TW201639874A - 豬胸膜肺炎放線桿菌重組毒素蛋白及其應用 - Google Patents
豬胸膜肺炎放線桿菌重組毒素蛋白及其應用 Download PDFInfo
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Abstract
本發明係關於一種豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
)重組毒素蛋白，包括至少一個豬胸膜肺炎放線桿菌毒素蛋白的抗原決定位；當該抗原決定位為複數個時，各抗原決定位之間可由連接子連接。該重組蛋白進一步可包含至少一個單元的補體裂解片段C3d的胺基酸序列，且該抗原決定位與該補體裂解片段C3d的胺基酸序列可以連接子連接。本發明並關於一種編碼如上述豬胸膜肺炎放線桿菌毒素蛋白的核苷酸序列，以及一種含有上述豬胸膜肺炎放線桿菌毒素蛋白的豬放線桿菌胸膜肺炎免疫組合物。
Description
本發明係關於豬胸膜肺炎放線桿菌重組蛋白的製備與應用，特別是關於使用豬胸膜肺炎放線桿菌重組毒素蛋白以預防豬隻感染豬胸膜肺炎放線桿菌。
豬放線桿菌胸膜肺炎(porcine pleuropneumonia)是一種由豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
, App.)所造成的高感染性豬隻呼吸疾病，其臨床症狀包括發燒、咳嗽、嘔吐、呼吸困難、抑鬱。感染本病後可能會引起急性肺炎造成驟死，或是慢性感染造成無症狀出現的帶原者。各種豬齡的豬隻均會感染豬放線桿菌胸膜肺炎，尤其是6月齡以下的小豬發病率和死亡率最高。
豬胸膜肺炎放線桿菌屬於革蘭氏陰性桿菌，具有二種生物型，生物型1需要b-菸鹼醯胺腺二核苷酸(b-nicotinamide adenine dinucleotide, b-NAD)，生物型2則不需要b-NAD。此外，根據莢膜多醣的差異，已有15種血清型的豬胸膜肺炎放線桿菌被確認，且所有的血清型皆具有致病能力。已知的毒力因子包括莢膜、脂多醣、外膜蛋白，以及最重要的細胞毒素(Apx)。
Apx毒素屬於結構重複毒素家族(repeats in structural toxin, RTX)的成員。豬胸膜肺炎放線桿菌的15種血清型菌株共可產生四種不同的Apx毒素。每種血清型的菌株最多可產生3種Apx毒素。ApxI由1、5、9、10、11、14血清型菌株所分泌；ApxII除了第10、14血清型以外之其他血清型都可分泌；ApxIII由2、3、4、6、8、15血清型所分泌；ApxIV由所有的血清型所分泌，但只在感染時才會表現。
豬放線桿菌胸膜肺炎在全世界的養豬業造成嚴重的經濟損失。豬胸膜肺炎放線桿菌感染的治療包括使用如阿莫西林(amoxicillin)、氨芐青黴素(ampicillin)、頭孢噻呋(ceftiofur)、恩諾沙星(enrofloxacin)、泰妙菌素(tiamulin)、青黴素(penicillin)以及青黴素/鏈黴素(penicillin/streptomycin)等的抗生素。然而，因為對抗生素產生抗性的問題日益嚴重且消費者對食物安全的需求日益增加，以疫苗預防豬胸膜肺炎放線桿菌感染已變得重要。大部分市售豬胸膜肺炎放線桿菌疫苗為全菌疫苗，這些疫苗可降低豬隻死亡率，但卻無法預防最初的感染及帶原狀態的發生。因此，發展更有效的豬胸膜肺炎放線桿菌疫苗是重要的。
於一方面，本發明提供一種豬胸膜肺炎放線桿菌重組毒素蛋白(recombinant Apx toxins, re-Apx)，係以下列之式(I)表示： (A)m-(C3d片段)n； 式(I)； 其中每一個A代表一個獨立的豬胸膜肺炎放線桿菌毒素蛋白的抗原決定位； 其中每一個C3d片段代表一個獨立的補體裂解片段C3d的胺基酸序列，每一個C3d片段係各自獨立選自於SEQ ID NOs: 22、23、24及25所組成的群組； 其中m是代表從1至約30的整數；以及 其中n是代表從0至約10的整數。
於另一方面，本發明提供一種編碼該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)的核苷酸序列。
於另一方面，本發明提供一種豬放線桿菌胸膜肺炎免疫組合物，包含該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)以及一藥學上可接受的載劑。
於又一方面，本發明提供一種動物對抗豬放線桿菌胸膜肺炎的方法，包含使用有效量之上述免疫組合物以施予一動物，以增強該動物對抗豬放線桿菌胸膜肺炎之免疫力。
於再一方面，本發明提供一種抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗體，係藉由該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)所製備或衍生而得。
於另一方面，本發明提供一種豬放線桿菌胸膜肺炎的檢測套組。該檢測套組係用以偵測檢驗樣本中是否含有豬胸膜肺炎放線桿菌毒素蛋白(Apx)，或偵測檢驗樣本內是否含有抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗體。
本發明所提供的豬放線桿菌胸膜肺炎免疫組合物，與其他習用技術相互比較時，更具有下列之優點：
本發明所提供的豬放線桿菌胸膜肺炎免疫組合物含有一豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)。該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)包含至少一個豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗原決定位(epitopes)以及補體裂解片段C3d的部份胺基酸序列。該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)遠比豬胸膜肺炎放線桿菌毒素蛋白(Apx)的全長胺基酸序列短，在生物表現系統中較容易表達，且重組蛋白的產率較高，可降低製造疫苗之成本。
此外，本發明所提供的豬放線桿菌胸膜肺炎免疫組合物所含的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)具有補體裂解片段C3d的部份胺基酸序列，因此可以增加特異性免疫反應，經試驗結果顯示，本發明所提供的豬放線桿菌胸膜肺炎免疫組合物可提高動物對抗豬胸膜肺炎放線桿菌感染的免疫性，顯著增加受感染動物的存活率。
本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。
本發明提供一種豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)，包含至少一個豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗原決定位(epitopes)，以誘導動物產生抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗體，並可進一步含補體裂解片段C3d的全長或部份胺基酸序列，以增加特異性免疫反應。本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)係以下列之式(I)表示： (A)m
-(C3d片段)n
； 式(I)； 其中每一個A代表一個獨立的豬胸膜肺炎放線桿菌毒素蛋白的抗原決定位； 其中每一個C3d片段代表一個獨立的補體裂解片段C3d的胺基酸序列； 其中m是代表從1至約30的整數；以及 其中n是代表從0至約10的整數。
於一實施例中，該補體裂解片段C3d的全長胺基酸序列為小鼠補體裂解片段C3d的全長序列(mC3d)，具有如SEQ ID NO: 25所示的胺基酸序列。於一較佳實施例中，該補體裂解片段C3d的部份胺基酸序列為小鼠補體裂解片段C3d第211個胺基酸至第238個胺基酸片段序列(mC3d-p28)，具有如SEQ ID NO: 24所示的胺基酸序列。於另一實施例中，該補體裂解片段C3d的全長胺基酸序列為豬補體裂解片段C3d的全長序列(pC3d)，具有如SEQ ID NO: 23所示的胺基酸序列。於另一較佳實施例中，該補體裂解片段C3d的部份胺基酸序列為豬補體裂解片段C3d第201個胺基酸至第231個胺基酸片段序列(pC3d-p31)，具有如SEQ ID NO: 22所示的胺基酸序列。本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)具有0至10個單元重複的上述補體裂解片段C3d的全長或部份胺基酸序列，該補體裂解片段C3d的全長或部份胺基酸序列較佳為1至重複10個單元，更佳為重複4至8個單元。
於一實施例中，每一個A之間進一步以一個連接子連接，每一個連接子係各自獨立選自於Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36。
於一實施例中，每一個C3d片段之間進一步以一個連接子連接，每一個連接子係各自獨立選自於Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36。
於一實施例中，A與C3d片段之間進一步以一連接子連接，該連接子係選自於Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36。
於一實施例中，該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白I (ApxI)，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)，且每一個A係各自獨立選自於SEQ ID NOs: 37、4、39、40、41、42、43、44、45、46、47、48、49、50及51所示之胺基酸序列，該豬胸膜肺炎放線桿菌毒素蛋白I (ApxI)的抗原決定位可為1至約30個，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，分別以五段較長的抗原決定位胺基酸序列涵蓋部分上述豬胸膜肺炎放線桿菌毒素蛋白I (ApxI)的抗原決定位，該五段較長的抗原決定位胺基酸序列係分別如SEQ ID NOs: 2、3、4、5、6所示。於一較佳實施例中，本發明之豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)含有二個以上該較長的抗原決定位胺基酸序列，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，該豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)包含如SEQ ID NO: 7或8所示的胺基酸序列。
於一實施例中，該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白II (ApxII)，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)，且每一個A係各自獨立選自於SEQ ID NOs: 14、52、53、54、55、56、57、58、59、60、61、62、63、64、65、67及68所示之胺基酸序列，該豬胸膜肺炎放線桿菌毒素蛋白II (ApxII)的抗原決定位可為1至約30個，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，分別以五段較長的抗原決定位胺基酸序列涵蓋部分上述豬胸膜肺炎放線桿菌毒素蛋白II (ApxII)的抗原決定位，該五段較長的抗原決定位胺基酸序列係分別如SEQ ID NOs: 10、11、12、13、14所示。於一較佳實施例中，本發明之豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)含有二個以上該較長的抗原決定位胺基酸序列，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，該豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)包含如SEQ ID NO: 15或16所示的胺基酸序列。
於一實施例中，該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白III (ApxIII)，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)，且每一個A係各自獨立選自於SEQ ID NOs: 69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87及88所示之胺基酸序列，該豬胸膜肺炎放線桿菌毒素蛋白III (ApxIII)的抗原決定位可為1至約30個，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，分別以二段較長的抗原決定位胺基酸序列涵蓋部分上述豬胸膜肺炎放線桿菌毒素蛋白III (ApxIII)的抗原決定位，該二段較長的抗原決定位胺基酸序列係分別如SEQ ID NOs: 18、19所示。於一較佳實施例中，本發明之豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)含有該二個該較長的抗原決定位胺基酸序列，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，該豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)包含如SEQ ID NO: 20或21所示的胺基酸序列。
於一實施例中，該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白IV (ApxIV)，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)，且每一個A係各自獨立選自於SEQ ID NOs: 93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116及117所示之胺基酸序列，該豬胸膜肺炎放線桿菌毒素蛋白IV (ApxIV)的抗原決定位可為1至約30個，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，分別以三段較長的抗原決定位胺基酸序列涵蓋部分上述豬胸膜肺炎放線桿菌毒素蛋白IV (ApxIV)的抗原決定位，該三段較長的抗原決定位胺基酸序列係分別如SEQ ID NOs: 66、89、90所示。於一較佳實施例中，本發明之豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)含有二個以上該較長的抗原決定位胺基酸序列，且各抗原決定位胺基酸序列自蛋白質N端到C端的排列順序並不限於依照序列辨識號(SEQ ID NO)之順序排列。於一較佳實施例中，該豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)包含如SEQ ID NO: 91或92所示的胺基酸序列。
於部分實施態樣中，本發明所提供之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)與上述式(I)所表示之胺基酸序列具有至少大約80%序列同源性，較佳者，具有大約85%序列同源性，更佳者，具有大約90%序列同源性，甚至是大約91%、大約92%、大約93%、大約94%、大約95%、大約96%、大約97%、大約98%、大約99%序列同源性。
本發明亦提供一種編碼豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)的核苷酸序列。該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)包含至少一個豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗原決定位，以及重複0至10個單元的補體裂解片段C3d的部份胺基酸序列。該編碼本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)的核苷酸序列，係由本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)的胺基酸序列衍生而來。將本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)胺基酸序列上的各個胺基酸置換為遺傳密碼表(genetic code table)所列之編碼該胺基酸的核苷酸序列(包含各種簡併密碼子(degenerate codons，或稱同義密碼子，synonymous codons))，即可得到本發明所提供之該核苷酸序列。例如，本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)胺基酸序列上的絲胺酸(serine)可由TCT、TCC、TCA、TCG、AGT、AGC等核苷酸序列所編碼。
此外，本發明提供一種豬放線桿菌胸膜肺炎免疫組合物。該豬放線桿菌胸膜肺炎免疫組合物包含本發明之豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)以及一藥學上可接受的載劑。於一實施例中，該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)為豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)、豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)、豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)、豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)至少其中一種。於一較佳實施例中，該豬放線桿菌胸膜肺炎免疫組合物包含豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)、豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)，與豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)，以及一藥學上可接受的載劑。於一較佳實施例中，該豬放線桿菌胸膜肺炎免疫組合物包含豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)、豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)、豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)，與豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)，以及一藥學上可接受的載劑。
於一實施例中，該豬放線桿菌胸膜肺炎免疫組合物可進一步包含至少一個豬胸膜肺炎放線桿菌血清型全菌。該豬胸膜肺炎放線桿菌血清型全菌包括，但不限於，豬胸膜肺炎放線桿菌血清型1、2、3、4、5、6、7、8、9、10、11、12、13、14、15。於一較佳實施例中，該豬放線桿菌胸膜肺炎免疫組合物進一步包含豬胸膜肺炎放線桿菌血清型1、2、5三型。
於一實施例中，本發明所提供之豬放線桿菌胸膜肺炎免疫組合物，進一步包含其他病原抗原，該病原抗原包括，但不限於，豬環狀病毒第二型(PCV2)抗原、豬流感病毒(SIV)抗原、豬繁殖與呼吸症候群病毒(PRRSV)抗原、豬黴漿菌(Mycoplasma)、豬小病毒 (Parvovirus, PPV)、豬丹毒(Erysipelas)、支氣管敗血性博德氏桿菌(Bordetella bronchiseptica
)、敗血性巴氏桿菌(Pasteurella multocida
)，以及偽狂犬病(Aujeszky's disease)。
另，本發明所提供的豬放線桿菌胸膜肺炎免疫組合物可進一步包含一或多種選自於下列藥學上可接受的載劑，包括：溶劑、乳化劑、懸浮劑、分解劑、黏結劑、賦形劑、安定劑、螯合劑、稀釋劑、膠凝劑、防腐劑、潤滑劑、界面活性劑、佐劑、生物型載體等。
該藥學上可接受的載劑包含一或多種選自於下列的試劑：溶劑(solvent)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤滑劑(lubricant)、界面活性劑(surfactant)、佐劑(adjuvant)，及其他類似或適用本發明之載劑。
該藥學上可接受的賦形劑可為適合於腸外、腸內或滴鼻施用的藥學上可接受之有機或無機載體物質，且該賦形劑不會與活性組合物產生有害的反應。適合的賦形劑包含但不限於水、鹽類溶液、蔬菜油、聚乙二醇、明膠、直鏈澱粉、乳糖、硬脂酸鎂、滑石、矽酸、黏性石蠟、脂肪酸單甘酯和甘油、脂肪酸酯、羥甲基纖維素、聚乙烯吡咯烷酮等。
該藥學上可接受的佐劑包含但不限於水性氫氧化鋁膠、明礬、Freund氏不完全佐劑、油質佐劑、水溶性佐劑、或水包油包水雙相佐劑(water-in-oil-in-water, W/O/W)；於一實施例中，該佐劑為水性氫氧化鋁膠。
進一步地，本發明提供一種動物對抗豬放線桿菌胸膜肺炎的方法，包含使用有效量之上述免疫組合物以施予一動物，以增強該動物對抗豬放線桿菌胸膜肺炎之免疫力，增加感染後的存活率。
本發明並提供一種抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗體，係藉由本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)所製備或衍生而得；該抗體包括但不限於：單株抗體、多株抗體，以及經基因重組之抗體。於一實施例中，該抗體係為經由將本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)施打於一動物體內而得到之多株抗體。
於另一方面，本發明提供一種豬放線桿菌胸膜肺炎的檢測套組。該檢測套組係用以偵測檢驗樣本中是否含有豬胸膜肺炎放線桿菌毒素蛋白(Apx)，或偵測檢驗樣本內是否含有抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)的抗體。該檢測套組包含但不限於：(1)一抗原，該抗原係為本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)，於一實施例中，該抗原係置於一抗原盤上；及/或(2)一抗體，該抗體係由該本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)所衍生、製備而得之單株抗體或多株抗體。
該檢測套組之形式包含但不限於：酵素連結免疫分析(enzyme-linked immunosorbent assay, ELISA)套組、微晶片檢驗套組(Microchip kit)、免疫螢光分析法(immuno fluorescent assay, IFA)檢測套組、或其他藉由該豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)所製得之檢測套組。於一實施例中，該檢測套組至少包含一含有本發明所提供的豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)之抗原盤，可用以檢驗樣本中是否含有抗豬胸膜肺炎放線桿菌毒素蛋白(Apx)之抗體。
於本發明中所使用之單數形式「一」、及「該」包含複數形式，除非文中另有清楚指明者。因此，例如，當提及「一樣本」時，包含複數個該等樣本及對該領域具有通常技藝者所知之同等物。
本文所使用的「約」、「大約」或「近乎」一詞實質上代表所述之數值或範圍位於20%以內，較佳為於10%以內，以及更佳者為於5%以內。於本文所提供之數字化的量為近似值，意旨若術語「約」、「大約」或「近乎」沒有被使用時亦可被推得。
本說明書中所述的所有技術性及科學術語，除非另外有所定義，皆為該所屬領域具有通常技藝者可共同瞭解的意義。
本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。
實施例一 豬胸膜肺炎放線桿菌重組毒素蛋白
I (re-ApxI)
之構築
自豬胸膜肺炎放線桿菌毒素蛋白I (ApxI)全長胺基酸序列(如SEQ ID NO: 1所示)選出5個抗原決定位(epitopes)片段，分別為： 抗原決定位片段ApxI-1: ELAGITRKGADAKSGK (SEQ ID NO: 2)； 抗原決定位片段ApxI-2: PAGVGAAA (SEQ ID NO: 3)； 抗原決定位片段ApxI-3: DILYGSDGTNLFDGGVGNDKIYGG (SEQ ID NO: 4)； 抗原決定位片段ApxI-4: EHGQVLVGAGGPLAYSNSPNSIPNAF (SEQ ID NO: 5)； 抗原決定位片段ApxI-5: RAKDELHSVEEIIGSNRKDKFFGSRFTDIFHGAKGDDEIYGNDGHDILYGDDGNDVIHGGDGNDHLVGGNGNDRLIGGKGNNFLNGGDGDDELLQVFE (SEQ ID NO: 6)； 這些抗原決定位片段中包含，但不限於，以下抗原決定位： RKGADAKSGK (SEQ ID NO: 37)； DILYGSDGTNLFDGGVGNDKIYGG (SEQ ID NO: 4)； GGPLAYSNSPNSIPNA (SEQ ID NO: 39)； GAGGPLAYSNSPNS (SEQ ID NO: 40)； LVGAGGPLAYSNSPNSIPNA (SEQ ID NO: 41)； GSNRKD (SEQ ID NO: 42)； GAKGDDEIYGNDGHDILYGDDGNDVIHGGDGNDHLVGGNGNDRLIGG (SEQ ID NO: 43； NNFLNGGDGDDEL (SEQ ID NO: 44)； GSRFTDIFHGAKGDDEIYGN (SEQ ID NO: 45)； DVIHGGDGNDHLVGGNGNDR (SEQ ID NO: 46)； IGGKGNNFLNGGDGDDELQV (SEQ ID NO: 47)； HGAKGDDEIYGNDG (SEQ ID NO: 48)； GGKGNNFLNGGDGD (SEQ ID NO: 49)； DGHDILYGDDGNDVIHGGDG (SEQ ID NO: 50)； RLIGGKGNNFLNGGDGDDEL (SEQ ID NO: 51)。
