TWI649331B - 鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原及其抗體以及編碼該抗原之核酸 - Google Patents
鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原及其抗體以及編碼該抗原之核酸 Download PDFInfo
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- TWI649331B TWI649331B TW104118940A TW104118940A TWI649331B TW I649331 B TWI649331 B TW I649331B TW 104118940 A TW104118940 A TW 104118940A TW 104118940 A TW104118940 A TW 104118940A TW I649331 B TWI649331 B TW I649331B
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- acinetobacter baumannii
- amino acid
- antigen
- acinetobacter
- acid sequence
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- Oncology (AREA)
Abstract
本發明係關於一種鮑氏不動桿菌(Acinetobacter baumannii)的多胜肽抗原,該多胜肽抗原包括至少一選自由下列所組成之群組中之胺基酸序列:(a)SEQ ID NO:1至5之胺基酸序列;(b)依據SEQ ID NO:1、3、4、5進行20%以下胺基酸取代、刪除或添加之胺基酸序列;(c)依據SEQ ID NO:2進行40%以下胺基酸取代、刪除或添加之胺基酸序列;以及(d)依據(a)至(c)胺基酸序列之片段;其中,依據(b)至(d)胺基酸序列之多胜肽係具有免疫刺激活性。藉由本發明所提供鮑氏不動桿菌的共通抗原,可應用於製備預防鮑氏不動桿菌感染之通用疫苗,並透過相對應抗體之製備可應用於鮑氏不動桿菌之檢測與治療。
Description
本發明係關於一種用以產生免疫反應的抗原,尤其是關於一種鮑氏不動桿菌多胜肽抗原。本發明亦係關於包括該鮑氏不動桿菌多胜肽抗原的疫苗組成物,以及可專一性結合鮑氏不動桿菌多胜肽抗原的抗體。同時,本發明亦係關於編碼該鮑氏不動桿菌多胜肽抗原之單離核酸,包括該單離核酸之表現載體以及包括該表現載體之宿主細胞。
不動桿菌屬(Acinetobacter spp.)細菌係廣泛地分佈於自然界當中,其大約分有20個種,屬於革蘭氏陰性桿菌。其中,鮑氏不動桿菌(Acinetobacter baumannii)是最常見且被研究的菌種,他們能夠在各種濕潤或乾燥的物體表面上生存,目前已是醫院環境裡最常引起院內感染的病原之一。鮑氏不動桿菌係一伺機性病原菌,其對於免疫力正常的宿主而言是無害的,但在宿主免疫力下降時,此細菌就容易引起感染,造成疾病。
舉凡手套、針筒、針頭、推車、呼吸器、床鋪、櫃架、水槽、地板、空調出風迴風口,甚至連聽診器、病歷都可能存有鮑氏不動桿菌,而在潮濕溫暖的飲水、食物以及和排水道中,或是人體上,例如皮膚、腋下、結膜、口腔、上呼吸道、鼻咽及腸胃道、尿道等,更是容易見到鮑氏不動桿菌的蹤跡。接觸到這些細菌的病患或家屬,經常就在抵抗力變弱時成為發病的宿主。特別的是,伺機性病原菌在進行侵入性治療例如插管、手術時,更增加了院內感染的機率,成為病人健康與醫護的一大隱憂。
一般對於細菌之感染,其治療方法係使用抗生素。然而,由
於抗生素的濫用,該些細菌逐漸產生抗藥性而難以消滅,目前,為了對抗越來越多院內感染而對抗生素具有抗藥性的細菌,患者必須更換抗生素種類或使用昂貴的新型抗生素,若細菌的抗藥性繼續發展演化,將來恐怕會面臨無有效抗生素可用的狀況。關於造成院內感染的鮑氏不動桿菌,目前臨床上已分離出具有多重抗藥性的菌株,由於其在物體表面上仍可存活相當一段時日,因此若無適當的治療或預防方式,未來勢必成為院內感染防治的棘手難題。
如前所述,鮑氏不動桿菌之無所不在與其抗藥性已逐漸成為院內感染防治的重大難題,因此若能對人體施打疫苗產生預防力,即可有效避免鮑氏不動桿菌的感染。一般疫苗係利用失去活性或減毒的病原體,注入人體內,藉由人體免疫系統產生體液性(例如抗體)與細胞性(例如:細胞毒性、輔助型或調節型T細胞)等免疫反應,而可於將來病原體進入宿主生物體後將該病原體中和後清除。然而,利用失去活性或減毒的病原體疫苗,對於某些疾病的預防是危險的,尤其是缺乏該些病原體相關的病理條件與減毒特性時。因此,利用病原體上能夠產生免疫刺激反應的抗原作為疫苗組成物,而不是引入整個的致病病原體,已是目前製備較具安全性疫苗之主要方法之一。
