上，經sortase作用將LPXTG motif中的蘇氨酸、甘氨酸與胜肽聚醣共價結合，(4)細胞壁與細胞壁結合蛋白之非共價結合；如存在於各種細菌的lysin motif(LysM)，及(5)表層蛋白結合。在實驗所製備GEM 顆粒經填塞在管柱及灌流初乳後，以SDS-PAGE分析會與菌膜結合的未知蛋白，試驗中發現初乳中會與菌膜結的蛋白應不只一個，過程中合計分離及胺基酸定序者有七個（第1圖，C1~C7），其中之一為已知的乳鐵蛋白，而C7則經NCBI序列比對後似為胜肽聚醣辨識蛋白家族一員（C7: RecName: Peptidoglycan recognition protein; Flags: Precursor，Nominal mass (Mr): 21024; Calculated pI value: 9.62，Variable modifications: Carbamidomethyl (C)，Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P，Sequence Coverage: 11%）。
由於市面上缺乏豬PGRP抗體，因此實驗先以純化後的C7蛋白並注射於小鼠腹部，再以誘發免疫的腹水檢視豬初乳與常乳中該蛋白的表現模式，結果如下圖，豬的乳清在此我們的研究中也被發現具有PGRP，並且豬初乳的乳清（第2圖，分娩後2–4天，標示*為PGRP 約17 kDa）中PGRP含量明顯高於豬常乳乳清的PGRP含量。然而，如第3圖所示，實驗過程也發現該蛋白極不穩定，同樣樣品在低溫（-20℃）保存兩天後該蛋白旋即消失，此種不穩定性會使得該蛋白無法實際應用於產業。因此，於後續實施例中，係對其蛋白質進行修飾，使該蛋白三級結構更趨穩定，並依其特性命名為病原辨識蛋白（pathological recognition protein, PRP），即本發明之經修飾之初乳蛋白質。
豬病原辨識蛋白基因選殖、酵母菌表現及產量監測：根據以往開發商用重組蛋白的前例，雖實驗室可以研發具市場價值的重組蛋白，但在蛋白表現量、蛋白穩定度及生產成本上是較難克服的問題，有鑒於此，豬病原辨識蛋白基因將轉殖於酵母菌表現系統，雖培養條件較難設定，但由於酵母菌在商用上有諸多優點，因此實驗還是決定以酵母菌為表現豬病原辨識蛋白的系統（第4圖）。經由多次更換培養培養基配方、溫度、溶氧率及發酵時間後，現已可穩定表現出豬病原辨識蛋白，月產量約可供配製280噸的哺乳豬飼料，每次發酵饋菌時旋即收取樣品1ml，待發酵結束時再取樣一次，兩次樣品將以Western blotting確定病原辨識蛋白的表現與產量。第5圖為其中一次蛋白表現的監測結果，圖中不同時間所收集的發酵樣品在抗體呈現下，箭頭（約17 kDa 處）指出在誘導發酵八小時後可獲取最佳的表現量，如此的監測將於每批發酵後立即進行，用以確認發酵條件及菌株是否在最佳狀態。而發酵後所收集的酵母菌將離心去懸浮液後，固形物將立即冷凍並於-50℃環境乾燥，乾燥後避免潮濕備用，待配製飼料前先行與飼料基質預混均勻後，再於其他成分混合。
同時參照第6-8圖，ICR（Institute for Cancer Research）品系小鼠以管餵方式給予體重的1/100病原辨識蛋白，兩天管餵一次歷時六天；而對照組將僅餵與同體積之二次滅菌水。實驗結束時犧牲動物取小腸（胃下1.5~2.5 cm）進行分析。每次實驗時對照組與受測組各為25隻，試驗中若因管餵導致動物死亡則將剔除該數據，在總菌數上經餵予豬病原辨識蛋白者約較對照組下降15-50%（藍色曲線為給予PRP的測試組，當時間拉長時PRP約可抑制50%的菌數），而菌相的鑑別上已知減少的菌以格蘭氏陽性菌為主。此外，由於病原辨識蛋白具有黏著於菌膜的特性，因此試驗將酵母菌表現後純化的病原辨識蛋白與大腸桿菌混合及更換培養液兩次後，以電子顯微鏡觀察菌膜上是否有病原辨識蛋白的結合，圈選處為可明顯看見蛋白結合處。
以下為本發明之經修飾之初乳蛋白質與動物飼量混合後，並投予小豬之試驗：
同期比較：16頭4週齡小豬，8頭一組飼養於相鄰二欄豬舍，抽血檢測PRRS抗體力價(IgG)，並於餵飼添加PRP0.02%飼糧4週後（8週齡），檢測血液中PRRS抗體力價(IgG)。
結果：表二中顯示豬隻於試驗前無論試驗組或對照組血液中免疫球蛋白IgG皆呈現陰性反應，然而8週齡時添加PRP處理組仍呈陰性，而對照組全部呈現曾被感染的陽性反應。由此證明PRP可輔助免疫球蛋白IgA的生成，以將於豬場中欲穿透豬隻黏膜進入體內之PRRS病毒於黏膜中和，因而可減少PRRS病毒穿透黏膜，而刺激淋巴免疫系統產生免疫球蛋白IgG。 【表2】 4週齡小豬血液中免疫球蛋白IgG
註.抗體力價0.4以下為陰性 【表3】 8週齡小豬血液中免疫球蛋白IgG
註.抗體力價0.4以下為陰性