由N端至C端依序連接抗原決定位片段ApxI-1、抗原決定位片段ApxI-2、抗原決定位片段ApxI-3、抗原決定位片段ApxI-4、抗原決定位片段ApxI-5，且分別在各抗原決定位片段之間以一個以上的連接子連接，該連接子具有如SEQ ID NO: 26所示的胺基酸序列；並且在抗原決定位片段ApxI-5的C端加上6個pC3d-p31生物佐劑(bioadjuvant)序列(如SEQ ID NO: 22所示)，每個pC3d-p31生物佐劑序列皆以一個以上的連接子(SEQ ID NO: 26)連接；得到的豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)胺基酸序列如SEQ ID NO: 7所示。該胺基酸序列可以合成儀合成。或者，先合成編碼該胺基酸序列的核酸序列，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。
此外，亦可以基因選殖方式構築編碼豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)的核酸序列，以限制酶切位連接DNA片段，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。於本實施例中，以Hind
III限制酶切位連接編碼豬胸膜肺炎放線桿菌毒素蛋白I的抗原決定位片段的DNA序列以及編碼pC3d-p31生物佐劑片段的DNA序列，經過選殖後得到之豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)的胺基酸序列如SEQ ID NO: 8所示。以鎳親和管柱(nickel affinity chromatography)以及離子交換層析管柱(ion exchange chromatography)純化後的豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)再以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)以及西方墨點法(western blot)確認，結果分別如圖1 (第1道)及圖2 (第2道)所示。豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)的分子量如預期的為約46kDa。
實施例二 豬胸膜肺炎放線桿菌重組毒素蛋白
II (re-ApxII)
之構築
自豬胸膜肺炎放線桿菌毒素蛋白II (ApxII)全長胺基酸序列(如SEQ ID NO: 9所示)選出5個抗原決定位片段，分別為： 抗原決定位片段ApxII-1: GNGVDTIDGNDGDDHLFGGAGDDVIDGGNGNNFLVGGTGNDIISGGKDNDIYVHKTGDGNDSITDSGGQDKL (SEQ ID NO: 10)； 抗原決定位片段ApxII-2: KVGNDYNTSKDRQNV (SEQ ID NO: 11)； 抗原決定位片段ApxII-3: LTPGEENRERIQEGKNSYITKLHIQRVDSWTVTDGDASSSV (SEQ ID NO: 12)； 抗原決定位片段ApxII-4: ILYIPQGYDSGQGNGVQDLV (SEQ ID NO: 13)； 抗原決定位片段ApxII-5: ATHPTNVGNREEKIEYRREDDRFH (SEQ ID NO: 14)； 這些抗原決定位片段中包含，但不限於，以下抗原決定位： GTGNDIISGGKDNDI (SEQ ID NO: 52)； HKTGDGNDSITDSGGQDKL (SEQ ID NO: 53)； FGGAGDDVIDGGNGNNFLVG (SEQ ID NO: 54)； YVHKTGDGNDSITDSGGQDK (SEQ ID NO: 55)； GGAGDDVIDGGNGN (SEQ ID NO: 56)； GGTGNDIISGGKDN (SEQ ID NO: 57)； KTGDGNDSITDSGG (SEQ ID NO: 58)； AGDDVIDGGNGNNFLVGGTG (SEQ ID NO: 59)； DIYVHKTGDGNDSITDSGGQ (SEQ ID NO: 60)； LTPGEENRERIQEGKNS (SEQ ID NO: 61)； WTVTDGDASSSV (SEQ ID NO: 62)； TPGEENRERIQEGKNSYITK (SEQ ID NO: 63)； LFRTPLLTPGEENR (SEQ ID NO: 64)； QGYDSGQGNGVQ (SEQ ID NO: 65)； ATHPTNVGNREEKIEYRREDDRFH (SEQ ID NO: 14)； NREEKIEYRREDDR (SEQ ID NO: 67)； PTNVGNREEKIEYRREDDRF (SEQ ID NO: 68)。
由N端至C端依序連接抗原決定位片段ApxII-1、抗原決定位片段ApxII-2、抗原決定位片段ApxII-3、抗原決定位片段ApxII-4、抗原決定位片段ApxII-5，且分別在各抗原決定位片段之間以一個以上的連接子連接，該連接子具有如SEQ ID NO: 26所示的胺基酸序列；並且在抗原決定位片段ApxII-5的C端加上6個pC3d-p31生物佐劑序列(如SEQ ID NO: 22所示)，每個pC3d-p31生物佐劑序列皆以一個以上的連接子(SEQ ID NO: 26)連接；得到的豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)胺基酸序列如SEQ ID NO: 15所示。該胺基酸序列可以合成儀合成。或者，先合成編碼該胺基酸序列的核酸序列，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。
此外，亦可以基因選殖方式構築編碼豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)的核酸序列，以限制酶切位連接DNA片段，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。於本實施例中，以Hind
III限制酶切位連接編碼豬胸膜肺炎放線桿菌毒素蛋白II的抗原決定位片段的DNA序列以及編碼pC3d-p31生物佐劑片段的DNA序列，經過選殖後得到之豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxII)的胺基酸序列如SEQ ID NO: 16所示。以鎳親和管柱(nickel affinity chromatography)以及離子交換層析管柱(ion exchange chromatography)純化後的豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)再以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)以及西方墨點法(western blot)確認，結果分別如圖1 (第2道)及圖2 (第3道)所示。豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)的分子量如預期的為約47kDa。
實施例三 豬胸膜肺炎放線桿菌重組毒素蛋白
III (re-ApxIII)
之構築
自豬胸膜肺炎放線桿菌毒素蛋白III (ApxIII)全長胺基酸序列(如SEQ ID NO: 17所示)選出2個抗原決定位片段，分別為： 抗原決定位片段ApxIII-1: ADGDDLLNGNDGDDILYGDKGNDELRGDNGNDQLYGGEGNDKLLGGNGNNYLSGGDGNDELQVLGNGFNVLRAGKGDDKLYGSSGSDLLDGGEGNDYLEGGDGSDFYVYRSTSGNHTIYDQGKSSDL (SEQ ID NO: 18)； 抗原決定位片段ApxIII-2: GELAGITGKGDKLSSGKAYVDYFQEGKLLEKKPDDFSKVVFDPTKGEIDISNSQTSTLLKFVTPLLTPGTESRERTQTGK (SEQ ID NO: 19)； 這些抗原決定位片段中包含，但不限於，以下抗原決定位： GADGDDLLNGNDGDDILYGDKGNDELRGDNGNDQLYGGEGNDKLLGGNGNNYLSGGDGNDEL (SEQ ID NO: 69)； AGKGDDKLYGSSGSDLLDGGEGNDYLEGGDGSD (SEQ ID NO: 70)； STSGNHTIYDQGKSSD (SEQ ID NO: 71)； GDKGNDELRGDNGNDQLYGG (SEQ ID NO: 72)； LLGGNGNNYLSGGDGNDELQ (SEQ ID NO: 73)； GDKGNDELRGDNGN (SEQ ID NO: 74)； GGNGNNYLSGGDGN (SEQ ID NO: 75)； DGDDILYGDKGNDELRGDNG (SEQ ID NO: 76)； LLGGNGNNYLSGGDGNDELQ (SEQ ID NO: 77)； NGFNVLRAGKGDDKLYGSSG (SEQ ID NO: 78)； GITGKGDKLSSGKA (SEQ ID NO: 79)； LLEKKPDDF (SEQ ID NO: 80)； FDPTKGEIDISNSQT (SEQ ID NO: 81)； GITGKGDKLSSGKAYVDYFQ (SEQ ID NO: 82)； VFDPTKGEIDISNSQTSTLL (SEQ ID NO: 83)； KGDKLSSGKAYVDY (SEQ ID NO: 84)； EKKPDDFSKVVFDP (SEQ ID NO: 85)； FVTPLLTPGTESRE (SEQ ID NO: 86)； ELAGITGKGDKLSSGKAYVD (SEQ ID NO: 87)； TLLKFVTPLLTPGTESRERT (SEQ ID NO: 88)。
由N端至C端依序連接抗原決定位片段ApxIII-1與抗原決定位片段ApxIII-2，且分別在各抗原決定位片段之間以一個以上的連接子連接，該連接子具有如SEQ ID NO: 26所示的胺基酸序列；並且在抗原決定位片段ApxIII-2的C端加上6個pC3d-p31生物佐劑序列(如SEQ ID NO: 22所示)，每個pC3d-p31生物佐劑序列皆以一個以上的連接子(SEQ ID NO: 26)連接；得到的豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)胺基酸序列如SEQ ID NO: 20所示。該胺基酸序列可以合成儀合成。或者，先合成編碼該胺基酸序列的核酸序列，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。
此外，亦可以基因選殖方式構築編碼豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)的核酸序列，以限制酶切位連接DNA片段，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。於本實施例中，以Hind
III限制酶切位連接編碼豬胸膜肺炎放線桿菌毒素蛋白III的抗原決定位片段的DNA序列以及編碼pC3d-p31生物佐劑片段的DNA序列，經過選殖後得到之豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)的胺基酸序列如SEQ ID NO: 21所示。以鎳親和管柱(nickel affinity chromatography)以及離子交換層析管柱(ion exchange chromatography)純化後的豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)再以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)以及西方墨點法(western blot)確認，結果分別如圖1 (第3道)及圖2 (第4道)所示。豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)的分子量如預期的為約49kDa。
實施例四 豬胸膜肺炎放線桿菌重組毒素蛋白
IV (re-ApxIV)
之構築
自豬胸膜肺炎放線桿菌毒素蛋白IV (ApxIV)全長胺基酸序列(如SEQ ID NO: 38所示)選出3個抗原決定位片段，分別為： 抗原決定位片段ApxIV-1: VIDAGAGNDTVNGGNGDDTLIGGKGNDILRGGYGADTYIFSKGHGQDIVYEDTNNDNRAR (SEQ ID NO: 66)； 抗原決定位片段ApxIV-2: EGKDTGFYGHAFYIERKNGGGSKNNSSGAGNSKDWGGNGHGNHRNNASDLNKPDGNNGNNQNNGSNQDNNSDVNAPNNPGRNYD (SEQ ID NO: 89)； 抗原決定位片段ApxIV-3: VIDAGAGNDTINGGYGDDTLIGGKGNDILKGSYGADTYIFSKGHGQDIVYEDTNNDNRARDIDTLK (SEQ ID NO: 90)； 這些抗原決定位片段中包含，但不限於，以下抗原決定位： VIDAGAGNDTVNGGNGDDTLIGGKGNDILR (SEQ ID NO: 93)； GYGA (SEQ ID NO: 94)； AGAGNDTVNGGNGDDTLIGG (SEQ ID NO: 95)； SKGHGQDIVYEDTNNDNRAR (SEQ ID NO: 96)； DAGAGNDTVNGGNG (SEQ ID NO: 97)； DIVYEDTNNDNRAR (SEQ ID NO: 98)； SKGHGQDIVYEDTNNDNRAR (SEQ ID NO: 99)； KNGGGSKNNSSGAGNSKDWG (SEQ ID NO: 100)； HRNNASDLNKPDGNNGNNQN (SEQ ID NO: 101)； GSNQDNNSDVNAPNNPGRNY (SEQ ID NO: 102)； ERKNGGGSKNNSSG (SEQ ID NO: 103)； GNSKDWGGNGHGNH (SEQ ID NO: 104)； KPDGNNGNNQNNGS (SEQ ID NO: 105)； DNNSDVNAPNNPGR (SEQ ID NO: 106)； NGGGSKNNSSGAGNSKDWGG (SEQ ID NO: 107)； NQNNGSNQDNNSDVNAPNNP (SEQ ID NO: 108)；VIDAGAGNDTINGGYGDDTLIGGKGNDILK (SEQ ID NO: 109)； SYGA (SEQ ID NO: 110)； GHGQDIVYEDTNNDNRARD (SEQ ID NO: 111)； DAGAGNDTINGGYGDDTLIG (SEQ ID NO: 112)； SKGHGQDIVYEDTNNDNRAR (SEQ ID NO: 113)； DAGAGNDTINGGYG (SEQ ID NO: 114)； DIVYEDTNNDNRAR (SEQ ID NO: 115)； NGGYGDDTLIGGKGNDILKG (SEQ ID NO: 116)； SKGHGQDIVYEDTNNDNRAR (SEQ ID NO: 117)。
由N端至C端依序連接抗原決定位片段ApxIV-1、抗原決定位片段ApxIV-2、抗原決定位片段ApxIV-3，且分別在各抗原決定位片段之間以一個以上的連接子連接，該連接子具有如SEQ ID NO: 26所示的胺基酸序列；並且在抗原決定位片段ApxIV-3的C端加上6個pC3d-p31生物佐劑序列(如SEQ ID NO: 22所示)，每個pC3d-p31生物佐劑序列皆以一個以上的連接子(SEQ ID NO: 26)連接；得到的豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)胺基酸序列如SEQ ID NO: 91所示。該胺基酸序列可以合成儀合成。或者，先合成編碼該胺基酸序列的核酸序列，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。
此外，亦可以基因選殖方式構築編碼豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)的核酸序列，以限制酶切位連接DNA片段，並將該核酸序列選殖至表現載體中，於生物表現宿主中表現該胺基酸序列並純化。於本實施例中，以Hind
III限制酶切位連接編碼豬胸膜肺炎放線桿菌毒素蛋白IV的抗原決定位片段的DNA序列以及編碼pC3d-p31生物佐劑片段的DNA序列，經過選殖後得到之豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)的胺基酸序列如SEQ ID NO: 92所示。以鎳親和管柱(nickel affinity chromatography)以及離子交換層析管柱(ion exchange chromatography)純化後的豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)再以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)以及西方墨點法(western blot)確認，結果分別如圖1 (第4道)及圖2 (第5道)所示。豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)的分子量如預期的為約50kDa。
實施例五 豬胸膜肺炎放線桿菌毒素萃取
1. 豬胸膜肺炎放線桿菌的培養
將具有生產毒素能力的豬胸膜肺炎放線桿菌血清型第1型(台灣野外分離株，分泌ApxI、ApxII、ApxIV毒素)、第2型(台灣野外分離株，分泌ApxII、ApxIII、ApxIV毒素)全菌接種於含有0.01% (v/v)煙醯胺腺嘌呤二核苷酸(β-Nicotinamide adenine dinucleotide, β-NAD)以及10% (v/v) 馬血清的腦心浸出液(BHI)液體培養基內(BD公司，美國)，於37°C、5% CO2
下培養隔夜。
2. 豬胸膜肺炎放線桿菌毒素的製備
將上述豬胸膜肺炎放線桿菌血清型第1、2型菌液以超音波振盪器(SONOPULS，Bandelin公司，德國)處理以將細菌擊碎，再以高速離心機(KUBOTA公司，日本)離心後，取上清液以0.22 mm孔徑過濾膜(Millipore公司，美國)過濾後，此過濾液即為豬胸膜肺炎放線桿菌粗萃毒素蛋白ApxI~IV (crude extracted ApxI~IV)，分裝後保存於-80°C備用。
3. 不活化豬胸膜肺炎放線桿菌的製備
將具有生產毒素能力的豬胸膜肺炎放線桿菌血清型第1型(台灣野外分離株)、第2型(台灣野外分離株)、第5型(台灣野外分離株)全菌接種於含有0.01% (v/v)煙醯胺腺嘌呤二核苷酸(β-NAD)以及10% (v/v) 馬血清的腦心浸出液(BHI)液體培養基內(BD公司，美國)，於37°C、5% CO2
下培養至少隔夜，接著加入甲醛以進行不活化作用，得到不活化豬胸膜肺炎放線桿菌。
實施例六
豬胸膜肺炎放線桿菌疫苗之配製
1. 豬胸膜肺炎放線桿菌重組毒素蛋白次單位疫苗(re-ApxI、re-ApxII、re-ApxIII、re-ApxIV)的配製
分別將實施例一至實施例四所得到的豬胸膜肺炎放線桿菌重組毒素蛋白re-ApxI (SEQ ID NO: 8)(最終濃度為500 µg/ml)、re-ApxII (SEQ ID NO: 16)(最終濃度為500 µg/ml)、re-ApxIII (SEQ ID NO: 21)(最終濃度為500 µg/ml) 、re-ApxIV (SEQ ID NO: 92)(最終濃度為500 µg/ml)以磷酸鹽緩衝溶液(phosphate buffered solution, PBS)混和均勻，並加入鋁膠[最終濃度為30% (v/v)]作為佐劑，以配製成為豬胸膜肺炎放線桿菌重組蛋白次單位疫苗。
2. 豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5)的配製
將實施例五所得到的不活化豬胸膜肺炎放線桿菌血清型第1, 2, 5型全菌(各菌株最終濃度皆為1 x 109
cfu/ml)以及磷酸鹽緩衝溶液(PBS)混和均勻，並加入鋁膠[最終濃度為30% (v/v)]作為佐劑，以配製成為豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5)。
3. 豬胸膜肺炎放線桿菌全菌及重組毒素蛋白混合多價疫苗(App 1, 2, 5 + re-ApxI~III)的配製
將實施例一至實施例三所得到的豬胸膜肺炎放線桿菌重組毒素蛋白re-ApxI (SEQ ID NO: 8)(最終濃度為20 µg/ml)、re-ApxII (SEQ ID NO: 16)(最終濃度為20 µg/ml)、re-ApxIII (SEQ ID NO: 21)(最終濃度為20 µg/ml)、實施例五所得到的不活化豬胸膜肺炎放線桿菌血清型第1, 2, 5型全菌(各菌株最終濃度皆為1 x 109
cfu/ml)以及磷酸鹽緩衝溶液(PBS)混和均勻，並加入鋁膠[最終濃度為30% (v/v)]作為佐劑，以配製成為豬胸膜肺炎放線桿菌全菌及重組毒素蛋白混合多價疫苗(App 1, 2, 5 + re-ApxI~III)。
實施例七 豬胸膜肺炎放線桿菌重組毒素蛋白疫苗有效性分析
1. 小鼠免疫及以豬胸膜肺炎放線桿菌粗萃毒素蛋白攻毒試驗
取豬胸膜肺炎放線桿菌抗體陰性的3~4週齡健康ICR小鼠(國家實驗動物中心，台灣) 60隻，隨機分為6組，每組10隻；第1組為負對照組，第2~6組為免疫試驗組；各組每隻小鼠分別以腹腔注射(ip.) 0.2 ml的以下物質： 第1組：含30% (v/v)鋁膠的PBS緩衝溶液(PBS組)； 第2組：含實施例一所得的豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI) (SEQ ID NO: 8)的次單位疫苗(re-ApxI組)； 第3組：含實施例二所得的豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII) (SEQ ID NO: 16)的次單位疫苗(re-ApxII組)； 第4組：含實施例三所得的豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII) (SEQ ID NO: 21)的次單位疫苗(re-ApxIII組)； 第5組：含實施例四所得的豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV) (SEQ ID NO: 92)的次單位疫苗(re-ApxIV組)；以及 第6組：含實施例六所得的豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5組)。
每隻小鼠第一次免疫後第14天以相同免疫劑量再進行第二次免疫，第二次免疫後第10天再對每隻小鼠注射0.1 ml的實施例五所得的豬胸膜肺炎放線桿菌粗萃毒素蛋白ApxI~IV以進行攻毒。以該全菌粗萃毒素蛋白之LD90
劑量(萃取APP1菌量6.5x109
cfu/ml及APP2菌量9.65x1010
cfu/ml)進行攻毒，十天後紀錄小鼠死亡數，以計算各組存活率。
2. 統計方法
小鼠存活率以Kaplan-Meier存活分析(Kaplan-Meier Survival Analysis: Log-Rank test)進行統計，並以p
＜0.05視為具有統計上的顯著差異。
3. 結果
小鼠攻毒試驗結果如表1所示，各別含有豬胸膜肺炎放線桿菌重組毒素蛋白re-ApxI、re-ApxII、re-ApxIII、re-ApxIV的次單位疫苗(即第2~5組)相較於負對照組(第1組，存活率0%)確實能誘發小鼠產生保護效力，並耐過豬胸膜肺炎放線桿菌粗萃毒素蛋白ApxI~IV的攻毒，提高存活率並且具有統計上的顯著差異。 【表1】小鼠攻毒試驗存活率
* 表示相對於負對照組有統計上的顯著差異(p
＜0.05)。 ** 表示相對於負對照組有統計上的顯著差異(p
＜0.01)。
實施例八 豬胸膜肺炎放線桿菌多價疫苗對豬胸膜肺炎放線桿菌血清型第
2
型菌株
(App 2)
之保護效力的分析
1. 小鼠免疫及以豬胸膜肺炎放線桿菌血清型第2型菌株(App2)攻毒試驗
取豬胸膜肺炎放線桿菌抗體陰性3~4週齡健康的ICR小鼠(國家實驗動物中心，台灣) 40隻，隨機分為4組，每組10隻；第1組為負對照組，第2~4組為免疫試驗組；各組每隻小鼠分別以腹腔注射(ip.)注射0.2 ml的以下物質： 第1組：含30% (v/v)鋁膠的PBS緩衝溶液(PBS組)； 第2組：實施例六所得的豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5組)； 第3組：實施例六所得的豬胸膜肺炎放線桿菌全菌及重組毒素蛋白混合多價疫苗(App 1, 2, 5 + re-ApxI~III組)；以及 第4組：市售豬放線桿菌胸膜肺炎不活化疫苗，含有豬胸膜肺炎放線桿菌血清型第1, 2, 3, 4, 5, 7型全菌(App 1, 2, 3, 4, 5, 7組)。
每隻小鼠第一次免疫後第14天以相同免疫劑量再進行第二次免疫，第二次免疫後第10天再對每隻小鼠注射0.1 ml的LD90
劑量(7.5x108
cfu/ml)的豬胸膜肺炎放線桿菌血清型第2型全菌(App 2)以進行攻毒，觀察小鼠10天並紀錄死亡數，以計算各組存活率。
2. 統計方法同實施例七所述。
3. 結果
小鼠攻毒試驗結果如表2所示，相較於負對照組(第1組，存活率為20%)，各免疫試驗組的存活率皆顯著增加，並具有統計上的差異。尤其是含有本發明重組毒素蛋白re-ApxI~III的多價疫苗(App 1, 2, 5 + re-ApxI~III組)對小鼠的保護效果最好，存活率最高。 【表2】以豬胸膜肺炎放線桿菌血清型第2型菌株(App 2)對小鼠攻毒試驗的存活率
* 表示相對於負對照組有統計上的顯著差異(p
＜0.05)。 ** 表示相對於負對照組有統計上的顯著差異(p
＜0.01)。
實施例九 豬胸膜肺炎放線桿菌多價疫苗對豬胸膜肺炎放線桿菌血清型第
5
型菌株
(App 5)
之保護效力的分析
小鼠免疫及以豬胸膜肺炎放線桿菌血清型第5型菌株(App5)攻毒試驗
取豬胸膜肺炎放線桿菌抗體陰性3~4週齡健康的ICR小鼠(國家實驗動物中心，台灣) 50隻，隨機分為4組；第1組為負對照組，第2~4組為免疫試驗組；各組每隻小鼠分別以腹腔注射(ip.)注射0.2 ml的以下物質： 第1組：含30% (v/v)鋁膠的PBS緩衝溶液(PBS組)(n = 15)； 第2組：實施例六所得的豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5組) (n = 15)； 第3組：實施例六所得的豬胸膜肺炎放線桿菌全菌及重組毒素蛋白混合多價疫苗(App 1, 2, 5 + re-ApxI~III組) (n = 10)；以及 第4組：市售豬放線桿菌胸膜肺炎不活化疫苗，含有豬胸膜肺炎放線桿菌血清型第1, 2, 3, 4, 5, 7型全菌(App 1, 2, 3, 4, 5, 7組) (n = 10)。