然而,如何找尋到鮑氏不動桿菌中具特異性的抗原而用以製作疫苗卻是困難的。雖然在先前的研究中,Pantophlet等人曾利用不動桿菌脂多醣體的O抗原和相對應的抗體,用以鑑定不動桿菌菌株(Pantophlet R.et al.,Clinical and Diagnostic Laboratory Immunology,9,60-65,2002),但其並未揭露其他抗原以及任何關於疫苗的應用,而美國公告第6,562,958號雖然也公開了大約4000種與鮑氏不動桿菌相關之核苷酸和胺基酸序列,但這些序列極大部分並不清楚其是否為可表現的基因,也不知其功能為何。因此,為了預防鮑氏不動桿菌之感染同時建立起精確之檢測診斷方式,找尋並利用能有效產生免疫反應之抗原目標以及具專一性的檢測、治療用抗體,有其迫切的需求。
本發明之目的在於提供一種鮑氏不動桿菌間共有之多胜肽抗原,用以製備疫苗組成物以預防鮑氏不動桿菌或具有相類似抗原病原菌之感染。本發明之另一目標則同時提供一種可專一性對抗該多胜肽抗原之抗體,以做為檢測或治療之用。
為了達成前述之目的,本發明提供一種單離的鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原,該多胜肽抗原包括至少一選自由下列所組成之群組中之胺基酸序列:(a)SEQ ID NO:1至5之胺基酸序列;(b)依據SEQ ID NO:1、3、4、5進行20%以下胺基酸取代、刪除或添加之胺基酸序列;(c)依據SEQ ID NO:2進行40%以下胺基酸取代、刪除或添加之胺基酸序列;以及(d)依據(a)至(c)胺基酸序列之片段;其中,依據(b)至(d)胺基酸序列之多胜肽係具有免疫刺激活性。前述SEQ ID NO:1至5之胺基酸序列請參見序列表。
在一實施例中,本發明提供一種對抗鮑氏不動桿菌的抗體,該抗體係可專一性地與前述之多胜肽抗原相結合。其中,該抗體可為多株抗體或單株抗體。此外,該抗體除了具二輕鏈、二重鏈之一般完整抗體結構外,其亦可為經重組選殖但具有相同抗原識別專一性之抗體片段(antibody fragment),例如Fab、F(ab’)2、Fv或ScFv片段抗體。
在另一實施例中,本發明提供一種醫藥組成物,包括前述對抗鮑氏不動桿菌多胜肽抗原的抗體以及一藥學上可接受之載劑。
在本發明再一實施例中,提供一種檢測鮑氏不動桿菌的方法,其步驟包括:提供一如前述可專一性地與前述之多胜肽抗原相結合之抗體,將其與待測樣品混合後,利用偵測試劑或儀器,分析該抗體之結合狀況,以判定待測樣品中是否存有鮑氏不動桿菌。該檢測方式可為西方墨點法或ELISA等常用之免疫反應檢測方法。
在本發明一實施例中,同時提供一種檢測鮑氏不動桿菌的套組,其包括如前所述之抗體。
在另一實施例中,本發明進一步提供一種利用如前所述之鮑氏不動桿菌多胜肽抗原作為疫苗之用途,以及一種包括至少一前述鮑氏不
動桿菌多胜肽抗原的疫苗組成物。該疫苗組成物,可進一步包括藥學上可接受載劑與/或佐劑。其中,「藥學上可接受載劑」係指不影響有效成分藥理活性且對於投予的生物體無毒性之任何載劑,除依據劑型種類所選用之賦形劑外,可包括但不限於:稀釋劑、穩定劑、防腐劑、抗氧化劑、分散劑、增溶劑、抗菌劑、抗真菌劑與佐劑等免疫刺激劑,以及以上之組合。「佐劑」則係指不同於疫苗所使用之抗原組成分,但可增加抗原反應之物質。佐劑可為但不限於戈布佐劑(Gerbu adjuvant)、分枝桿菌或棒狀桿菌、霍亂毒素、破傷風類毒素或各種油水乳化物(例如:IDEC-AF)。此外該佐劑也可包括礦物鹽或礦物凝膠(例如:氫氧化鋁、磷酸鋁和鈣磷酸鹽)、表面活性物質(例如:卵磷脂)以及免疫刺激物質(例如:皂甙)、寡核苷酸、白細胞介素-2等。
為了製備如前所述之多胜肽抗原,本發明於一實施例中,更提供一種單離的核酸,其係編碼至少一選自由下列所組成之群組中之胺基酸序列,該胺基酸序列係為鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原:(a)SEQ ID NO:1至5之胺基酸序列;(b)依據SEQ ID NO:1、3、4、5進行20%以下胺基酸取代、刪除或添加之胺基酸序列;(c)依據SEQ ID NO:2進行40%以下胺基酸取代、刪除或添加之胺基酸序列;以及(d)依據(a)至(c)胺基酸序列之片段;其中,依據(b)至(d)胺基酸序列之多胜肽係具有免疫刺激活性。
在一實施例中,前述之單離的核酸可包括至少一選自由SEQ ID NO:6至10所組成之群組之序列。
進一步,本發明提供一種表現載體,可將前述之核酸選殖於該載體中,並連接有一可調控其表現之轉錄調節因子,藉以表現多胜肽抗原。此處「調控」可直接調控、調節啟動子(例如lac operon)以啟動DNA之轉錄以及後續之轉譯,或是間接調控、調節轉錄或轉譯過程所需酵素(例如聚合酶)或其他調節子之表現,以使DNA之轉錄或轉譯能夠進行,而最後表現出多胜肽抗原。
在一實施例中,本發明再提供一種宿主細胞,該宿主細胞可轉染以前述之表現載體或是導入前述可表現多胜肽抗原之核酸,而使該宿
