TWI527829B - 經分離之噬菌體溶菌蛋白 - Google Patents
經分離之噬菌體溶菌蛋白 Download PDFInfo
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
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- Bioinformatics & Cheminformatics (AREA)
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Description
本發明係關於一種經分離之蛋白,特別係關於一種經分離之噬菌體溶菌蛋白。
鮑氏不動桿菌(Acinetobacter baumannii)屬於伺機性病原菌(opportunistic pathogen),在醫院中之推車、醫療器具、水槽及病床等皆可以被偵測到。鮑氏不動桿菌容易對抗生素產生抗藥性,衍生具多重抗藥性(multi-drug resistance)或全抗藥性(pan-drug resistance)之菌株,且在醫院常爆發嚴重之院內感染。
噬菌體溶菌蛋白(lysin或endolysin)為噬菌體所產生之水解酶,於噬菌體感染宿主細胞(即,細菌)後,噬菌體溶菌蛋白可以裂解宿主細胞之細胞壁,而造成宿主細胞之死亡。惟,鮑氏不動桿菌隸屬於革蘭氏陰性菌(gram negative bacteria),其具有外膜結構(outer membrane),使習知噬菌體溶菌蛋白無法作用於肽聚醣(peptidoglycan),因而須先對鮑氏不動桿菌進行氯仿(chloroform)、乙二胺四乙酸(EDTA)等前處理以提高其外膜通透性,而後才能夠以該習知噬菌體溶菌蛋白溶裂鮑氏不動桿菌。
然而,上述之前處理不僅無法適用於醫療治療,亦難以於環境、器具之殺菌使用,因而該習知噬菌體溶菌蛋白應用於抑制鮑氏不動桿菌仍有改善之空間。
本發明係提供一種經分離之噬菌體溶菌蛋白,可以直接溶裂
未經前處理之鮑氏不動桿菌者。
本發明係提供一種經分離之噬菌體溶菌蛋白,係包含:一第一胜肽片段,具有如SEQ ID NO:4所示之序列;及一第二胜肽片段,具有如SEQ ID NO:5所示之胺基酸序列。
本發明經分離之噬菌體溶菌蛋白,係具有如SEQ ID NO:6所示之胺基酸序列。
本發明經分離之噬菌體溶菌蛋白,其中,該經分離之噬菌體溶菌蛋白係由一大腸桿菌所表現。
本發明經分離之噬菌體溶菌蛋白,其中,該大腸桿菌係藉由表現一質體以獲得該經分離之噬菌體溶菌蛋白,該質體係包含如SEQ ID NO:1及2所示之核苷酸序列。
本發明經分離之噬菌體溶菌蛋白,其中,該質體係包含如SEQ ID NO:3所示之核苷酸序列。
本發明經分離之噬菌體溶菌蛋白,其中,包含該質體之該大腸桿菌係寄存於財團法人食品工業發展研究所,寄存編號BCRC940658。
本發明之噬菌體溶菌蛋白可以被表現及純化,且透過該第一、第二胜肽片段之功能區,該噬菌體溶菌蛋白可以辨識並穿過鮑氏不動桿菌之外膜,進而可以直接溶裂未經前處理之鮑氏不動桿菌,故而該噬菌體溶菌蛋白可以作為藥劑使用,達成治療鮑氏不動桿菌感染之功效。
本發明之噬菌體溶菌蛋白係可以耐受25~50℃及pH值3~12之環境,仍保有溶裂鮑氏不動桿菌之能力,故而該噬菌體溶菌蛋白可以製成殺菌劑,用於環境、器具等,達成殺菌之功效。
第1圖:係本發明噬菌體溶菌蛋白之表現、純化SDS-PAGE圖。
第2圖:係本發明噬菌體溶菌蛋白之最低抑菌濃度分析結果圖。
第3圖:係本發明噬菌體溶菌蛋白之最小抑菌濃度測試長條圖。
第4圖:係本發明噬菌體溶菌蛋白之耐熱活性分析結果圖。
第5圖:係本發明噬菌體溶菌蛋白之耐酸鹼活性分析結果圖。
為讓本發明之上述及其他目的、特徵及優點能更明顯易懂,下文特舉本發明之較佳實施例,並配合所附圖式,作詳細說明如下:本發明一實施例經分離之噬菌體溶菌蛋白,係包含一第一胜肽片段及一第二胜肽片段。該第一胜肽片段係為噬菌體T7尾纖維蛋白(Phage T7 tail fiber protein)之功能區,該噬菌體溶菌蛋白係可以利用T7尾纖維蛋白之功能區,辨認鮑氏不動桿菌外膜之膜蛋白,並專一地結合於鮑氏不動桿菌。此外,該第一胜肽片段可以協助該噬菌體溶菌蛋白穿過鮑氏不動桿菌之外膜。該第二胜肽片段係為果膠裂解酶(Pectate lyase superfamily protein)之功能區,使該噬菌體溶菌蛋白可以利用果膠裂解酶之功能區,,以溶裂鮑氏不動桿菌之細胞壁,而造成鮑氏不動桿菌死亡。
該噬菌體溶菌蛋白可以透過表現一質體所獲得,例如以該質體轉型(transform)進入一大腸桿菌,續以該大腸桿菌表現該質體,並經純化以獲得該噬菌體溶菌蛋白。該質體包含一第一核苷酸片段,對應該第一胜肽片段;另包含一第二核苷酸片段,對應該第二胜肽片段;此外,該質體較佳係為大腸桿菌之密碼使用(codon usage)。