每隻小鼠分別於第一次免疫前24小時採血保存，第一次免疫後第14天以相同免疫劑量再進行第二次免疫，第二次免疫後第10天採血，並分離血液樣本中的血清，以進行毒素抗體的酵素連結免疫分析(ELISA)；採血後，對每隻小鼠注射0.1 ml的LD90
劑量(7.32x108
cfu/ml)的豬胸膜肺炎放線桿菌血清型第5型全菌(App 5)以進行攻毒，觀察小鼠10天並紀錄死亡數，以計算各組存活率。
2. 毒素抗體的酵素連結免疫分析(ELISA)
以豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII，SEQ ID NO: 16)作為抗原，並將抗原塗佈(coating)於ELISA用96孔盤(Thermo公司，美國)(100 ng/孔)，於4°C下靜置16小時。去除多餘抗原後加入清洗緩衝液(wash buffer；0.9% NaCl；0.1 % Tween20)，清洗3次後倒乾。接著加入阻隔緩衝液(blocking buffer；含有1% BSA的wash buffer)，於室溫下靜置1小時後，以清洗緩衝液清洗，接著將上述各組小鼠採集到的血清樣品以PBS緩衝溶液稀釋後，每孔加入稀釋(1:100)的小鼠血清，於室溫下靜置1小時後，去除血清樣品，並以清洗緩衝液清洗，然後加入辣根過氧化酵素(Horseradish peroxidase, HRP)標定的山羊抗小鼠的二級抗體(goat anti-mouse conjugated HRP，Gene Tex公司，美國)，該二級抗體先以阻隔緩衝液稀釋5,000倍後再加入96孔盤(100 ml/孔)，於室溫下靜置1小時後，去除二級抗體，並以清洗緩衝液清洗後，每孔加入100 ml 3, 3’, 5, 5’-四甲基聯苯胺二鹽酸(3, 3’, 5, 5’-tetramethylbenzidine, TMB，KPL公司，美國)溶液避光呈色10分鐘，並以酵素連結免疫分析測讀儀(SpectraMax® M2/M2 ELISA Reader，Molecular Devices公司，美國)讀取波長650 nm的吸光值(OD650 nm
)。
3. 統計方法
小鼠存活率以Kaplan-Meier存活分析(Kaplan-Meier Survival Analysis: Log-Rank test)進行統計，並以p
＜0.05視為具有統計上的顯著差異。酵素連結免疫分析(ELISA)結果以Student Newman-Keuls Method方法進行統計，並以p
＜0.05視為具有統計上的顯著差異。
4. 結果
小鼠攻毒試驗結果如表3所示，相較於負對照組(第1組，存活率為6.7%)，各免疫試驗組的存活率皆顯著增加，並具有統計上的差異。尤其是含有本發明重組毒素蛋白re-ApxI~III的多價疫苗(App 1, 2, 5 + re-ApxI~III組)對小鼠的保護效果最好，存活率最高。 【表3】以豬胸膜肺炎放線桿菌血清型第5型菌株(App 5)對小鼠攻毒試驗的存活率
* 表示相對於負對照組有統計上的顯著差異(p
＜0.05)。 ** 表示相對於負對照組有統計上的顯著差異(p
＜0.01)。
酵素連結免疫分析(ELISA)結果如圖3所示，免疫含有本發明之豬胸膜肺炎放線桿菌全菌及重組蛋白混合多價疫苗的小鼠(第3組，即App 1, 2, 5 + ApxI~III組)，在第二次免疫第10天後，其血清中的抗豬胸膜肺炎放線桿菌毒素蛋白抗體力價顯著高於負對照組小鼠(第1組，即PBS組，p
＜0.01)、免疫豬胸膜肺炎放線桿菌全菌多價疫苗(第2組，即App 1, 2, 5組，p
＜0.01)、免疫市售豬放線桿菌胸膜肺炎不活化疫苗(第4組，即市售App 1, 2, 3, 4, 5, 7組，p
＜0.01)的小鼠的抗體力價。由此可知，本發明所提供之豬胸膜肺炎放線桿菌全菌及重組蛋白混合多價疫苗可以在動物體內有效地誘導出抗豬胸膜肺炎放線桿菌毒素蛋白抗體，具免疫原性，且效果顯著優於習知疫苗。
實施例十 抗豬胸膜肺炎放線桿菌毒素
I~IV (ApxI~IV)
多株抗體之製備
分別將實施例一至實施例四所得到的豬胸膜肺炎放線桿菌重組蛋白re-ApxI (SEQ ID NO: 8)、re-ApxII (SEQ ID NO: 16)、re-ApxIII (SEQ ID NO: 21)、re-ApxIV (SEQ ID NO: 92)與佛氏完全佐劑(Freund’s complete adjuvant, FCA，Sigma)充分混合乳化後，再以腹腔注射方式施與Balb/c小鼠(0.2 ml/隻)以進行初級免疫，3週後以相同方式進行第二次免疫，隔週採集免疫小鼠之血清，即製得抗豬胸膜肺炎放線桿菌毒素I~IV (ApxI~IV)之多株抗體，該多株抗體係用於西方墨點法進行抗原分析。
上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，均應包含於本案之專利範圍中。
圖1所示為根據實施例一至四，以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)確認純化的本發明豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)；M：蛋白質分子量標記；第1道：豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)；第2道：豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)；第3道：豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)；第4道：豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)。
圖2所示為根據實施例一至四，以西方墨點法確認純化的本發明豬胸膜肺炎放線桿菌重組毒素蛋白(re-Apx)；M：蛋白質分子量標記；第1道：豬胸膜肺炎放線桿菌重組毒素蛋白I (re-ApxI)；第2道：豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)；第3道：豬胸膜肺炎放線桿菌重組毒素蛋白III (re-ApxIII)；第4道：豬胸膜肺炎放線桿菌重組毒素蛋白IV (re-ApxIV)；一級抗體為鹼性磷酸酶綴合的小鼠抗His標記單株抗體(Alkaline phosphatase-conjugated mouse anti-His，Invitrogen)。
圖3為根據實施例九，以酵素連結免疫分析(ELISA)測定抗豬胸膜肺炎放線桿菌重組毒素蛋白II (re-ApxII)抗體力價的結果；第1組為負對照組(PBS組)；第2組為含有實施例六所得的豬胸膜肺炎放線桿菌全菌多價疫苗(App 1, 2, 5組)；第3組為實施例六所得的豬胸膜肺炎放線桿菌全菌及重組毒素蛋白混合多價疫苗(App 1, 2, 5 + re-ApxI~III組)；第4組為市售豬放線桿菌胸膜肺炎不活化疫苗，含有豬胸膜肺炎放線桿菌血清型第1, 2, 3, 4, 5, 7型全菌(App 1, 2, 3, 4, 5, 7組)。符號**代表該組與負對照組之間具有顯著差異(p
＜0.01)；符號##代表兩組之間具有顯著差異(p
＜0.01)。
＜110＞ 金協國際實業有限公司 ＜120＞ 豬胸膜肺炎放線桿菌重組毒素蛋白及其應用 ＜130＞ P16-0074 ＜150＞ US 62/153711 ＜151＞ 2015-04-28 ＜160＞ 117 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 1022 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 1 Met Ala Asn Ser Gln Leu Asp Arg Val Lys Gly Leu Ile Asp Ser Leu 1 5 10 15 Asn Gln His Thr Lys Ser Ala Ala Lys Ser Gly Ala Gly Ala Leu Lys 20 25 30 Asn Gly Leu Gly Gln Val Lys Gln Ala Gly Gln Lys Leu Ile Leu Tyr 35 40 45 Ile Pro Lys Asp Tyr Gln Ala Ser Thr Gly Ser Ser Leu Asn Asp Leu 50 55 60 Val Lys Ala Ala Glu Ala Leu Gly Ile Glu Val His Arg Ser Glu Lys 65 70 75 80 Asn Gly Thr Ala Leu Ala Lys Glu Leu Phe Gly Thr Thr Glu Lys Leu 85 90 95 Leu Gly Phe Ser Glu Arg Gly Ile Ala Leu Phe Ala Pro Gln Phe Asp 100 105 110 Lys Leu Leu Asn Lys Asn Gln Lys Leu Ser Lys Ser Leu Gly Gly Ser 115 120 125 Ser Glu Ala Leu Gly Gln Arg Leu Asn Lys Thr Gln Thr Ala Leu Ser 130 135 140 Ala Leu Gln Ser Phe Leu Gly Thr Ala Ile Ala Gly Met Asp Leu Asp 145 150 155 160 Ser Leu Leu Arg Arg Arg Arg Asn Gly Glu Asp Val Ser Gly Ser Glu 165 170 175 Leu Ala Lys Ala Gly Val Asp Leu Ala Ala Gln Leu Val Asp Asn Ile 180 185 190 Ala Ser Ala Thr Gly Thr Val Asp Ala Phe Ala Glu Gln Leu Gly Lys 195 200 205 Leu Gly Asn Ala Leu Ser Asn Thr Arg Leu Ser Gly Leu Ala Ser Lys 210 215 220 Leu Asn Asn Leu Pro Asp Leu Ser Leu Ala Gly Pro Gly Phe Asp Ala 225 230 235 240 Val Ser Gly Ile Leu Ser Val Val Ser Ala Ser Phe Ile Leu Ser Asn 245 250 255 Lys Asp Ala Asp Ala Gly Thr Lys Ala Ala Ala Gly Ile Glu Ile Ser 260 265 270 Thr Lys Ile Leu Gly Asn Ile Gly Lys Ala Val Ser Gln Tyr Ile Ile 275 280 285 Ala Gln Arg Val Ala Ala Gly Leu Ser Thr Thr Ala Ala Thr Arg Trp 290 295 300 Phe Asn Arg Ser Val Val Ala Leu Ala Ile Ser Pro Leu Ser Phe Leu 305 310 315 320 Asn Val Ala Asp Lys Phe Glu Arg Ala Lys Gln Leu Glu Gln Tyr Ser 325 330 335 Glu Arg Phe Lys Lys Phe Gly Tyr Glu Gly Asp Ser Leu Leu Ala Ser 340 345 350 Phe Tyr Arg Glu Thr Gly Ala Ile Glu Ala Ala Leu Thr Thr Ile Asn 355 360 365 Ser Val Leu Ser Ala Ala Pro Ala Gly Val Gly Ala Ala Ala Thr Gly 370 375 380 Ser Leu Val Gly Ala Pro Val Ala Ala Leu Val Ser Ala Ile Thr Gly 385 390 395 400 Ile Ile Ser Gly Ile Leu Asp Ala Ser Lys Gln Ala Ile Phe Glu Arg 405 410 415 Val Ala Thr Lys Leu Ala Asn Lys Ile Asp Glu Trp Glu Lys Lys His 420 425 430 Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg His Ser Ala Phe 435 440 445 Leu Glu Asp Thr Phe Glu Leu Leu Ser Gln Tyr Asn Lys Glu Tyr Ser 450 455 460 Val Glu Arg Val Val Ala Ile Thr Gln Gln Arg Trp Asp Val Asn Ile 465 470 475 480 Gly Glu Leu Ala Gly Ile Thr Arg Lys Gly Ala Asp Ala Lys Ser Gly 485 490 495 Lys Ala Tyr Val Asp Phe Phe Glu Glu Gly Lys Leu Leu Glu Lys Asp 500 505 510 Pro Asp Arg Phe Asp Lys Lys Val Phe Asp Pro Leu Glu Gly Lys Ile 515 520 525 Asp Leu Ser Ser Ile Asn Lys Thr Thr Leu Leu Lys Phe Ile Thr Pro 530 535 540 Ala Phe Thr Ala Gly Glu Glu Ile Arg Glu Arg Lys Gln Thr Gly Lys 545 550 555 560 Tyr Glu Tyr Met Thr Glu Leu Phe Val Lys Gly Lys Glu Lys Trp Val 565 570 575 Val Thr Gly Val Gln Ser His Asn Ala Ile Tyr Asp Tyr Thr Asn Leu 580 585 590 Ile Gln Leu Ala Ile Asp Lys Lys Gly Glu Lys Arg Gln Val Thr Ile 595 600 605 Glu Ser His Leu Gly Glu Lys Asn Asp Arg Ile Tyr Leu Ser Ser Gly 610 615 620 Ser Ser Ile Glu Tyr Ala Gly Asn Gly His Asp Val Ala Tyr Tyr Asp 625 630 635 640 Lys Thr Asp Thr Gly Tyr Leu Thr Phe Asp Gly Gln Ser Ala Gln Lys 645 650 655 Ala Gly Glu Tyr Ile Val Thr Lys Glu Leu Lys Ala Asp Val Lys Val 660 665 670 Leu Lys Glu Val Val Lys Thr Gln Asp Ile Ser Val Gly Lys Arg Ser 675 680 685 Glu Lys Leu Glu Tyr Arg Asp Tyr Glu Leu Ser Pro Phe Glu Leu Gly 690 695 700 Asn Gly Ile Arg Ala Lys Asp Glu Leu His Ser Val Glu Glu Ile Ile 705 710 715 720 Gly Ser Asn Arg Lys Asp Lys Phe Phe Gly Ser Arg Phe Thr Asp Ile 725 730 735 Phe His Gly Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly His 740 745 750 Asp Ile Leu Tyr Gly Asp Asp Gly Asn Asp Val Ile His Gly Gly Asp 755 760 765 Gly Asn Asp His Leu Val Gly Gly Asn Gly Asn Asp Arg Leu Ile Gly 770 775 780 Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu Leu 785 790 795 800 Gln Val Phe Glu Cys Gln Tyr Asn Val Leu Leu Gly Gly Ala Gly Asn 805 810 815 Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Leu Phe Asp Gly Gly Val 820 825 830 Gly Asn Asp Lys Ile Tyr Gly Gly Leu Gly Lys Asp Ile Tyr Arg Tyr 835 840 845 Ser Lys Glu Tyr Gly Arg His Ile Ile Ile Glu Lys Gly Gly Asp Asp 850 855 860 Asp Thr Leu Leu Leu Ser Asp Leu Ser Phe Lys Asp Val Gly Phe Ile 865 870 875 880 Arg Ile Gly Asp Asp Leu Leu Val Asn Lys Arg Ile Gly Gly Thr Leu 885 890 895 Tyr Tyr His Glu Asp Tyr Asn Gly Asn Ala Leu Thr Ile Lys Asp Trp 900 905 910 Phe Lys Glu Gly Lys Glu Gly Gln Asn Asn Lys Val Glu Lys Ile Val 915 920 925 Asp Lys Asp Gly Ala Tyr Val Leu Ser Gln Tyr Leu Thr Glu Leu Thr 930 935 940 Ala Pro Gly Arg Gly Ile Asn Tyr Phe Asn Gly Leu Glu Glu Lys Leu 945 950 955 960 Tyr Tyr Gly Glu Gly Tyr Asn Ala Leu Pro Gln Leu Arg Lys Asp Ile 965 970 975 Glu Gln Ile Ile Ser Ser Thr Gly Ala Leu Thr Gly Glu His Gly Gln 980 985 990 Val Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser Asn Ser Pro Asn 995 1000 1005 Ser Ile Pro Asn Ala Phe Ser Asn Tyr Leu Thr Gln Ser Ala 1010 1015 1020 ＜210＞ 2 ＜211＞ 16 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 2 Glu Leu Ala Gly Ile Thr Arg Lys Gly Ala Asp Ala Lys Ser Gly Lys 1 5 10 15 ＜210＞ 3 ＜211＞ 8 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
)＜400＞ 3 Pro Ala Gly Val Gly Ala Ala Ala 1 5 ＜210＞ 4 ＜211＞ 24 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 4 Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Leu Phe Asp Gly Gly Val 1 5 10 15 Gly Asn Asp Lys Ile Tyr Gly Gly 20 ＜210＞ 5 ＜211＞ 26 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 5 Glu His Gly Gln Val Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser 1 5 10 15 Asn Ser Pro Asn Ser Ile Pro Asn Ala Phe 20 25 ＜210＞ 6 ＜211＞ 97 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 6 Arg Ala Lys Asp Glu Leu His Ser Val Glu Glu Ile Ile Gly Ser Asn 1 5 10 15 Arg Lys Asp Lys Phe Phe Gly Ser Arg Phe Thr Asp Ile Phe His Gly 20 25 30 Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly His Asp Ile Leu 35 40 45 Tyr Gly Asp Asp Gly Asn Asp Val Ile His Gly Gly Asp Gly Asn Asp 50 55 60 His Leu Val Gly Gly Asn Gly Asn Asp Arg Leu Ile Gly Gly Lys Gly 65 70 75 80 Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu Leu Gln Val Phe 85 90 95 Glu ＜210＞ 7 ＜211＞ 410 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 重組ApxI毒素 ＜400＞ 7 Glu Leu Ala Gly Ile Thr Arg Lys Gly Ala Asp Ala Lys Ser Gly Lys 1 5 10 15 Gly Ser Thr Ala Pro Ala Gly Val Gly Ala Ala Ala Gly Ser Thr Ala 20 25 30 Gly Ser Thr Ala Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Leu Phe 35 40 45 Asp Gly Gly Val Gly Asn Asp Lys Ile Tyr Gly Gly Gly Ser Thr Ala 50 55 60 Glu His Gly Gln Val Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser 65 70 75 80 Asn Ser Pro Asn Ser Ile Pro Asn Ala Phe Gly Ser Thr Ala Arg Ala 85 90 95 Lys Asp Glu Leu His Ser Val Glu Glu Ile Ile Gly Ser Asn Arg Lys 100 105 110 Asp Lys Phe Phe Gly Ser Arg Phe Thr Asp Ile Phe His Gly Ala Lys 115 120 125 Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly His Asp Ile Leu Tyr Gly 130 135 140 Asp Asp Gly Asn Asp Val Ile His Gly Gly Asp Gly Asn Asp His Leu 145 150 155 160 Val Gly Gly Asn Gly Asn Asp Arg Leu Ile Gly Gly Lys Gly Asn Asn 165 170 175 Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu Leu Leu Gln Val Phe Glu 180 185 190 Gly Ser Thr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr 195 200 205 Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn 210 215 220 Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu 225 230 235 240 Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys 245 250 255 Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu 260 265 270 Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro 275 280 285 Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr 290 295 300 Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp 305 310 315 320 Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 325 330 335 Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg 340 345 350 Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr 355 360 365 Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala 370 375 380 Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val 385 390 395 400 Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 405 410 ＜210＞ 8 ＜211＞ 412 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 帶有一個HindIII切位的重組ApxI毒素 ＜400＞ 8 Glu Leu Ala Gly Ile Thr Arg Lys Gly Ala Asp Ala Lys Ser Gly Lys 1 5 10 15 Gly Ser Thr Ala Pro Ala Gly Val Gly Ala Ala Ala Gly Ser Thr Ala 20 25 30 Gly Ser Thr Ala Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Leu Phe 