主細胞表現出所需之多胜肽抗原。
在另一實施例中,本發明再提供一種檢測鮑氏不動桿菌的套組,其包括一引子對,該引子對可經由聚合酶鏈鎖反應針對含有鮑氏不動桿菌的待測樣品中之模板核酸增幅產生前述之單離的核酸或該核酸之片段,亦即,該引子對可增幅該核酸之全長或其中之片段。藉此,除前述以抗體偵測鮑氏不動桿菌菌體的存在外,亦可從分子階層檢測是否存有鮑氏不動桿菌的核酸,而達到篩檢鮑氏不動桿菌的目的。
以下將配合圖式進一步說明本發明的實施方式,下述所列舉的實施例係用以闡明本發明,並非用以限定本發明之範圍,任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
第一圖係本發明實施例1中,經二維電泳後利用西方墨點法與銀染偵測鮑氏不動桿菌外膜具免疫誘導活性之抗原之結果圖。其中,(a)與(b)圖係分別以不同的人類血清進行反應,各圖中左側係西方墨點法分析結果圖,右側則為銀染結果圖,而箭頭所指處表示相對應之蛋白位置。
第二圖係本發明實施例1中pET-NcsP與pET-TonB-R質體表現之重組蛋白質經以孔雀藍染色與人體血清進行西方墨點法分析之結果圖。其中,(a)圖係pET-NcsP質體的表現;(b)圖係pET-TonB-R質體的表現;「M」表示蛋白質分子量標記;「-」表示未加入IPTG,「+」表示有加入IPTG以誘導後續蛋白質的表現;CB表示孔雀藍;HS-AB表示人類血清;「*」表示表現之重組蛋白。
第三圖係本發明實施例3中分別以抗原NcsP、TonB-R、Pase1與Pase2免疫小鼠後之存活率測試結果之比較圖。其中,(a)為施打TonB-R,(b)為NcsP,(c)為Pase1,而(d)則為Pase2,其中PBS為對照組。
第四圖係本發明實施例4中多胜肽抗原關於MTT測試之結果圖。
第五圖係本發明實施例6中anti-GP1抗血清之殺菌力測試結果圖。
第六圖係本發明實施例6中以人體血清對重組GP1抗原進行西方墨點法分析之結果圖。其中,行1為鮑氏不動桿菌外膜蛋白A(控制組),行2為TonB-R抗原,行3為GP1抗原。左圖為正常人血清(對照組),右圖為鮑氏不動桿菌感染的病人血清。
本發明所提供之單離的鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原,其中,該多胜肽抗原包括至少一選自由下列所組成之群組中之胺基酸序列:(a)SEQ ID NO:1至5之胺基酸序列;(b)依據SEQ ID NO:1、3、4、5進行20%以下胺基酸取代、刪除或添加之胺基酸序列;(c)依據SEQ ID NO:2進行40%以下胺基酸取代、刪除或添加之胺基酸序列;以及(d)依據(a)至(c)胺基酸序列之片段;其中,依據(b)至(d)胺基酸序列之多胜肽係具有免疫刺激活性。
前述關於多胜肽抗原的胺基酸序列以及相對應之核苷酸序列之序列識別號(SEQ ID NO),係對照以多胜肽抗原名稱如下述表1所示。各序列識別號之胺基酸或核苷酸序列,請參見序列表。
其中,SEQ ID NO:1至5係本發明實施例中可作為多胜肽抗原之胺基酸序列,而SEQ ID NO:6至10則係其分別相對應之核苷酸序列。作
為本發明鮑氏不動桿菌多胜肽抗原的實施例,該多胜肽抗原可為(a)至(d)胺基酸序列群組中任一胺基酸序列或其組合之具有免疫刺激活性的多胜肽抗原,其中,除可包括SEQ ID NO:1至5中任一胺基酸序列外,亦可包括進行20%以下序列修飾的SEQ ID NO:1、3、4、5,或是進行40%以下序列修飾的SEQ ID NO:2,抑或是SEQ ID NO:1至5或前述經修飾序列之片段。關於胺基酸序列選用之方式或組合及其比例,於此僅為例示,並未設有特別的限制。
本專利說明書中所稱之「單離的」,係指該物質之型態與所伴隨之組份與自然界中所存在者並不相同,該物質係經分離或純化而與自然存在形態下所伴隨之組份分離,或是藉由人工方式產生或製造者。
本說明書中所提及之「抗原」,係指一種能夠刺激生物體產生免疫反應的物質,亦即,係一種具有「免疫刺激活性」(immunostimulatory activity)的物質。「免疫刺激活性」,則係指該抗原進入生物體後,能夠引起體液性(humoral)反應產生抗體,或是產生細胞性(cellular)反應啟動T細胞之作用,而使其能辨識並與該抗原結合作用,以及產生相關免疫分子等免疫反應之特性。此等免疫反應可由該抗原逕自誘導產生,或是透過佐劑的輔助而引起。因此,「鮑氏不動桿菌多胜肽抗原」,係指一種能在生物體內針對鮑氏不動桿菌多胜肽或與該多胜肽類似的胺基酸序列,而引起上述免疫反應之抗原,亦即該抗原可使受鮑氏不動桿菌感染之個體產生有效免疫反應,而對該個體產生免疫保護力,且該抗原係一種多胜肽(polypeptide)。