第1圖為顯示經分離與菌膜結合的初乳蛋白的結果； 第2圖為顯示於豬的乳清中的PGRP的結果； 第3圖為顯示根據本發明的實施例的經修飾之初乳蛋白質以及野生型初乳蛋白質經於培養液中添加50 μg /ml放線菌酮（cycloheximide, CHX）處理的折線圖； 第4圖為顯示以酵母菌表現豬病原辨識蛋白的系統； 第5圖為顯示根據本發明的實施例的經修飾之初乳蛋白質的西方墨點圖； 第6圖為顯示根據本發明的實施例的經修飾之初乳蛋白質與大腸桿菌混合時的電子顯微鏡圖； 第7圖為顯示根據本發明的實施例的經修飾之初乳蛋白質於管餵小鼠後腸道總菌數的折線圖； 第8圖為顯示根據本發明的實施例的經修飾之初乳蛋白質於管餵小鼠後的腸道免疫染色圖。
SEQUENCE LISTING ＜110＞ 虹廣生物 ＜120＞ 經修飾之初乳蛋白質及其應用 ＜130＞ 2016 ＜160＞ 3 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 196 ＜212＞ PRT ＜213＞ human ＜400＞ 1 Met Ser Arg Arg Ser Met Leu Leu Ala Trp Ala Leu Pro Ser Leu Leu 1 5 10 15 Arg Leu Gly Ala Ala Gln Glu Thr Glu Asp Pro Ala Cys Cys Ser Pro 20 25 30 Ala Val Pro Arg Asn Glu Trp Lys Ala Leu Ala Ser Glu Cys Ala Gln 35 40 45 His Leu Ser Leu Pro Leu Arg Tyr Val Val Val Ser His Thr Ala Gly 50 55 60 Ser Ser Cys Asn Thr Pro Ala Ser Cys Gln Gln Gln Ala Arg Asn Val 65 70 75 80 Gln His Tyr His Met Lys Thr Leu Gly Trp Cys Asp Val Gly Tyr Asn 85 90 95 Phe Leu Ile Gly Cys Asp Gly Leu Val Tyr Glu Gly Arg Gly Trp Asn 100 105 110 Phe Thr Gly Ala His Ser Gly His Leu Trp Asn Pro Met Ser Ile Gly 115 120 125 Ile Ser Phe Met Gly Asn Tyr Met Asp Arg Val Pro Thr Pro Gln Ala 130 135 140 Ile Arg Ala Ala Gln Gly Leu Leu Ala Cys Gly Val Ala Gln Gly Ala 145 150 155 160 Leu Arg Ser Asn Tyr Val Leu Lys Gly His Arg Asp Val Gln Cys Thr 165 170 175 Leu Ser Pro Gly Asn Gln Leu Tyr His Leu Ile Gln Asn Trp Pro His 180 185 190 Tyr Arg Ser Pro 195 ＜210＞ 2 ＜211＞ 196 ＜212＞ PRT ＜213＞ human ＜400＞ 2 Met Ser Arg Arg Ser Met Leu Leu Ala Trp Ala Leu Pro Ser Leu Leu 1 5 10 15 Arg Leu Gly Ala Ala Gln Glu Thr Glu Asp Pro Ala Cys Cys Ser Pro 20 25 30 Ile Val Pro Arg Asn Glu Trp Lys Ala Leu Ala Ser Glu Cys Ala Gln 35 40 45 His Leu Ser Leu Pro Leu Arg Tyr Val Val Val Ser His Thr Ala Gly 50 55 60 Ser Ser Cys Asn Thr Pro Ala Ser Cys Gln Gln Gln Ala Arg Asn Val 65 70 75 80 Gln His Tyr His Met Lys Thr Leu Gly Trp Cys Asp Val Gly Tyr Asn 85 90 95 Phe Leu Ile Gly Glu Asp Gly Leu Val Tyr Glu Gly Arg Gly Trp Asn 100 105 110 Phe Thr Gly Ala His Ser Gly His Leu Trp Asn Pro Met Ser Ile Gly 115 120 125 Ile Ser Phe Met