35 40 45 Asp Gly Gly Val Gly Asn Asp Lys Ile Tyr Gly Gly Gly Ser Thr Ala 50 55 60 Glu His Gly Gln Val Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser 65 70 75 80 Asn Ser Pro Asn Ser Ile Pro Asn Ala Phe Gly Ser Thr Ala Arg Ala 85 90 95 Lys Asp Glu Leu His Ser Val Glu Glu Ile Ile Gly Ser Asn Arg Lys 100 105 110 Asp Lys Phe Phe Gly Ser Arg Phe Thr Asp Ile Phe His Gly Ala Lys 115 120 125 Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly His Asp Ile Leu Tyr Gly 130 135 140 Asp Asp Gly Asn Asp Val Ile His Gly Gly Asp Gly Asn Asp His Leu 145 150 155 160 Val Gly Gly Asn Gly Asn Asp Arg Leu Ile Gly Gly Lys Gly Asn Asn 165 170 175 Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu Leu Leu Gln Val Phe Glu 180 185 190 Gly Ser Thr Ala Lys Leu Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu 195 200 205 Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu 210 215 220 Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn 225 230 235 240 Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly 245 250 255 Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 260 265 270 Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu 275 280 285 Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly 290 295 300 Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn 305 310 315 320 Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser 325 330 335 Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys 340 345 350 Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu 355 360 365 Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser 370 375 380 Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr 385 390 395 400 Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 405 410 ＜210＞ 9 ＜211＞ 956 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 9 Met Ser Lys Ile Thr Leu Ser Ser Leu Lys Ser Ser Leu Gln Gln Gly 1 5 10 15 Leu Lys Asn Gly Lys Asn Lys Leu Asn Gln Ala Gly Thr Thr Leu Lys 20 25 30 Asn Gly Leu Thr Gln Thr Gly His Ser Leu Gln Asn Gly Ala Lys Lys 35 40 45 Leu Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly 50 55 60 Val Gln Asp Leu Val Lys Ala Ala Asn Asp Leu Gly Ile Glu Val Trp 65 70 75 80 Arg Glu Glu Arg Ser Asn Leu Asp Ile Ala Lys Thr Ser Phe Asp Thr 85 90 95 Thr Gln Lys Ile Leu Gly Phe Thr Asp Arg Gly Ile Val Leu Phe Ala 100 105 110 Pro Gln Leu Asp Asn Leu Leu Lys Lys Asn Pro Lys Ile Gly Asn Thr 115 120 125 Leu Gly Ser Ala Ser Ser Ile Ser Gln Asn Ile Gly Lys Ala Asn Thr 130 135 140 Val Leu Gly Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ser Gly Val 145 150 155 160 Asn Leu Asn Glu Leu Leu Gln Asn Lys Asp Pro Asn Gln Leu Glu Leu 165 170 175 Ala Lys Ala Gly Leu Glu Leu Thr Asn Glu Leu Val Gly Asn Ile Ala 180 185 190 Ser Ser Val Gln Thr Val Asp Ala Phe Ala Glu Gln Ile Ser Lys Leu 195 200 205 Gly Ser His Leu Gln Asn Val Lys Gly Leu Gly Gly Leu Ser Asn Lys 210 215 220 Leu Gln Asn Leu Pro Asp Leu Gly Lys Ala Ser Leu Gly Leu Asp Ile 225 230 235 240 Ile Ser Gly Leu Leu Ser Gly Ala Ser Ala Gly Leu Ile Leu Ala Asp 245 250 255 Lys Glu Ala Ser Thr Glu Lys Lys Ala Ala Ala Gly Val Glu Phe Ala 260 265 270 Asn Gln Ile Ile Gly Asn Val Thr Lys Ala Val Ser Ser Tyr Ile Leu 275 280 285 Ala Gln Arg Val Ala Ser Gly Leu Ser Ser Thr Gly Pro Val Ala Ala 290 295 300 Leu Ile Ala Ser Thr Val Ala Leu Ala Val Ser Pro Leu Ser Phe Leu 305 310 315 320 Asn Val Ala Asp Lys Phe Lys Gln Ala Asp Leu Ile Lys Ser Tyr Ser 325 330 335 Glu Arg Phe Gln Lys Leu Gly Tyr Asp Gly Asp Arg Leu Leu Ala Asp 340 345 350 Phe His Arg Glu Thr Gly Thr Ile Asp Ala Ser Val Thr Thr Ile Asn 355 360 365 Thr Ala Leu Ala Ala Ile Ser Gly Gly Val Gly Ala Ala Ser Ala Gly 370 375 380 Ser Leu Val Gly Ala Pro Val Ala Leu Leu Val Ala Gly Val Thr Gly 385 390 395 400 Leu Ile Thr Thr Ile Leu Glu Tyr Ser Lys Gln Ala Met Phe Glu His 405 410 415 Val Ala Asn Lys Val His Asp Arg Ile Val Glu Trp Glu Lys Lys His 420 425 430 Asn Lys Asn Tyr Phe Glu Gln Gly Tyr Asp Ser Arg His Leu Ala Asp 435 440 445 Leu Gln Asp Asn Met Lys Phe Leu Ile Asn Leu Asn Lys Glu Leu Gln 450 455 460 Ala Glu Arg Val Val Ala Ile Thr Gln Gln Arg Trp Asp Asn Gln Ile 465 470 475 480 Gly Asp Leu Ala Ala Ile Ser Arg Arg Thr Asp Lys Ile Ser Ser Gly 485 490 495 Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Gln His Gln Ser Tyr Asp 500 505 510 Ser Ser Val Gln Leu Asp Asn Lys Asn Gly Ile Ile Asn Ile Ser Asn 515 520 525 Thr Asn Arg Lys Thr Gln Ser Val Leu Phe Arg Thr Pro Leu Leu Thr 530 535 540 Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn Ser Tyr 545 550 555 560 Ile Thr Lys Leu His Ile Gln Arg Val Asp Ser Trp Thr Val Thr Asp 565 570 575 Gly Asp Ala Ser Ser Ser Val Asp Phe Thr Asn Val Val Gln Arg Ile 580 585 590 Ala Val Lys Phe Asp Asp Ala Gly Asn Ile Ile Glu Ser Lys Asp Thr 595 600 605 Lys Ile Ile Ala Asn Leu Gly Ala Gly Asn Asp Asn Val Phe Val Gly 610 615 620 Ser Ser Thr Thr Val Ile Asp Gly Gly Asp Gly His Asp Arg Val His 625 630 635 640 Tyr Ser Arg Gly Glu Tyr Gly Ala Leu Val Ile Asp Ala Thr Ala Glu 645 650 655 Thr Glu Lys Gly Ser Tyr Ser Val Lys Arg Tyr Val Gly Asp Ser Lys 660 665 670 Ala Leu His Glu Thr Ile Ala Thr His Pro Thr Asn Val Gly Asn Arg 675 680 685 Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp Asp Arg Phe His Thr Gly 690 695 700 Tyr Thr Val Thr Asp Ser Leu Lys Ser Val Glu Glu Ile Ile Gly Ser 705 710 715 720 Gln Phe Asn Asp Ile Phe Lys Gly Ser Gln Phe Asp Asp Val Phe His 725 730 735 Gly Gly Asn Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asp Asp His 740 745 750 Leu Phe Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn 755 760 765 Asn Phe Leu Val Gly Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys 770 775 780 Asp Asn Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile 785 790 795 800 Thr Asp Ser Gly Gly Gln Asp Lys Leu Ala Phe Ser Asp Val Asn Leu 805 810 815 Lys Asp Leu Thr Phe Lys Lys Val Asp Ser Ser Leu Glu Ile Ile Asn 820 825 830 Gln Lys Gly Glu Lys Val Arg Ile Gly Asn Trp Phe Leu Lys Asn Asp 835 840 845 Leu Ala Ser Thr Val Ala Asn Tyr Lys Ala Thr Asn Asp Arg Lys Ile 850 855 860 Glu Glu Ile Ile Gly Lys Gly Gly Glu Arg Ile Thr Ser Lys Gln Val 865 870 875 880 Asp Lys Leu Ile Lys Glu Gly Asn Asn Gln Ile Ser Ala Lys Ala Leu 885 890 895 Ser Lys Val Gly Asn Asp Tyr Asn Thr Ser Lys Asp Arg Gln Asn Val 900 905 910 Ser Asn Ser Leu Ala Lys Leu Ile Ser Ser Val Glu Ser Phe Thr Ser 915 920 925 Ser Ser Asn Phe Arg Asn Asn Leu Gly Ala Tyr Val Pro Ser Ser Ile 930 935 940 Asn Val Ser Asn Asn Ile Gln Leu Ala Arg Ala Ala 945 950 955 ＜210＞ 10 ＜211＞ 72 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 10 Gly Asn Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asp Asp His Leu 1 5 10 15 Phe Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn Asn 20 25 30 Phe Leu Val Gly Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys Asp 35 40 45 Asn Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr 50 55 60 Asp Ser Gly Gly Gln Asp Lys Leu 65 70 ＜210＞ 11 ＜211＞ 15 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 11 Lys Val Gly Asn Asp Tyr Asn Thr Ser Lys Asp Arg Gln Asn Val 1 5 10 15 ＜210＞ 12 ＜211＞ 41 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 12 Leu Thr Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn 1 5 10 15 Ser Tyr Ile Thr Lys Leu His Ile Gln Arg Val Asp Ser Trp Thr Val 20 25 30 Thr Asp Gly Asp Ala Ser Ser Ser Val 35 40 ＜210＞ 13 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 13 Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly Val 1 5 10 15 Gln Asp Leu Val 20 ＜210＞ 14 ＜211＞ 24 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 14 Ala Thr His Pro Thr Asn Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr 1 5 10 15 Arg Arg Glu Asp Asp Arg Phe His 20 ＜210＞ 15 ＜211＞ 410 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 重組ApxII毒素 ＜400＞ 15 Gly Asn Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asp Asp His Leu 1 5 10 15 Phe Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn Asn 20 25 30 Phe Leu Val Gly Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys Asp 35 40 45 Asn Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr 50 55 60 Asp Ser Gly Gly Gln Asp Lys Leu Gly Ser Thr Ala Lys Val Gly Asn 65 70 75 80 Asp Tyr Asn Thr Ser Lys Asp Arg Gln Asn Val Gly Ser Thr Ala Gly 85 90 95 Ser Thr Ala Leu Thr Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu 100 105 110 Gly Lys Asn Ser Tyr Ile Thr Lys Leu His Ile Gln Arg Val Asp Ser 115 120 125 Trp Thr Val Thr Asp Gly Asp Ala Ser Ser Ser Val Gly Ser Thr Ala 130 135 140 Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly Val 145 150 155 160 Gln Asp Leu Val Gly Ser Thr Ala Ala Thr His Pro Thr Asn Val Gly 165 170 175 Asn Arg Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp Asp Arg Phe His 180 185 190 Gly Ser Thr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr 195 200 205 Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn 210 215 220 Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu 225 230 235 240 Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys 245 250 255 Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu 260 265 270 Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro 275 280 285 Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr 290 295 300 Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp 305 310 315 320 Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 325 330 335 Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg 340 345 350 Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr 355 360 365 Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala 370 375 380 Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val 385 390 395 400 Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 405 410 ＜210＞ 16 ＜211＞ 412 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 帶有一個HindIII切位的重組ApxII毒素 ＜400＞ 16 Gly Asn Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asp Asp His Leu 1 5 10 15 Phe Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn Asn 20 25 30 Phe Leu Val Gly Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys Asp 35 40 45 Asn Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr 50 55 60 Asp Ser Gly Gly Gln Asp Lys Leu Gly Ser Thr Ala Lys Val Gly Asn 65 70 75 80 Asp Tyr Asn Thr Ser Lys Asp Arg Gln Asn Val Gly Ser Thr Ala Gly 85 90 95 Ser Thr Ala Leu Thr Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu 100 105 110 Gly Lys Asn Ser Tyr Ile Thr Lys Leu His Ile Gln Arg Val Asp Ser 115 120 125 Trp Thr Val Thr Asp Gly Asp Ala Ser Ser Ser Val Gly Ser Thr Ala 130 135 140 Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly Val 145 150 155 160 Gln Asp Leu Val Gly Ser Thr Ala Ala Thr His Pro Thr Asn Val Gly 165 170 175 Asn Arg Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp Asp Arg Phe His 180 185 190 Gly Ser Thr Ala Lys Leu Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu 195 200 205 Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu 210 215 220 Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn 225 230 235 240 Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly 245 250 255 Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 260 265 270 Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu 275 280 285 Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly 290 295 300 Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn 305 310 315 320 Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser 325 330 335 Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys 340 345 350 Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu 355 360 365 Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser 370 375 380 Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr 385 390 395 400 Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 405 410 ＜210＞ 17 ＜211＞ 1052 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 17 Met Ser Thr Trp Ser Ser Met Leu Ala Asp Leu Lys Lys Arg Ala Glu 1 5 10 