本專利說明書中所稱之「胺基酸取代、刪除或添加」,係指以其他胺基酸取代原本胺基酸序列中之胺基酸,或刪除某些位置的胺基酸序列,或於某些位置插入一個或數個胺基酸使序列變長,或以上任何修飾後組合之類似物。該些類似物與原胺基酸序列經比對後具有一定比例之相似度。其中,依據SEQ ID NO:1、3、4、5可進行20%以下胺基酸之取代、刪除或添加,較佳為10%以下,最佳為5%以下,更加為1至5個胺基酸的取代、刪除或添加,使具有80%以上之相似度。而於SEQ ID NO:2則可進行40%以下胺基酸之取代、刪除或添加,較佳為20%以下,最佳為5%以下,更加為1至5個胺基酸的取代、刪除或添加,使具有60%以上之相似度。
本專利說明書中所稱之「胺基酸序列之片段」,係指原胺基酸序列中所擷取之某區段部分,其可為原序列中任何區域之擷取,或擷取後之組合,其態樣於此不包括前述20%或40%以下胺基酸之刪除。
關於胺基酸序列之相似度,可利用習知電腦程式加以比對分析。例如Altschul等人(Nucleic Acids Res.25:3389-3402,1997)所描述之BLAST方式進行。BLAST分析軟體可透過美國國家生物技術資訊中心網站(National Center for Biotechnology Information,http://www.ncbi.nlm.nih.gov/blast/blast.cgi)執行之。
本發明係利用tBLAST進行胺基酸分析,比對時所設定之參數為:期望值(Expect threshold):10;字長(word size):3;查詢範圍內的最大匹配(Max matches in a query range):0;計分矩陣(Matrix):BLOSUM62;既存(Existence):11,延伸(Extension):1;組成調整(Compositional adjustments):依條件成分得分矩陣調整(conditional compositional score matrix adjustment);篩選(Filter):低複雜性區域選定(Low complexity regions selected);遮罩(Mask):未選擇(not selected)。
本專利說明書中所稱之「鮑氏不動桿菌(Acinetobacter baumannii)」,係指依據Acinetobacter Molecular Biology(Ed.:Ulrike Gerischer,Caister Academic Press,2008)所分類的Acinetobacter baumannii種,其可包括ATCC 17978以及下述表2中之6200、SDF...等菌株(各株菌詳細資料請參網址:http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=470&IvI=3&keep%20=1&srchmode=1&unlock)。
表2係本發明鮑氏不動桿菌中NcsP、TonB-R、Pase1、Pase2以及GP1五種多胜肽抗原與其他29株鮑氏不動桿菌菌株中同源多胜肽保留序列相似度之比較。由表2之相似度比較結果可發現,多胜肽抗原NcsP、TonB-R、Pase1、Pase2以及GP1,與大部分的鮑氏不動桿菌株之相似度高達98~99%以上,僅其中多胜肽抗原TonB-R少部分為60%以上之相似度。由於序列相似度極高,且經由後續殺菌力以及小鼠免疫保護力之測試,可證明本發明中之五種多胜肽抗原確實可做為鮑氏不動桿菌間共有之多胜肽抗
原,而可用以製備疫苗組成物以預防鮑氏不動桿菌之感染。
鮑氏不動桿菌抗原之多胜肽可藉由一般基因工程技術所製備。藉由聚合酶鏈鎖反應(polymerase chain reaction,PCR)增幅表現該胜肽相對應之DNA序列,再將其選殖至表現載體表現即可(可參Sambrook et al.,Molecular Cloning:A-Laboratory Manual,Cold Spring Harbor Press,N.Y.,
Second(1998)and Third(2000)Edition;Gene Expression Technology,Methods in Enzymology,Genetics and Molecular Biology,Methods,in Enzymology,Guthrie & Fink,Academic Press,San Diego,Calif.,1991,以及Hitzeman et al.,J.Biol.Chem.,255:12073-12080,1990)。此外亦可利用合成方式,製備出所需序列之胜肽,相關製備方式可利用所屬技術領域習知的方法為之。
本發明分別採用免疫蛋白質體技術與反向疫苗學之策略尋找出符合所需之鮑氏不動桿菌抗原。以下將進一步詳細說明鮑氏不動桿菌多胜肽抗原之篩選、確認,以及該抗原之免疫效果。
<實施例1>抗原NcsP與TonB-R之篩選與確認