Gly Asn Tyr Met Asp Arg Val Pro Thr Pro Gln Ala 130 135 140 Ile Arg Ala Ala Gln Gly Leu Leu Ala Cys Gly Val Ala Gln Gly Ala 145 150 155 160 Leu Arg Ser Asn Tyr Val Leu Lys Gly His Arg Asp Val Gln Arg Thr 165 170 175 Leu Ser Pro Gly Asn Gln Leu Tyr His Leu Ile Gln Asn Trp Pro His 180 185 190 Tyr Arg Ser Pro 195 ＜210＞ 3 ＜211＞ 590 ＜212＞ DNA ＜213＞ human ＜400＞ 3 atgtcccgcc gctctatgct gcttgcctgg gctctcccca gcctccttcg actcggagcg 60 gctcaggaga cagaagaccc ggcctgctgc agccccgccg tgccccggaa cgagtggaag 120 gccctggcat cagagtgcgc ccagcacctg agcctgccct tacgctatgt ggtggtatcg 180 cacacggcgg gcagcagctg caacaccccc gcctcgtgcc agcagcaggc ccggaatgtg 240 cagcactacc acatgaagac actgggctgg tgcgacgtgg gctacaactt cctgattgga 300 tgcgacgggc tcgtatacga gggccgtggc tggaacttca cgggtgccca ctcaggtcac 360 ttatggaacc ccatgtccat tggcatcagc ttcatgggca actacatgga tcgggtgccc 420 acaccccagg ccatccgggc agcccagggt ctactggcct gcggtgtggc tcagggagcc 480 ctgaggtcca actatgtgct caaaggacac cgggatgtgc agtgcacact ctctccaggc 540 aaccagctct accacctcat ccagaattgg ccacactacc gctccccctg 590
Claims (14)
- 一種經修飾之初乳蛋白質，其胺基酸序列係如SEQ ID NO：1所示之胺基酸序列，為自如SEQ ID NO：2所示之野生型初乳蛋白質之胺基酸序列之第33位置的異白胺酸被丙胺酸取代，第101位置的谷胺酸被半胱胺酸取代，以及第175位置的精胺酸被半胱胺酸取代而產生。
- 一種編碼如請求項1所述之經修飾之初乳蛋白質之胺基酸序列的DNA，具有SEQ ID NO：3所示之鹼基序列
- 一種口服劑型，包含如請求項1所述之經修飾之初乳蛋白質。
- 一種動物飼料組成物，包含如請求項1所述之經修飾之初乳蛋白質。
- 如請求項3所述之動物飼料組成物，其中該經修飾之初乳蛋白質係佔該動物飼料組成物0.01 wt%-0.02 wt%。
- 一種醫藥組成物，包含： 一醫藥載劑及疫苗佐劑；以及 如請求項1所述之經修飾之初乳蛋白質。
- 一種如請求項1所述的經修飾之初乳蛋白質用於製備增加一動物的免疫的飼料的應用。
- 如請求項7所述的應用，其中係透過增加該動物的免疫球蛋白IgA生成而增加該動物的免疫。
- 一種如請求項1所述的經修飾之初乳蛋白質用於製備投遞於一動物的飼料的應用。
- 一種如請求項1所述的經修飾之初乳蛋白質用於製備投遞於一動物的醫藥組成物的應用。
- 一種如請求項1所述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的飼料的應用。
- 如請求項11所述的應用，其中該疾病包括豬繁殖和呼吸障礙綜合症（porcine reproductive and respiratory syndrome, PRRS）、口蹄疫、豬流行性下痢病毒（porcine epidemic diarrhea, PED）及禽流感。
- 一種如請求項1所述的經修飾之初乳蛋白質用於製備預防或治療引發黏膜免疫之疾病的醫藥組成物的應用。
- 如請求項13所述的應用，其中該疾病包括豬繁殖和呼吸障礙綜合症（porcine reproductive and respiratory syndrome, PRRS）、口蹄疫、豬流行性下痢病毒（porcine epidemic diarrhea, PED）、禽流感及人類之流行性感冒。
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