15 Glu Ala Lys Arg Gln Ala Lys Lys Gly Tyr Asp Val Thr Lys Asn Gly 20 25 30 Leu Gln Tyr Gly Val Ser Gln Ala Lys Leu Gln Ala Leu Ala Ala Gly 35 40 45 Lys Ala Val Gln Lys Tyr Gly Asn Lys Leu Val Leu Val Ile Pro Lys 50 55 60 Glu Tyr Asp Gly Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys Ala 65 70 75 80 Ala Glu Glu Leu Gly Ile Gln Val Lys Tyr Val Asn Arg Asn Glu Leu 85 90 95 Glu Val Ala His Lys Ser Leu Gly Thr Ala Asp Gln Phe Leu Gly Leu 100 105 110 Thr Glu Arg Gly Leu Thr Leu Ser Ala Pro Gln Leu Asp Gln Phe Leu 115 120 125 Gln Lys His Ser Lys Ile Ser Asn Val Val Gly Ser Ser Thr Gly Asp 130 135 140 Ala Val Ser Lys Leu Ala Lys Ser Gln Thr Ile Ile Ser Gly Ile Gln 145 150 155 160 Ser Val Leu Gly Thr Val Leu Ala Gly Ile Asn Leu Asn Glu Ala Ile 165 170 175 Ile Ser Gly Gly Ser Glu Leu Glu Leu Ala Glu Ala Gly Val Ser Leu 180 185 190 Ala Ser Glu Leu Val Ser Asn Ile Ala Lys Gly Thr Thr Thr Ile Asp 195 200 205 Ala Phe Thr Thr Gln Ile Gln Asn Phe Gly Lys Leu Val Glu Asn Ala 210 215 220 Lys Gly Leu Gly Gly Val Gly Arg Gln Leu Gln Asn Ile Ser Gly Ser 225 230 235 240 Ala Leu Ser Lys Thr Gly Leu Gly Leu Asp Ile Ile Ser Ser Leu Leu 245 250 255 Ser Gly Val Thr Ala Ser Phe Ala Leu Ala Asn Lys Asn Ala Ser Thr 260 265 270 Ser Thr Lys Val Ala Ala Gly Phe Glu Leu Ser Asn Gln Val Ile Gly 275 280 285 Gly Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Leu Ala 290 295 300 Ala Gly Leu Ser Thr Thr Gly Pro Ala Ala Ala Leu Ile Ala Ser Ser 305 310 315 320 Ile Ser Leu Ala Ile Ser Pro Leu Ala Phe Leu Arg Val Ala Asp Asn 325 330 335 Phe Asn Arg Ser Lys Glu Ile Gly Glu Phe Ala Glu Arg Phe Lys Lys 340 345 350 Leu Gly Tyr Asp Gly Asp Lys Leu Leu Ser Glu Phe Tyr His Glu Ala 355 360 365 Gly Thr Ile Asp Ala Ser Ile Thr Thr Ile Ser Thr Ala Leu Ser Ala 370 375 380 Ile Ala Ala Gly Thr Ala Ala Ala Ser Ala Gly Ala Leu Val Gly Ala 385 390 395 400 Pro Ile Thr Leu Leu Val Thr Gly Ile Thr Gly Leu Ile Ser Gly Ile 405 410 415 Leu Glu Phe Ser Lys Gln Pro Met Leu Asp His Val Ala Ser Lys Ile 420 425 430 Gly Asn Lys Ile Asp Glu Trp Glu Lys Lys Tyr Gly Lys Asn Tyr Phe 435 440 445 Glu Asn Gly Tyr Asp Ala Arg His Lys Ala Phe Leu Glu Asp Ser Phe 450 455 460 Ser Leu Leu Ser Ser Phe Asn Lys Gln Tyr Glu Thr Glu Arg Ala Val 465 470 475 480 Leu Ile Thr Gln Gln Arg Trp Asp Glu Tyr Ile Gly Glu Leu Ala Gly 485 490 495 Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp 500 505 510 Tyr Phe Gln Glu Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser 515 520 525 Lys Val Val Phe Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser 530 535 540 Gln Thr Ser Thr Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly 545 550 555 560 Thr Glu Ser Arg Glu Arg Thr Gln Thr Gly Lys Tyr Glu Tyr Ile Thr 565 570 575 Lys Leu Val Val Lys Gly Lys Asp Lys Trp Val Val Asn Gly Val Lys 580 585 590 Asp Lys Gly Ala Val Tyr Asp Tyr Thr Asn Leu Ile Gln His Ala His 595 600 605 Ile Ser Ser Ser Val Ala Arg Gly Glu Glu Tyr Arg Glu Val Arg Leu 610 615 620 Val Ser His Leu Gly Asn Gly Asn Asp Lys Val Phe Leu Ala Ala Gly 625 630 635 640 Ser Ala Glu Ile His Ala Gly Glu Gly His Asp Val Val Tyr Tyr Asp 645 650 655 Lys Thr Asp Thr Gly Leu Leu Val Ile Asp Gly Thr Lys Ala Thr Glu 660 665 670 Gln Gly Arg Tyr Ser Val Thr Arg Glu Leu Ser Gly Ala Thr Lys Ile 675 680 685 Leu Arg Glu Val Ile Lys Asn Gln Lys Ser Ala Val Gly Lys Arg Glu 690 695 700 Glu Thr Leu Glu Tyr Arg Asp Tyr Glu Leu Thr Gln Ser Gly Asn Ser 705 710 715 720 Asn Leu Lys Ala His Asp Glu Leu His Ser Val Glu Glu Ile Ile Gly 725 730 735 Ser Asn Gln Arg Asp Glu Phe Lys Gly Ser Lys Phe Arg Asp Ile Phe 740 745 750 His Gly Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp 755 760 765 Ile Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly 770 775 780 Asn Asp Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly 785 790 795 800 Asn Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln 805 810 815 Val Leu Gly Asn Gly Phe Asn Val Leu Arg Ala Gly Lys Gly Asp Asp 820 825 830 Lys Leu Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly 835 840 845 Asn Asp Tyr Leu Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg 850 855 860 Ser Thr Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp 865 870 875 880 Leu Asp Lys Leu Tyr Leu Ser Asp Phe Ser Phe Asp Arg Leu Leu Val 885 890 895 Glu Lys Val Asp Asp Asn Leu Val Leu Arg Ser Asn Glu Ser Ser His 900 905 910 Asn Asn Arg Val Leu Thr Ile Lys Asp Trp Phe Lys Glu Gly Asn Lys 915 920 925 Tyr Asn His Lys Ile Glu Gln Ile Val Asp Lys Asn Gly Arg Lys Leu 930 935 940 Thr Ala Glu Asn Leu Gly Thr Tyr Phe Lys Asn Ala Pro Lys Ala Asp 945 950 955 960 Asn Leu Leu Asn Tyr Ala Thr Lys Glu Asp Gln Asn Glu Ser Asn Leu 965 970 975 Ser Ser Leu Lys Thr Glu Leu Ser Lys Ile Ile Thr Asn Ala Gly Asn 980 985 990 Phe Gly Val Ala Lys Gln Gly Asn Thr Gly Ile Asn Thr Ala Ala Leu 995 1000 1005 Asn Asn Glu Val Asn Lys Ile Ile Ser Ser Ala Asn Thr Phe Ala 1010 1015 1020 Thr Ser Gln Leu Gly Gly Ser Gly Met Gly Thr Leu Pro Ser Thr 1025 1030 1035 Asn Val Asn Ser Met Met Leu Gly Asn Leu Ala Arg Ala Ala 1040 1045 1050 ＜210＞ 18 ＜211＞ 127 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 18 Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp Ile Leu 1 5 10 15 Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp 20 25 30 Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly Asn Gly 35 40 45 Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln Val Leu 50 55 60 Gly Asn Gly Phe Asn Val Leu Arg Ala Gly Lys Gly Asp Asp Lys Leu 65 70 75 80 Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly Asn Asp 85 90 95 Tyr Leu Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg Ser Thr 100 105 110 Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp Leu 115 120 125 ＜210＞ 19 ＜211＞ 80 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 19 Gly Glu Leu Ala Gly Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly 1 5 10 15 Lys Ala Tyr Val Asp Tyr Phe Gln Glu Gly Lys Leu Leu Glu Lys Lys 20 25 30 Pro Asp Asp Phe Ser Lys Val Val Phe Asp Pro Thr Lys Gly Glu Ile 35 40 45 Asp Ile Ser Asn Ser Gln Thr Ser Thr Leu Leu Lys Phe Val Thr Pro 50 55 60 Leu Leu Thr Pro Gly Thr Glu Ser Arg Glu Arg Thr Gln Thr Gly Lys 65 70 75 80 ＜210＞ 20 ＜211＞ 433 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 重組ApxIII毒素 ＜400＞ 20 Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp Ile Leu 1 5 10 15 Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp 20 25 30 Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly Asn Gly 35 40 45 Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln Val Leu 50 55 60 Gly Asn Gly Phe Asn Val Leu Arg Ala Gly Lys Gly Asp Asp Lys Leu 65 70 75 80 Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly Asn Asp 85 90 95 Tyr Leu Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg Ser Thr 100 105 110 Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp Leu Gly 115 120 125 Ser Thr Ala Gly Ser Thr Ala Gly Glu Leu Ala Gly Ile Thr Gly Lys 130 135 140 Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp Tyr Phe Gln Glu 145 150 155 160 Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser Lys Val Val Phe 165 170 175 Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser Gln Thr Ser Thr 180 185 190 Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly Thr Glu Ser Arg 195 200 205 Glu Arg Thr Gln Thr Gly Lys Gly Ser Thr Ala Gly Ser Thr Ala Phe 210 215 220 Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu 225 230 235 240 Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser 245 250 255 Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg 260 265 270 Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr 275 280 285 Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu 290 295 300 Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala 305 310 315 320 Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr 325 330 335 Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn 340 345 350 Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu 355 360 365 Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys 370 375 380 Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu 385 390 395 400 Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro 405 410 415 Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr 420 425 430 Ala ＜210＞ 21 ＜211＞ 435 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 帶有一個HindIII切位的重組ApxIII毒素 ＜400＞ 21 Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp Ile Leu 1 5 10 15 Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp 20 25 30 Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly Asn Gly 35 40 45 Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln Val Leu 50 55 60 Gly Asn Gly Phe Asn Val Leu Arg Ala Gly Lys Gly Asp Asp Lys Leu 65 70 75 80 Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly Asn Asp 85 90 95 Tyr Leu Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg Ser Thr 100 105 110 Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp Leu Gly 115 120 125 Ser Thr Ala Gly Ser Thr Ala Gly Glu Leu Ala Gly Ile Thr Gly Lys 130 135 140 Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp Tyr Phe Gln Glu 145 150 155 160 Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser Lys Val Val Phe 165 170 175 Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser Gln Thr Ser Thr 180 185 190 Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly Thr Glu Ser Arg 195 200 205 Glu Arg Thr Gln Thr Gly Lys Gly Ser Thr Ala Lys Leu Gly Ser Thr 210 215 220 Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp 225 230 235 240 Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 245 250 255 Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg 260 265 270 Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr 275 280 285 Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala 290 295 300 Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val 305 310 315 320 Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu 325 330 335 Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu 340 345 350 Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn 355 360 365 Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly 370 375 380 Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 385 390 395 400 Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu 405 410 415 Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly 420 425 430 Ser Thr Ala 435 ＜210＞ 22 ＜211＞ 31 ＜212＞ PRT ＜213＞ 豬(Sus scrofa
) ＜300＞ ＜308＞ GenBank: ADG26759 ＜309＞ 2010-05-08 ＜313＞ (201)..(231) ＜400＞ 22 Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu 1 5 10 15 Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 20 25 30 ＜210＞ 23 ＜211＞ 296 ＜212＞ PRT ＜213＞ 豬(Sus scrofa
) ＜300＞ ＜308＞ GenBank: ADG26759 ＜309＞ 2010-05-08 ＜313＞ (1)..(296) ＜400＞ 23 Thr Pro Ser Gly Cys Gly Lys Gln Asn Met Ile Gly Met Thr Pro Thr 1 5 10 15 Val Ile Ala Val His Tyr Leu Asp Ser Thr Glu Gln Trp Glu Lys Phe 20 25 30 Gly Leu Glu Lys Arg Gln Glu Ala Leu Glu Leu Ile Lys Lys Gly Tyr 35 40 45 Thr Gln Gln Leu Ala Phe Arg Gln Lys Asn Ser Ala Phe Ala Ala Phe 50 55 60 Gln Asp Arg Leu Ser Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val 65 70 75 80 Phe Ala Met Ala Ala Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys 85 90 95 Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val 100 105 110 Phe Glu Glu Asn Gly Pro Val Ile His Gln Glu Met Ile Gly Gly Phe 115 120 125 Lys Asn Thr Glu Glu Lys Asp Val Ser Leu Thr Ala Phe Val Leu Ile 130 135 140 Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln Val Asn Ser Leu 145 150 155 160 Leu Arg Ser Ile Asn Lys Ala Arg Asp Phe Leu Ala Asp Tyr Tyr Leu 165 170 175 Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala 180 185 190 Leu Ser Asp Lys Leu Asp Glu Pro Phe Leu Asn Lys Leu Leu Ser Thr 195 200 205 Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn 210 215 220 Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu Val Val Lys Asp 225 230 235 240 Phe Asp Ser Val Pro Pro Ile Val Arg Trp Leu Asn Glu Gln Arg Tyr 245 250 255 Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln 260 265 270 Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His Lys Asp Leu Asn 275 280 285 Leu Asp Val Ser Ile His Leu Pro 290 295 ＜210＞ 24 ＜211＞ 28 ＜212＞ PRT ＜213＞ 小鼠(Mus musculus