本實施例係以免疫蛋白質體技術鑑定出抗原NcsP與TonB-R。首先,採取醫院中數個病患之血液,並分離出血清。以前述血清對台灣分離的鮑氏不動桿菌菌株之菌體總蛋白質進行西方墨點法分析(Western blot),初步篩選出內含對鮑氏不動桿菌具專一性的抗體的血清(以下稱:HS-AB)。另外,萃取、分離鮑氏不動桿菌之外膜蛋白質,進行二維電泳分析,一式兩份,然後將其中一份膠片進行銀染,另一份則以HS-AB進行免疫偵測,其結果如第一圖所示。對照免疫偵測墨點以及銀染的膠片,將相對應位置的蛋白質加以分離,再將該蛋白質進行質譜儀分析。分析結果中,該蛋白質樣品分數最高的目標蛋白質分別為AB SDF品系的「推定LysM結構域超族群(putative LysM domain superfamily)蛋白質」(YP_001707847)以及AB ACICU品系的「外膜受器(outer membrane receptor)」(YP_001845144)。依序列性質分析,推測YP_001707847應係一非典型的分泌性蛋白(nonclassical secretory protein),而YP_001845144則為推定鐵載體受體蛋白(putative ferric siderophore receptor protein)。
為進一步確認此二抗原確實能為HS-AB所辨識,遂進行以下基因選殖與表現蛋白之確認反應。首先,分別根據編碼YP_001707847與YP_001845144蛋白質的基因序列設計進行聚合酶鏈鎖反應(PCR)的引子,並以前述台灣的鮑氏不動桿菌臨床分離株為模板進行菌落-聚合酶鏈鎖反應(colony-PCR),將編碼此二抗原的DNA增幅後選殖於pGEM-T Easy載體後進
行定序分析。定序結果中,前者所增幅的DNA序列(SEQ ID NO:6)與YP_001707847基因序列間之相似度達98%,而後者所增幅的DNA序列(SEQ ID NO:7)與YP_001845144基因序列間之相似度則達99%。進一步比對前述DNA序列所轉譯後之胺基酸序列,前者(SEQ ID NO:1)與YP_001707847胺基酸序列間之相似度達99%,而後者(SEQ ID NO:2)與YP_001845144胺基酸序列間之相似度一樣高達99%。證明所篩選到可表現的蛋白質樣品應即為YP_001707847與YP_001845144,故以下將具有SEQ ID NO:1與2胺基酸序列之蛋白質分別稱為「NcsP」與「TonB-R」。
之後,另外將前述PCR增幅後的DNA片段再分別選殖於pET-21表現質體(選殖後分別命名為pET-NcsP與pET-TonB-R質體),送入大腸桿菌BL21(DE3)中,並以IPTG間接調控以進行重組蛋白的表現,最後再以西方墨點法分析,分別以人體血清HS-AB偵測,確認其表現狀況,其結果如第二圖所示。由圖中可知,由NcsP與TonB-R的基因所表現之重組蛋白質皆可為人體血清HS-AB所辨識,因此可證實NcsP與TonB-R確實為HS-AB的目標抗原。此結果更首度證實YP_001707847/NcsP與YP_001845144/TonB-R此二蛋白質確係能表現而存在於鮑氏不動桿菌中,且具抗原性而為病人抗血清所能辨識。
此外,為確認NcsP與TonB-R的基因是否存在於不同鮑氏不動桿菌菌株間,遂將台灣所蒐集數百株的臨床菌株進行西方墨點法分析,發現所有的菌株都可偵測到此蛋白質,從中隨機挑取十株菌進行PCR與定序分析,也確認該些菌株都有此二基因序列。進一步將NcsP與TonB-R的胺基酸序列分別與GenBank中的29株鮑氏不動桿菌的序列比較後,更確認了其序列於該些鮑氏不動桿菌間有著相當高的相似度。請參閱表2,NcsP(SEQ ID NO:1)間的比對有高達99%的相似度,而TonB-R(SEQ ID NO:2)間的比對也大多具有99%的相似度,僅其中部分為60%以上的相似度。因此,NcsP與TonB-R係普遍存在於不同鮑氏不動桿菌菌株間,且具有極高的相似度。
<實施例2>抗原Pase1與Pase2之篩選與確認
本實施例係以反向疫苗學策略篩選出抗原Pase1與Pase2。首
先,以protease為關鍵字搜尋ATCC 17978之基因體,發現兩個分別被註解為「分泌性類胰蛋白酶的絲氨酸蛋白酶(secreted trypsin-like serine protease)」的A1S_2546(以下稱「Pase1」)與「推定醣蛋白內切酶金屬蛋白酶(putative glycoprotein endopeptidase metalloprotease)」的A1S_0699(以下稱「Pase2」)。經比對,A1S_2546/Pase1與A1S_0699/Pase2在各個鮑氏不動桿菌之基因體中均有與其序列相似度達約96%以上之序列,但因功能未知,命名不盡相同。
為驗證此二DNA序列是否有表現,首先根據鮑氏不動桿菌ATCC 17978的基因組序列設計引子進行RT-PCR,將預期大小的產物選殖於pGEM-T Easy載體後進行定序分析。由於可以獲得RT-PCR產物,因此證實了此兩個依序列所註解的Pase1與Pase2的基因確實會被轉錄表現出來。
