TWI693231B - 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 PDF

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TWI693231B
TWI693231B TW106129199A TW106129199A TWI693231B TW I693231 B TWI693231 B TW I693231B TW 106129199 A TW106129199 A TW 106129199A TW 106129199 A TW106129199 A TW 106129199A TW I693231 B TWI693231 B TW I693231B
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楊瀅臻
郭村勇
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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

豬胸膜肺炎放線桿菌重組毒素蛋白及其應用
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
Figure 106129199-A0304-0001
 * 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
Figure 106129199-A0304-0002
 * 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
Figure 106129199-A0304-0003
 * 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＞  P17-0188   ＜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 (8)

  1. 一種豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae)重組毒素蛋白，實質上由一胺基酸序列所組成，該胺基酸序列係選自由SEQ ID NOs：15、16、20、21、91，以及92所組成之群組。
  2. 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。
  3. 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。
  4. 如申請專利範圍第3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SEQ ID NO：15或16所示之豬胸膜肺炎放線桿菌重組毒素蛋白II、一如SEQ ID NO：20或21所示之豬胸膜肺炎放線桿菌重組毒素蛋白III，以及一如SEQ ID NO：91或92所示之豬胸膜肺炎放線桿菌重組毒素蛋白IV。
  5. 如申請專利範圍第3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。
  6. 如申請專利範圍第3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)。
  7. 一種使用如申請專利範圍第1項所述之豬胸膜肺炎放線桿菌重組毒素蛋白於製造供動物對抗豬放線桿菌胸膜肺炎的疫苗之用途。
  8. 一種豬放線桿菌胸膜肺炎的檢測套組，包含一偵測單元，該偵測單元係為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。
TW106129199A 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 TWI693231B (zh)

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