) ＜300＞ ＜308＞ GenBank: ADL70201 ＜309＞ 2010-08-18 ＜313＞ (211)..(238) ＜400＞ 24 Lys Phe Leu Asn Thr Ala Lys Asp Arg Asn Arg Trp Glu Glu Pro Asp 1 5 10 15 Gln Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 20 25 ＜210＞ 25 ＜211＞ 312 ＜212＞ PRT ＜213＞ 小鼠(Mus musculus
) ＜300＞ ＜308＞ GenBank: ADL70201 ＜309＞ 2010-08-18 ＜313＞ (1)..(312) ＜400＞ 25 Arg Leu Lys His Leu Ile Val Thr Pro Ala Gly Cys Gly Glu Gln Asn 1 5 10 15 Met Ile Gly Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln 20 25 30 Thr Glu Gln Trp Glu Lys Phe Gly Ile Glu Lys Arg Gln Glu Ala Leu 35 40 45 Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Lys Gln Pro 50 55 60 Ser Ser Ala Tyr Ala Ala Phe Asn Asn Arg Pro Pro Ser Thr Trp Leu 65 70 75 80 Thr Ala Tyr Val Val Lys Val Phe Ser Leu Ala Ala Asn Leu Ile Ala 85 90 95 Ile Asp Ser His Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu 100 105 110 Lys Gln Lys Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His 115 120 125 Gln Glu Met Ile Gly Gly Phe Arg Asn Ala Lys Glu Ala Asp Val Ser 130 135 140 Leu Thr Ala Phe Val Leu Ile Ala Leu Gln Glu Ala Arg Asp Ile Cys 145 150 155 160 Glu Gly Gln Val Asn Ser Leu Pro Gly Ser Ile Asn Lys Ala Gly Glu 165 170 175 Tyr Ile Glu Ala Ser Tyr Met Asn Leu Gln Arg Pro Tyr Thr Val Ala 180 185 190 Ile Ala Gly Tyr Ala Leu Ala Leu Met Asn Lys Leu Glu Glu Pro Tyr 195 200 205 Leu Gly Lys Phe Leu Asn Thr Ala Lys Asp Arg Asn Arg Trp Glu Glu 210 215 220 Pro Asp Gln Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu 225 230 235 240 Ala Leu Leu Leu Leu Lys Asp Phe Asp Ser Val Pro Pro Val Val Arg 245 250 255 Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln 260 265 270 Ala Thr Phe Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Thr Asp Val 275 280 285 Pro Asp His Lys Asp Leu Asn Met Asp Val Ser Phe His Leu Pro Ser 290 295 300 Cys Ser Ser Ala Thr Thr Phe Arg 305 310 ＜210＞ 26 ＜211＞ 4 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GSTA連接子的胺基酸序列 ＜400＞ 26 Gly Ser Thr Ala 1 ＜210＞ 27 ＜211＞ 9 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GS連接子的胺基酸序列 ＜400＞ 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 ＜210＞ 28 ＜211＞ 14 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GSGGG連接子的胺基酸序列 ＜400＞ 28 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser 1 5 10 ＜210＞ 29 ＜211＞ 6 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ (GS)3連接子的胺基酸序列 ＜400＞ 29 Gly Ser Gly Ser Gly Ser 1 5 ＜210＞ 30 ＜211＞ 15 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ (GGGGS)3連接子的胺基酸序列 ＜400＞ 30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 ＜210＞ 31 ＜211＞ 4 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GGSG連接子的胺基酸序列 ＜400＞ 31 Gly Gly Ser Gly 1 ＜210＞ 32 ＜211＞ 8 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ (GGSG)2連接子的胺基酸序列 ＜400＞ 32 Gly Gly Ser Gly Gly Gly Ser Gly 1 5 ＜210＞ 33 ＜211＞ 12 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ (GGSG)3連接子的胺基酸序列 ＜400＞ 33 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 ＜210＞ 34 ＜211＞ 10 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GENLYFQSGG連接子的胺基酸序列 ＜400＞ 34 Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly 1 5 10 ＜210＞ 35 ＜211＞ 36 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ GAST連接子的胺基酸序列 ＜400＞ 35 Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser 1 5 10 15 Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly 20 25 30 Thr Gly Ser Gly 35 ＜210＞ 36 ＜211＞ 36 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ SEG連接子的胺基酸序列 ＜400＞ 36 Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 1 5 10 15 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser 35 ＜210＞ 37 ＜211＞ 10 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 37 Arg Lys Gly Ala Asp Ala Lys Ser Gly Lys 1 5 10 ＜210＞ 38 ＜211＞ 1951 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 38 Met Thr Lys Leu Thr Met Gln Asp Val Thr Asn Leu Tyr Leu Tyr Lys 1 5 10 15 Thr Lys Thr Leu Pro Lys Asp Arg Leu Asp Asp Ser Leu Ile Ser Glu 20 25 30 Ile Gly Lys Gly Asp Asp Asp Ile Asp Arg Lys Glu Phe Met Val Gly 35 40 45 Pro Gly Arg Phe Val Thr Ala Asp Asn Phe Ser Val Val Arg Asp Phe 50 55 60 Phe Asn Ala Gly Lys Ser Arg Ile Ile Ala Pro Gln Val Pro Pro Ile 65 70 75 80 Arg Ser Gln Gln Glu Lys Ile Leu Val Gly Leu Lys Pro Gly Lys Tyr 85 90 95 Ser Lys Ala Gln Ile Leu Glu Met Leu Gly Tyr Thr Lys Gly Gly Glu 100 105 110 Val Val Asn Gly Met Phe Ala Gly Glu Val Gln Thr Leu Gly Phe Tyr 115 120 125 Asp Asp Gly Lys Gly Asp Leu Leu Glu Arg Ala Tyr Ile Trp Asn Thr 130 135 140 Thr Gly Phe Lys Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser 145 150 155 160 Gly Lys Arg Tyr Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln 165 170 175 Glu Asp Phe Asp Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg 180 185 190 Gly Leu Glu Ser Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn 195 200 205 Leu Asn Phe Thr Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg 210 215 220 Phe Lys Ala Glu Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala 225 230 235 240 Ala Lys Val Tyr Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp 245 250 255 Ser Gly Ser Ile Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu 260 265 270 Lys Pro Ile Ile Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly 275 280 285 Thr Lys Ile Thr Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile 290 295 300 Ala Asn Gln Lys Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser 305 310 315 320 Val Ser Gly Ser Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro 325 330 335 Thr Leu Asn Arg Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp 340 345 350 Ser Leu Ala Lys Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly 355 360 365 Val Val Tyr Val Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu 370 375 380 His Glu Asp Leu Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala 385 390 395 400 Arg Val Gly Asp Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr 405 410 415 Phe Val Arg Glu Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu 420 425 430 Phe Ser Glu Tyr Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly 435 440 445 Leu Leu Thr Tyr Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr 450 455 460 Ala Thr Phe Ala His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr 465 470 475 480 Thr Asn Tyr Arg Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys 485 490 495 Lys Leu Lys Ser Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val 500 505 510 Thr Pro Ile Asp Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr 515 520 525 Val Lys Asn Phe Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro 530 535 540 Asn Ser Tyr Tyr Tyr Phe Leu Glu Gly Lys Asp Thr Gly Phe Tyr Gly 545 550 555 560 His Ala Phe Tyr Ile Glu Arg Lys Asn Gly Gly Gly Ser Lys Asn Asn 565 570 575 Ser Ser Gly Ala Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly 580 585 590 Asn His Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn 595 600 605 Gly Asn Asn Gln Asn Asn Gly Ser Asn Gln Asp Asn Asn Ser Asp Val 610 615 620 Asn Ala Pro Asn Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu 625 630 635 640 Ala Leu Asp Leu Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly 645 650 655 Arg Gln Gly Ala Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala 660 665 670 Thr Gly Trp Leu Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn 675 680 685 Gln Asp Gly Ile Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn 690 695 700 Gln Leu Ser Asp Gly Ser Ile Ser Ala His Gly Phe Ala Ala Leu Ala 705 710 715 720 Asp Leu Asp Thr Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu 725 730 735 Phe Ser Lys Leu Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser 740 745 750 Glu Ala Asn Glu Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu 755 760 765 His Thr Ala Tyr Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile 770 775 780 Leu Ala Gln Leu Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Ala Gln 785 790 795 800 Met Gly Asp Leu Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr 805 810 815 Glu Ala Leu Asn Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr 820 825 830 Gly Thr Gly Arg Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu 835 840 845 Glu Leu Ala Ala Leu Leu Gln Gln Tyr Thr Lys Ala Ser Asp Phe Gln 850 855 860 Ala Gln Arg Glu Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr 865 870 875 880 Asp Leu Gln Tyr Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Leu Glu 885 890 895 Ser Thr Asp Ser Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu 900 905 910 Ser Ser Ile Arg Asn Val Lys His Asp Pro Thr Val Met Gln Asn Phe 915 920 925 Glu Gln Ser Lys Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu 930 935 940 Asn Ile Asp Gln Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile 945 950 955 960 Thr Asp Lys Val Asn Asn Met Tyr Gln Thr Thr Val Glu Leu Ala Tyr 965 970 975 Arg Ser Leu Leu Leu Gln Thr Arg Leu Lys Lys Tyr Val Tyr Ser Val 980 985 990 Asn Ala Lys Gln Phe Glu Gly Lys Trp Val Ala Asp Tyr Ser Arg Thr 995 1000 1005 Glu Ala Leu Phe Asn Ser Thr Phe Lys Gln Ser Pro Glu Asn Ala 1010 1015 1020 Leu Tyr Asp Leu Ser Glu Tyr Leu Ser Phe Phe Asn Asp Pro Thr 1025 1030 1035 Glu Trp Lys Glu Gly Leu Leu Leu Leu Ser Arg Tyr Ile Asp Tyr 1040 1045 1050 Ala Lys Ala Gln Gly Phe Tyr Glu Asn Trp Ala Thr Thr Ser Asn 1055 1060 1065 Leu Thr Ile Ala Arg Leu Arg Glu Ala Gly Val Ile Phe Ala Glu 1070 1075 1080 Ser Thr Asp Leu Lys Gly Asp Glu Lys Asn Asn Ile Leu Leu Gly 1085 1090 1095 Ser Gln Lys Asp Asn Asn Leu Ser Gly Ser Ala Gly Asp Asp Leu 1100 1105 1110 Leu Ile Gly Gly Glu Gly Asn Asp Thr Leu Lys Gly Ser Tyr Gly 1115 1120 1125 Ala Asp Thr Tyr Leu Phe Ser Lys Gly His Gly Gln Asp Val Ile 1130 1135 1140 Tyr Glu Tyr Ser Asp Ser Ala Asn Ser Lys Ser Asp Ile Asp Thr 1145 1150 1155 Leu Lys Phe Thr Asp Ile Asn Tyr Ala Glu Val Lys Phe Arg Arg 1160 1165 1170 Val Gly Asp Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp Ser 1175 1180 1185 Val Thr Val Lys Ser Phe Tyr Asn His Glu Tyr Tyr Gln Phe Glu 1190 1195 1200 Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Gly 1205 1210 1215 Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asp Ile 1220 1225 1230 Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn 1235 1240 1245 Asp Thr Ile Asn Gly Ser Tyr Gly Asp Asp Thr Leu Ile Gly Gly 1250 1255 1260 Thr Gly Asn Asp Ile Leu Lys Gly Ser Tyr Gly Ala Asp Thr Tyr 1265 1270 1275 Leu Phe Ser Lys Gly His Gly Gln Asp Val Ile Tyr Glu Tyr Ser 1280 1285 1290 Asp Ser Ala Asn Ser Lys Arg Asp Ile Asp Thr Leu Lys Phe Thr 1295 1300 1305 Asp Val Asn Tyr Ala Glu Val Lys Phe Arg Arg Val Gly Asp Asp 1310 1315 1320 Leu Met Leu Phe Gly Tyr His Asp Thr Asp Ser Val Thr Val Lys 1325 1330 1335 Ser Phe Tyr Asp His Glu Tyr Tyr Gln Phe Glu Lys Leu Glu Phe 1340 1345 1350 Ala Asp Arg Ser Ile Ser Arg Asp Glu Leu Ile Lys Ala Gly Leu 1355 1360 1365 His Leu Tyr Gly Thr Asp Gly Asn Asp Glu Ile Asn Asp His Ala 1370 1375 1380 Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly Asn Asp Ile Leu 1385 1390 1395 Arg Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His 1400 1405 1410 Gly Gln Asp Val Ile Tyr Glu Tyr Ser Asp Ser Ala Asn Ser Lys 1415 1420 1425 Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu 1430 1435 1440 Val Lys Phe Arg Arg Val Asp Asp Asp Leu Met Leu Phe Gly Tyr 1445 1450 1455 His Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Asp His Glu 1460 1465 1470 Tyr Tyr Gln Phe Glu Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr 1475 1480 1485 Arg Asp Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp 1490 1495 1500 