進一步將Pase1與Pase2其胺基酸序列分別與GenBank中的29株鮑氏不動桿菌的序列相比較,發現其序列於該些鮑氏不動桿菌間也有著相當高的相似度。請參閱表2,Pase1(SEQ ID NO:3)間的比對有高達95%~99%的相似度,而Pase2(SEQ ID NO:4)間的比對則具有92~100%的相似度。因此,Pase1與Pase2同樣普遍存在於不同鮑氏不動桿菌菌株間,且有極高的相似度。
之後,將前述PCR增幅後的DNA片段分別選殖於pET-21表現質體(選殖後分別命名為pET-Pase1與pET-Pase2質體)中,於下述實施例中表現該些基因。
<實施例3>以抗原NcsP、TonB-R、Pase1與Pase2免疫小鼠之存活率測試
為了評估上述四個鮑氏不動桿菌的蛋白質是否可做為鮑氏不動桿菌疫苗的組成分,將分別選殖有pET-NcsP、pET-TonB-R、pET-Pase1與pET-Pase2表現載體的大腸桿菌BL21(DE3),培養至OD600達0.8~1之間時,加入異丙基-β-D-硫代半乳糖苷(IPTG)間接誘導以進行重組蛋白表現。
之後以Ni2+-親和管柱純化前述表現載體所表現的重組蛋白質(NcsP、TonB-R、Pase1與Pase2),並以SDS-PAGE檢測其純度後,利用於免疫小鼠。另外,則以西方墨點法及酵素免疫分析法確定抗血清對各重組蛋白質的專一性後,以鮑氏不動桿菌ATCC 17978進行肺部攻毒試驗,觀察
小鼠之存活情形,其存活率結果如第三圖所示。結果顯示,施打本發明實施例抗原之小鼠存活率明顯高於僅施打PBS的對照組,而各組之五天存活率分別為:PBS(10~30%)、NcsP(90%)、TonB-R(80%)、Pase1(80%)、Pase2(90%)。顯見本發明的多胜肽抗原能夠對施打的小鼠產生優秀的免疫保護力,故其係足以作為疫苗組成物之多胜肽抗原。特別的是,雖然攻毒之菌株ATCC 17978其相當於TonB-R的胺基酸序列與施打之重組TonB-R序列之相似度只有61%,仍具有顯著的保護效果。
<實施例4>抗原NcsP、TonB-R、Pase1與Pase2的安全性測試
為確認本發明多胜肽抗原是否對生物體具有毒性,遂取先前所純化之重組蛋白質NcsP、TonB-R、Pase1與Pase2進行細胞存活率測試(MTT assay)。MTT測試係以一般所屬技術領域所常用之方法進行,例如利用前揭Sambrook等人所著Molecular cloning書中所描述之方法,並以人類A549細胞為受測細胞,其結果如第四圖所示。結果顯示上述四種蛋白對人類A549細胞並沒有毒性,因此是具安全性的抗原胜肽,適於作為疫苗的組成物,而得應用於鮑氏不動桿菌感染的預防。
此外,雖然Pase1和Pase2被註解為「tyrpsin-like protease(類胰蛋白酶的絲氨酸蛋白酶)」,但以QuantiCleaveTM檢測套組(Thermo Scientific Pierce)進行測試後發現,在0.5mg/mL的濃度下仍未偵測到此二蛋白質具有蛋白酶酵素的活性,以此結果配合前述MTT測試結果,更可確認此二蛋白質並不具細胞毒性。
綜合以上結果,可確認NcsP、TonB-R、Pase1與Pase2這四個多胜肽抗原於各鮑氏不動桿菌菌株中應係可表現的蛋白,且由於序列之相似度極高,對於不同鮑氏不動桿菌的感染應皆同具有極佳的保護效果,加上其不具細胞毒性的高安全性,顯示此四個多胜肽均足以作為廣效性鮑氏不動桿菌疫苗的優良抗原。
<實施例5>抗原GP1之篩選與確認
本實施例同樣係以反向疫苗學策略篩選出抗原GP1。首先,以細菌脂蛋白資料庫(DOLOP)分析A1S_0556(以下稱GP1),顯示其N端帶有
一個脂化訊號的前導序列,應係一脂蛋白,除此之外,Iwashkiw等人(Iwashkiw JA,Seper2 A,Weber BS,.Scott NE,Vinogradov E,Stratilo C,Reiz Bet al.,2012.Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.PLoS Pathogens,2012)發現在鮑氏不動桿菌ATCC 17978中此蛋白是具有醣化修飾的。本實施例以PCR增幅GP1中第39-313胺基酸編碼的DNA片段,再將其選殖於pET21表現質體中,送入E.coli BL21(DE3)後以IPTG間接誘導重組蛋白的表現,再以Ni2+-親和管柱純化之重組蛋白質對小鼠進行免疫注射,以取得之抗血清對鮑氏不動桿菌的菌體蛋白質進行西方墨點法分析,結果發現所有測試的菌株都可偵測到GP1蛋白質。此外,將GP1胺基酸序列與GenBank中的29株鮑氏不動桿菌的序列相比較,發現該些菌株的GP1間有高達95%~99%的相似度(請參閱表2),證明GP1蛋白質也是鮑氏不動桿菌之保守抗原。
<實施例6>以抗原GP1免疫小鼠後取得之抗血清之殺菌力分析
為了評估GP1所表現之多胜肽抗原是否可做為鮑氏不動桿菌疫苗的組成分,除前述可利用鮑氏不動桿菌直接感染小鼠進行存活率分析之體內試驗外,於生物體外利用該抗原所誘發之抗體,對鮑氏不動桿菌進行殺菌力測試,分析其存活率,亦是一確認疫苗有效性常用之方式。
因此,於本實施例中,係以經Ni2+-親和管柱純化後的重組蛋白GP1免疫小鼠後,以取得的抗血清(anti-GP1)進行殺菌力分析。測試時,取約100CFU的鮑氏不動桿菌置於含有50%抗血清的PBS中,於37℃下培養4小時,觀察存活之菌數,其結果如第五圖所示。以免疫前之血清為對照組,可發現anti-GP1處理後之菌數存活率明顯低於對照組,其效果甚至高於anti-NcsP,而僅剩18%,顯示anti-GP1具有優異的殺菌效果,可證明GP-1能誘發具有保護效果的免疫反應,因此可做為一個有效的疫苗抗原。此外,以鮑氏不動桿菌感染之病人血清對此重組蛋白進行西方墨點法分析後,如第六圖所示,可發現有專一辨識訊號,顯示此蛋白質的確可誘發人類的免疫反應,更進一步證明GP1是一可用做疫苗組成分之多胜肽抗原。
<實施例7>單株抗體之製備
將前述各重組蛋白/胜肽抗原,分別以Ni2+管柱純化後,取之進行BALB/c小鼠的免疫注射,每個月間隔進行3次,每次劑量為10-15mg。首次免疫時係加入以佛氏完全佐劑(Freund’s complete adjuvant),後續則以佛氏不完全佐劑進行。在最後一次注射的4天後,將小鼠之脾臟取出,再將其中之脾臟細胞與Sp2/0-Ag14骨髓瘤(myeloma)細胞融合。進行篩選,對於存活之融合細胞以ELISA以及西方墨點法進一步篩選出具專一性之細胞株。
該些個別針對本發明胜肽抗原所篩選出之單株抗體,可進一步作為套組的組成,用以檢測鮑氏不動桿菌的存在。檢測時,可利用所屬技術領域所熟知之方式進行,將前述之單株抗體與待測樣品混合後,利用偵測試劑之呈色或儀器檢測吸光值、螢光值,分析該抗體之結合狀況,以判定待測樣品中是否存有鮑氏不動桿菌。
前述套組中之單株抗體,可使用針對本發明中其中一種多胜肽抗原之單一單株抗體,亦可使用兩種以上針對其中一種多胜肽抗原或兩種以上多胜肽抗原所產生製備的多種單株抗體,以能多重確認辨識關係,增加檢測之準確性。
另一方面,無論是針對本發明多胜肽抗原之單株抗體,或是多株抗體或是其抗體片段,亦可用於鮑氏不動桿菌感染的治療,以所屬技術領域所熟知之一般方法,加上適當的藥學上可接受之載劑,製備成可治療鮑氏不動桿菌感染所引起疾病的醫藥組成物。
利用於治療鮑氏不動桿菌感染的醫藥組成物時,該些抗體以單株抗體為較佳。同樣地,所使用的單株抗體可為針對本發明中其中一種多胜肽抗原之單一單株抗體,亦可使用兩種以上針對其中一種多胜肽抗原或兩種以上多胜肽抗原所產生製備的多種單株抗體加以組合治療。
<110> 國立中興大學
<120> 鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原及其抗體以及編碼該抗原之核苷酸
<130> 104B0201-I1
<160> 10
<210> 1
<211> 157
<212> PRT
<213> Acinetobacter baumannii
<400> 1
<210> 2
<211> 743
<212> PRT
<213> Acinetobacter baumannii
<400> 2
<210> 3
<211> 132
<212> PRT
<213> Acinetobacter baumannii
<400> 3
<210> 4
<211> 180
<212> PRT
<213> Acinetobacter baumannii
<400> 4
<210> 5
<211> 275
<212> PRT
<213> Acinetobacter baumannii
<400> 5
<210> 6
<211> 471
<212> DNA
<213> Acinetobacter baumannii
<400> 6
<210> 7
<211> 2229
<212> DNA
<213> Acinetobacter baumannii
<400> 7
<210> 8
<211> 396
<212> DNA
<213> Acinetobacter baumannii
<400> 8
<210> 9
<211> 540
<212> DNA
<213> Acinetobacter baumannii
<400> 9
<210> 10
<211> 825
<212> DNA
<213> Acinetobacter baumannii
<400> 10
Claims (4)
- 一種單離的鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原,該多胜肽抗原包括SEQ ID NO:5之胺基酸序列,該多胜肽抗原並可使受鮑氏不動桿菌感染之個體產生有效免疫反應。
- 一種利用如申請專利範圍第1項所述之多胜肽抗原作為疫苗之用途。
- 一種鮑氏不動桿菌疫苗組成物,其包括至少一如申請專利範圍第1項所述之多胜肽抗原。