Gly Asp Asp Asp Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp 1505 1510 1515 Ala Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly Asp Asp 1520 1525 1530 Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Lys Gly Ser Tyr 1535 1540 1545 Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile 1550 1555 1560 Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp 1565 1570 1575 Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe Arg 1580 1585 1590 Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp 1595 1600 1605 Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln Phe 1610 1615 1620 Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu 1625 1630 1635 Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp 1640 1645 1650 Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys 1655 1660 1665 Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile 1670 1675 1680 Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn 1685 1690 1695 Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp 1700 1705 1710 Val Asn Tyr Ala Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu 1715 1720 1725 Met Leu Phe Gly Tyr His Asp Thr Asp Ser Val Thr Ile Lys Ser 1730 1735 1740 Phe Tyr Asn His Val Asp Tyr Gln Phe Asp Lys Leu Asp Phe Ala 1745 1750 1755 Asp Arg Ser Ile Thr Arg Asp Glu Leu Gly Lys Gln Gly Met Ala 1760 1765 1770 Leu Phe Gly Thr Asp Gly Asp Asp Asn Ile Asn Asp Trp Gly Arg 1775 1780 1785 Asn Ser Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly 1790 1795 1800 Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile 1805 1810 1815 Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly 1820 1825 1830 His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 1835 1840 1845 Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Ile Asn Leu Ser 1850 1855 1860 Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu Ile Ile Lys Ser 1865 1870 1875 Leu Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp Tyr Ser His 1880 1885 1890 Gln Asp His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu Gln Thr 1895 1900 1905 Leu Val Ser Thr Gln Val Glu Lys Met Val Glu Ser Met Ala Gly 1910 1915 1920 Phe Ala Gln Lys His Gly Gly Glu Ile Ser Leu Ala Ser Pro Glu 1925 1930 1935 Glu Val Lys Gln Tyr Ile Asn Ser Leu Thr Ala Ala Leu 1940 1945 1950 ＜210＞ 39 ＜211＞ 16 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 39 Gly Gly Pro Leu Ala Tyr Ser Asn Ser Pro Asn Ser Ile Pro Asn Ala 1 5 10 15 ＜210＞ 40 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 40 Gly Ala Gly Gly Pro Leu Ala Tyr Ser Asn Ser Pro Asn Ser 1 5 10 ＜210＞ 41 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 41 Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser Asn Ser Pro Asn Ser 1 5 10 15 Ile Pro Asn Ala 20 ＜210＞ 42 ＜211＞ 6 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 42 Gly Ser Asn Arg Lys Asp 1 5 ＜210＞ 43 ＜211＞ 47 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 43 Gly Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly His Asp Ile 1 5 10 15 Leu Tyr Gly Asp Asp Gly Asn Asp Val Ile His Gly Gly Asp Gly Asn 20 25 30 Asp His Leu Val Gly Gly Asn Gly Asn Asp Arg Leu Ile Gly Gly 35 40 45 ＜210＞ 44 ＜211＞ 13 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 44 Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu Leu 1 5 10 ＜210＞ 45 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 45 Gly Ser Arg Phe Thr Asp Ile Phe His Gly Ala Lys Gly Asp Asp Glu 1 5 10 15 Ile Tyr Gly Asn 20 ＜210＞ 46 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 46 Asp Val Ile His Gly Gly Asp Gly Asn Asp His Leu Val Gly Gly Asn 1 5 10 15 Gly Asn Asp Arg 20 ＜210＞ 47 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 47 Ile Gly Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp Asp 1 5 10 15 Glu Leu Gln Val 20 ＜210＞ 48 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 48 His Gly Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly 1 5 10 ＜210＞ 49 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 49 Gly Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp 1 5 10 ＜210＞ 50 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 50 Asp Gly His Asp Ile Leu Tyr Gly Asp Asp Gly Asn Asp Val Ile His 1 5 10 15 Gly Gly Asp Gly 20 ＜210＞ 51 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 51 Arg Leu Ile Gly Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly Asp Gly 1 5 10 15 Asp Asp Glu Leu 20 ＜210＞ 52 ＜211＞ 15 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 52 Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys Asp Asn Asp Ile 1 5 10 15 ＜210＞ 53 ＜211＞ 19 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 53 His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr Asp Ser Gly Gly Gln 1 5 10 15 Asp Lys Leu ＜210＞ 54 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 54 Phe Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn Asn 1 5 10 15 Phe Leu Val Gly 20 ＜210＞ 55 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 55 Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr Asp Ser Gly 1 5 10 15 Gly Gln Asp Lys 20 ＜210＞ 56 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 56 Gly Gly Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn 1 5 10 ＜210＞ 57 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 57 Gly Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Lys Asp Asn 1 5 10 ＜210＞ 58 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 58 Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr Asp Ser Gly Gly 1 5 10 ＜210＞ 59 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 59 Ala Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn Asn Phe Leu Val 1 5 10 15 Gly Gly Thr Gly 20 ＜210＞ 60 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 60 Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile Thr Asp 1 5 10 15 Ser Gly Gly Gln 20 ＜210＞ 61 ＜211＞ 17 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 61 Leu Thr Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn 1 5 10 15 Ser ＜210＞ 62 ＜211＞ 12 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 62 Trp Thr Val Thr Asp Gly Asp Ala Ser Ser Ser Val 1 5 10 ＜210＞ 63 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 63 Thr Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn Ser 1 5 10 15 Tyr Ile Thr Lys 20 ＜210＞ 64 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 64 Leu Phe Arg Thr Pro Leu Leu Thr Pro Gly Glu Glu Asn Arg 1 5 10 ＜210＞ 65 ＜211＞ 12 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 65 Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly Val Gln 1 5 10 ＜210＞ 66 ＜211＞ 60 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 66 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly 20 25 30 Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile 35 40 45 Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg 50 55 60 ＜210＞ 67 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 67 Asn Arg Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp Asp Arg 1 5 10 ＜210＞ 68 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 68 Pro Thr Asn Val Gly Asn Arg Glu Glu Lys Ile Glu Tyr Arg Arg Glu 1 5 10 15 Asp Asp Arg Phe 20 ＜210＞ 69 ＜211＞ 62 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 69 Gly Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp Ile 1 5 10 15 Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn 20 25 30 Asp Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly Asn 35 40 45 Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu 50 55 60 ＜210＞ 70 ＜211＞ 33 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 70 Ala Gly Lys Gly Asp Asp Lys Leu Tyr Gly Ser Ser Gly Ser Asp Leu 1 5 10 15 Leu Asp Gly Gly Glu Gly Asn Asp Tyr Leu Glu Gly Gly Asp Gly Ser 20 25 30 Asp ＜210＞ 71 ＜211＞ 16 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 71 Ser Thr Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp 1 5 10 15 ＜210＞ 72 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 72 Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp Gln 1 5 10 15 Leu Tyr Gly Gly 20 ＜210＞ 73 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 73 Leu Leu Gly Gly Asn Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn 1 5 10 15 Asp Glu Leu Gln 20 ＜210＞ 74 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 74 Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn 1 5 10 ＜210＞ 75 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 75 Gly Gly Asn Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn 1 5 10 ＜210＞ 76 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 76 Asp Gly Asp Asp Ile Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg 1 5 10 15 Gly Asp Asn Gly 20 ＜210＞ 77 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 77 Leu Leu Gly Gly Asn Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn 1 5 10 15 Asp Glu Leu Gln 20 ＜210＞ 78 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 78 Asn Gly Phe Asn Val Leu Arg Ala Gly Lys Gly Asp Asp Lys Leu Tyr 1 5 10 15 Gly Ser Ser Gly 20 ＜210＞ 79 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 79 Gly Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala 1 5 10 ＜210＞ 80 ＜211＞ 9 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 80 Leu Leu Glu Lys Lys Pro Asp Asp Phe 1 5 ＜210＞ 81 ＜211＞ 15 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 81 Phe Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser Gln Thr 1 5 10 15 ＜210＞ 82 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 82 Gly Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val 1 5 10 15 Asp Tyr Phe Gln 20 ＜210＞ 83 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 83 Val Phe Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser Gln Thr 1 5 10 15 Ser Thr Leu Leu 20 ＜210＞ 84 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 84 Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp Tyr 1 5 10 ＜210＞ 85 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 85 Glu Lys Lys Pro Asp Asp Phe Ser Lys Val Val Phe Asp Pro 1 5 10 ＜210＞ 86 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 86 Phe Val Thr Pro Leu Leu Thr Pro Gly Thr Glu Ser Arg Glu 1 5 10 ＜210＞ 87 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 87 Glu Leu Ala Gly Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys 1 5 10 15 Ala Tyr Val Asp 20 ＜210＞ 88 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 88 Thr Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly Thr Glu Ser 1 5 10 15 Arg Glu Arg Thr 20 ＜210＞ 89 ＜211＞ 84 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 89 Glu Gly Lys Asp Thr Gly Phe Tyr Gly His Ala Phe Tyr Ile Glu Arg 1 5 10 15 Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala Gly Asn Ser 20 25 30 Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg Asn Asn Ala Ser 35 40 45 Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln Asn Asn Gly 50 55 60 Ser Asn Gln Asp Asn Asn Ser Asp Val Asn Ala Pro Asn Asn Pro Gly 65 70 75 80 Arg Asn Tyr Asp ＜210＞ 90 ＜211＞ 66 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 90 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Lys Gly Ser 20 25 30 Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile 35 40 45 Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr 50 55 60 Leu Lys 65 ＜210＞ 91 ＜211＞ 440 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 重組ApxIV毒素 ＜400＞ 91 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly 20 25 30 Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile 35 40 45 Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Gly Ser Thr Ala 50 55 60 Glu Gly Lys Asp Thr Gly Phe Tyr Gly His Ala Phe Tyr Ile Glu Arg 65 70 75 80 Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala Gly Asn Ser 85 90 95 Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg Asn Asn Ala Ser 100 105 110 Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln Asn Asn Gly 115 120 125 Ser Asn Gln Asp Asn Asn Ser Asp Val Asn Ala Pro Asn Asn Pro Gly 130 135 140 Arg Asn Tyr Asp Gly Ser Thr Ala Gly Ser Thr Ala Val Ile Asp Ala 145 150 155 160 Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly Asp Asp Thr Leu 165 170 175 Ile Gly Gly Lys Gly Asn Asp Ile Leu Lys Gly Ser Tyr Gly Ala Asp 180 185 190 Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp 195 200 205 Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Gly Ser 210 215 220 Thr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys 225 230 235 240 Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu 245 250 255 Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser 260 265 270 Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr 275 280 285 Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys 290 295 300 Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln 305 310 315 320 Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe 325 330 335 Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu 340 345 350 Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser 355 360 365 Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg 370 375 380 Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr 385 390 395 400 Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu 405 410 415 Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala 420 425 430 Thr Ser Tyr Ala Gly Ser Thr Ala 435 440 ＜210＞ 92 ＜211＞ 442 ＜212＞ PRT ＜213＞ 人工序列 ＜220＞ ＜223＞ 帶有一個HindIII切位的重組ApxIV毒素 ＜400＞ 92 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly 20 25 30 Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile 35 40 45 Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Gly Ser Thr Ala 50 55 60 Glu Gly Lys Asp Thr Gly Phe Tyr Gly His Ala Phe Tyr Ile Glu Arg 65 70 75 80 Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala Gly Asn Ser 85 90 95 Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg Asn Asn Ala Ser 100 105 110 Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln Asn Asn Gly 115 120 125 Ser Asn Gln Asp Asn Asn Ser Asp Val Asn Ala Pro Asn Asn Pro Gly 130 135 140 Arg Asn Tyr Asp Gly Ser Thr Ala Gly Ser Thr Ala Val Ile Asp Ala 145 150 155 160 Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly Asp Asp Thr Leu 165 170 175 Ile Gly Gly Lys Gly Asn Asp Ile Leu Lys Gly Ser Tyr Gly Ala Asp 180 185 190 Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp 195 200 205 Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Gly Ser 210 215 220 Thr Ala Lys Leu Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr 225 230 235 240 Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn 245 250 255 Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu 260 265 270 Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys 275 280 285 Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala Phe Leu 290 295 300 Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro 305 310 315 320 Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Gly Ser Thr 325 330 335 Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp 340 345 350 Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala 355 360 365 Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala Lys Glu Arg 370 375 380 Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr 385 390 395 400 Ser Tyr Ala Gly Ser Thr Ala Phe Leu Asn Lys Leu Leu Ser Thr Ala 405 410 415 Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu Tyr Asn Val 420 425 430 Glu Ala Thr Ser Tyr Ala Gly Ser Thr Ala 435 440 ＜210＞ 93 ＜211＞ 30 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 93 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg 20 25 30 ＜210＞ 94 ＜211＞ 4 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 94 Gly Tyr Gly Ala 1 ＜210＞ 95 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 95 Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr 1 5 10 15 Leu Ile Gly Gly 20 ＜210＞ 96 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 96 Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp 1 5 10 15 Asn Arg Ala Arg 20 ＜210＞ 97 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 97 Asp Ala Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly 1 5 10 ＜210＞ 98 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 98 Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg 1 5 10 ＜210＞ 99 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 99 Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp 1 5 10 15 Asn Arg Ala Arg 20 ＜210＞ 100 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 100 Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala Gly Asn Ser 1 5 10 15 Lys Asp Trp Gly 20 ＜210＞ 101 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 101 His Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly 1 5 10 15 Asn Asn Gln Asn 20 ＜210＞ 102 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 102 His Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly 1 5 10 15 Asn Asn Gln Asn 20 ＜210＞ 103 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 103 Glu Arg Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly 1 5 10 ＜210＞ 104 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 104 Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly Asn His 1 5 10 ＜210＞ 105 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 105 Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln Asn Asn Gly Ser 1 5 10 ＜210＞ 106 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 106 Asp Asn Asn Ser Asp Val Asn Ala Pro Asn Asn Pro Gly Arg 1 5 10 ＜210＞ 107 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 107 Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala Gly Asn Ser Lys 1 5 10 15 Asp Trp Gly Gly 20 ＜210＞ 108 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 108 Asn Gln Asn Asn Gly Ser Asn Gln Asp Asn Asn Ser Asp Val Asn Ala 1 5 10 15 Pro Asn Asn Pro 20 ＜210＞ 109 ＜211＞ 30 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 109 Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly 1 5 10 15 Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp Ile Leu Lys 20 25 30 ＜210＞ 110 ＜211＞ 4 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 110 Ser Tyr Gly Ala 1 ＜210＞ 111 ＜211＞ 19 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 111 Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 1 5 10 15 Ala Arg Asp ＜210＞ 112 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 112 Asp Ala Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly Asp Asp 1 5 10 15 Thr Leu Ile Gly 20 ＜210＞ 113 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 113 Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp 1 5 10 15 Asn Arg Ala Arg 20 ＜210＞ 114 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 114 Asp Ala Gly Ala Gly Asn Asp Thr Ile Asn Gly Gly Tyr Gly 1 5 10 ＜210＞ 115 ＜211＞ 14 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 115 Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg 1 5 10 ＜210＞ 116 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 116 Asn Gly Gly Tyr Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp 1 5 10 15 Ile Leu Lys Gly 20 ＜210＞ 117 ＜211＞ 20 ＜212＞ PRT ＜213＞ 豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae
) ＜400＞ 117 Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp 1 5 10 15 Asn Arg Ala Arg 20
Claims (22)
- 一種豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae )重組毒素蛋白，其係以下列之式(I)表示： (A)m -(C3d片段)n ； 式(I)； 其中每一個A代表一個獨立的豬胸膜肺炎放線桿菌毒素蛋白的抗原決定位； 其中每一個C3d片段代表一個獨立的補體裂解片段C3d的胺基酸序列，該每一個C3d片段係各自獨立選自於由SEQ ID NOs: 22、23、24及25所組成的群組； 其中m是代表從1至約30的整數；以及 其中n是代表從0至約10的整數。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中每一個A之間以一個連接子連接，每一個連接子係各自獨立選自於由Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36所組成的群組。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中每一個C3d片段之間以一個連接子連接，每一個連接子係各自獨立選自於由Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36所組成的群組。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中A與C3d片段之間以一連接子連接，該連接子係選自於由Gly-Gly、Gly-Ser及SEQ ID NOs: 26、27、28、29、30、31、32、33、34、35及36所組成的群組。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白I，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白I，且每一個A係各自獨立選自於由SEQ ID NOs: 2、3、4、5、6、37、39、40、41、42、43、44、45、46、47、48、49、50及51所組成之群組。
- 如申請專利範圍第5項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌重組毒素蛋白I包含如SEQ ID NO: 7或8所示的胺基酸序列。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白II，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白II，且每一個A係各自獨立選自於由SEQ ID NOs: 10、11、12、13、14、52、53、54、55、56、57、58、59、60、61、62、63、64、65、67及68所組成之群組。
- 如申請專利範圍第7項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌重組毒素蛋白II包含如SEQ ID NO: 15或16所示的胺基酸序列。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白III，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白III，且每一個A係各自獨立選自於由SEQ ID NOs: 18、19、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87及88所組成之群組。
- 如申請專利範圍第9項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌重組毒素蛋白III包含如SEQ ID NO: 20或21所示的胺基酸序列。
- 如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌毒素蛋白為豬胸膜肺炎放線桿菌毒素蛋白IV，該豬胸膜肺炎放線桿菌重組毒素蛋白為豬胸膜肺炎放線桿菌重組毒素蛋白IV，且每一個A係各自獨立選自於由SEQ ID NOs: 66、89、90、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116及117所組成之群組。
- 如申請專利範圍第11項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，其中該豬胸膜肺炎放線桿菌重組毒素蛋白IV包含如SEQ ID NO: 91或92所示的胺基酸序列。
- 一種編碼如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白的核苷酸序列。
- 一種豬放線桿菌胸膜肺炎免疫組合物，包含一如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白以及一藥學上可接受的載劑。
- 如申請專利範圍第14項所述之豬放線桿菌胸膜肺炎免疫組合物，其中該豬胸膜肺炎放線桿菌重組毒素蛋白係選自由下列群組所組成之至少一者：一如申請專利範圍第5項所述之豬胸膜肺炎放線桿菌重組毒素蛋白I、一如申請專利範圍第7項所述之豬胸膜肺炎放線桿菌重組毒素蛋白II、一如申請專利範圍第9項所述之豬胸膜肺炎放線桿菌重組毒素蛋白III，以及一如申請專利範圍第11項所述之豬胸膜肺炎放線桿菌重組毒素蛋白IV。
- 如申請專利範圍第14項所述之豬放線桿菌胸膜肺炎免疫組合物，進一步包含至少一個豬胸膜肺炎放線桿菌血清型全菌。
- 如申請專利範圍第14項所述之豬放線桿菌胸膜肺炎免疫組合物，進一步包含其他病原抗原，該病原抗原係選自由下列群組所組成者：豬環狀病毒第二型(PCV2)抗原、豬流感病毒(SIV)抗原、豬繁殖與呼吸症候群病毒(PRRSV)抗原、豬黴漿菌(Mycoplasma)、豬小病毒 (Parvovirus, PPV)、豬丹毒(Erysipelas)、支氣管敗血性博德氏桿菌(Bordetella bronchiseptica )、敗血性巴氏桿菌(Pasteurella multocida )，以及偽狂犬病(Aujeszky's disease)。
- 一種動物對抗豬放線桿菌胸膜肺炎的方法，包含使用如申請專利範圍第14項所述之免疫組合物以施予一動物，以增強該動物對抗豬放線桿菌胸膜肺炎之免疫力。
- 一種抗豬胸膜肺炎放線桿菌毒素蛋白的抗體，係藉由如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白所製備而得。
- 如申請專利範圍第19項所述之抗體，該抗體包含至少下列其中一種：一單株抗體、一多株抗體，以及一經基因重組之抗體。
- 一種豬放線桿菌胸膜肺炎的檢測套組，包含一偵測單元，該偵測單元係選自於下列群組所組成中至少一者：一如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白，以及一藉由如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白所製備之抗體。
- 如申請專利範圍第21項所述之檢測套組，其中該抗體包含至少下列其中一種：一單株抗體、一多株抗體，以及一經基因重組之抗體。
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| TW105113267A TWI693230B (zh) | 2015-04-28 | 2016-04-28 | 豬胸膜肺炎放線桿菌重組毒素蛋白及其應用 |
| TW106129199A TWI693231B (zh) | 2015-04-28 | 2016-04-28 | 豬胸膜肺炎放線桿菌重組毒素蛋白及其應用 |
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| CN112391352B (zh) * | 2020-10-26 | 2023-03-28 | 湖北省农业科学院畜牧兽医研究所 | 一种胸膜肺炎放线杆菌rApxⅣAN的单克隆抗体及其应用 |
| CN113980101B (zh) * | 2021-09-11 | 2023-06-30 | 江苏南农高科技股份有限公司 | 一种胸膜肺炎放线杆菌亚单位疫苗 |
| CN113980908B (zh) * | 2021-09-11 | 2023-07-21 | 南京农业大学 | 一种胸膜肺炎放线杆菌ApxIV蛋白单克隆抗体及其阻断ELISA试剂盒 |
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| CN1511844A (zh) * | 2002-07-31 | 2004-07-14 | 张甘楠 | 猪胸膜肺炎放线杆菌基因工程类毒素ApxI与死菌混合疫苗 |
| TWI548746B (zh) * | 2009-08-06 | 2016-09-11 | 英特威特國際股份有限公司 | 防備豬胸膜肺炎之疫苗及製備該疫苗之方法 |
| CN101691396A (zh) * | 2009-09-16 | 2010-04-07 | 天津农学院 | 一种猪胸膜肺炎放线杆菌的重组外膜蛋白、编码基因、表达载体及其制备方法 |
| CN101691369A (zh) * | 2009-10-09 | 2010-04-07 | 江苏大学 | 一种叶酸铬(ⅲ)配合物及其制备方法 |
| CN102925548B (zh) * | 2012-08-02 | 2014-12-24 | 四川农业大学 | 猪传染性胸膜肺炎放线杆菌lamp试剂盒及使用方法 |
| EA201591861A1 (ru) * | 2013-03-22 | 2016-03-31 | Сбк Вирбак Лимитед | Рекомбинантный токсин pasteurella multocida и его применение |
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| US20190048045A1 (en) | 2019-02-14 |
| TWI693230B (zh) | 2020-05-11 |
| TWI693231B (zh) | 2020-05-11 |
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| WO2016173504A1 (zh) | 2016-11-03 |
| CN107614534B (zh) | 2021-03-09 |
| CN107614534A (zh) | 2018-01-19 |
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