- 一種單離的核酸,係編碼申請專利範圍第1項所述之胺基酸序列,該胺基酸序列係為鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原,並可使受鮑氏不動桿菌感染之個體產生有效免疫反應。
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TW104118940A TWI649331B (zh) | 2015-06-11 | 2015-06-11 | 鮑氏不動桿菌(Acinetobacter baumannii)多胜肽抗原及其抗體以及編碼該抗原之核酸 |
EP16173055.1A EP3103471A3 (en) | 2015-06-11 | 2016-06-06 | Acinetobacter baumanii antigens and the uses thereof |
BR102016013224-0A BR102016013224A2 (pt) | 2015-06-11 | 2016-06-08 | Acinetobacter baumannii antigens and respective uses |
US15/177,184 US10124049B2 (en) | 2015-06-11 | 2016-06-08 | Acinetobacter baumannii antigens and the uses thereof |
JP2016114720A JP6334607B2 (ja) | 2015-06-11 | 2016-06-08 | アシネトバクター・バウマニ(Acinetobacter baumannii)ポリペプチド抗原及びその抗体及び該抗原をコーディングする核酸 |
CN201610404902.0A CN106243198A (zh) | 2015-06-11 | 2016-06-08 | 鲍氏不动杆菌多肽抗原及其抗体以及编码该抗原的核酸 |
JP2018084905A JP6488419B2 (ja) | 2015-06-11 | 2018-04-26 | アシネトバクター・バウマニ(Acinetobacter baumannii)ポリペプチド抗原及びその抗体及び該抗原をコーディングする核酸 |
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CN108503698B (zh) * | 2018-05-24 | 2021-03-05 | 中国人民解放军军事科学院军事医学研究院 | 鲍曼不动杆菌Ata蛋白的免疫原性 |
CN110713524B (zh) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | 一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒 |
CN110713523B (zh) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | 一种定性检测人血清中鲍曼不动杆菌特异性抗体的检测条 |
CN110724201B (zh) * | 2019-10-31 | 2023-01-24 | 湖北工业大学 | 基于多重表位融合抗原的鲍曼不动杆菌感染快速检测法 |
WO2022057854A1 (zh) * | 2020-09-16 | 2022-03-24 | 南京迈西可生物科技有限公司 | 病原体特异性核酸片段及其应用 |
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WO2011125015A2 (en) * | 2010-04-05 | 2011-10-13 | Bar-Ilan University | Protease-activatable pore-forming polypeptides |
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JP2018134103A (ja) | 2018-08-30 |
EP3103471A2 (en) | 2016-12-14 |
JP6488419B2 (ja) | 2019-03-20 |
US20160361406A1 (en) | 2016-12-15 |
JP2017008034A (ja) | 2017-01-12 |
BR102016013224A2 (pt) | 2017-09-26 |
US10124049B2 (en) | 2018-11-13 |
EP3103471A3 (en) | 2017-02-15 |
TW201643180A (zh) | 2016-12-16 |
JP6334607B2 (ja) | 2018-05-30 |
CN106243198A (zh) | 2016-12-21 |
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