詳言之,該質體包含之該第一、第二核苷酸片段,分別具有如SEQ ID NO:1、2所示核苷酸序列,對應具有如SEQ ID NO:4、5所示胺基酸序列之該第一、第二胜肽片段。於本實施例中,該噬菌體溶菌蛋白係篩選自Φkm18p噬菌體,該質體具有如SEQ ID NO:3所示之核苷酸序列,對應如SEQ ID NO:6所示之胺基酸序列。
該質體之構築、該噬菌體溶菌蛋白之表現及後續之純化,係為本發明所屬技術領域之通常知識,不以本實施例為限,例如:該質體較佳係可以被異丙基β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,簡稱IPTG)誘導而表現,且經該質體所表現之該噬菌體溶菌蛋白較佳係包含具有結合特異性之胜肽片段,以利於後續以親和力管柱(affinity column)分離之。由於該噬菌體溶菌蛋白亦有可能溶裂該大腸桿菌,故較佳係使用大腸桿菌C43(DE3)表現之。本實施例中,該質體係以如SEQ ID NO:3所示之核苷酸片段,選殖入pET21b載體,送入大腸桿菌C43(DE3)。包含該質體之該大腸桿菌係寄存於財團法人食品工業發展研究所,寄存編號BCRC940658。
前述之大腸桿菌係可以大量培養,續經IPTG誘導以表現該噬菌體溶菌蛋白,該噬菌體溶菌蛋白係具有一段連續組胺酸片段標記(His Tag),其能夠以二價鎳離子(Ni2+)等親合力管柱純化,以獲得該噬菌體溶菌蛋白。
該噬菌體溶菌蛋白,透過該第一、第二胜肽片段之功能區,可以辨識、穿過鮑氏不動桿菌之外膜,因而無須進行前處理,即可以直接溶裂鮑氏不動桿菌。
該噬菌體溶菌蛋白,可以用於治療鮑氏不動桿菌之感染,例如投予一所需生物體,以治療該所需生物體之感染。該噬菌體溶菌蛋白可以結合適當之載劑,製成錠劑或藥膏等製劑,以口服或塗抹等方式使用之。
該噬菌體溶菌蛋白,另可以製成殺菌劑,使用於環境、器具等之清潔殺菌。例如將該噬菌體溶於pH值介於3~12範圍內之適當溶劑,於25~50℃之溫度範圍下,噴灑於空氣中或以浸泡、塗抹等方式進行環境及器具之殺菌。
為證實本發明之噬菌體溶菌蛋白確實可以表現、純化,且可
以直接溶裂未經前處理的鮑氏不動桿菌,而能夠作為治療或殺菌之用途,遂使用台灣本土分離之全抗藥性Km18鮑氏不動桿菌菌株進行下述實驗:
(一)噬菌體溶菌蛋白之表現、純化分析
於本實驗中,係培養前述寄存於財團法人食品工業發展研究所、寄存編號BCRC940658之大腸桿菌菌株,續以IPTG誘導,以產生該噬菌體溶菌蛋白。本實驗係於收集菌液後,使用超音波破菌、離心取其上清液加入一結合緩衝液(binding buffer),續於4㊣℃下混合二價鎳離子樹脂1小時後倒入管柱,以該結合緩衝液洗出未結合於樹脂之雜蛋白,續以一流析緩衝液(elution buffer)置換結合於樹脂之蛋白,以獲得該噬菌體溶菌蛋白。該結合緩衝液含有pH8.2之50mM三羥甲基氨基甲烷-鹽酸(Tris-HC1)、15mM氯化鎂、20%(v/v)甘油、0.05%㊣巰基乙醇(β-mercaptoethanol,簡稱β-ME),及0.1mM苯甲基磺醯氟(PMSF);該流析緩衝液係於該結合緩衝液中另加入250mM咪唑㊣(imidazo此)。
分別取未經IPTG誘導之菌液(第A1組)、經IPTG誘導之菌液(第A2組)、經IPTG誘導且經破菌及離心之菌液沉澱(第A3組),及收集之二價鎳離子管柱流析緩衝液(第A4組)加入β-ME及經煮沸變性進行十二烷基磺酸鈉-聚丙烯酰胺膠體電泳(sodium dodecylsulphatepolyacrylgel electrophoresis,簡稱SDS-PAGE),其結果係如第1圖所示。對照蛋白質量標示(protein marker,簡稱PM),第A2~A4組皆有分子量約72kDa之片段出現,故可以證實該噬菌體溶菌蛋白確實可以經IPTG誘導表現及進行純化。
(二)噬菌體溶菌蛋白對於鮑氏不動桿菌之抑菌效果分析
以紙碇擴散法(paper disk diffusion method)進行噬菌體溶菌蛋白最低抑菌濃度分析,係分為第B1~B5組,各取1、5、1㊣0、50、100μg/20μl之噬菌體溶菌蛋白點於紙碇上,將紙碇放置於已接種㊣Km18鮑氏不
動桿菌之固態培養基進行培養。結果係如第2圖所示,於培養24小時後,第B2~B5組之該噬菌體溶菌蛋白皆可以產生抑菌圈(inhibition zone);且於培養72小時後,第B1~B5組之該噬菌體溶菌蛋白皆可以產生抑菌圈。
取隔夜培養之Km18鮑氏不動桿菌菌液,重新接種至新鮮含有50μg/ml氨比西林(Ampicillin)之LB培養液,培養至進入對數期,續經適當稀釋使起始菌量為106CFU/ml,分為第C0~C5組進行最小抑菌濃度測試(minimum inhibitory concentration)。將噬菌體溶菌蛋白分別加入各組,使第C1~C5組之噬菌體溶菌蛋白最終濃度為0.5、1.0、1.5、2.0、2.5mg/ml,而第C0組則不加入該噬菌體溶菌蛋白以作為對照。將第C0~C5組於37℃培養6小時後,分別取出各組菌液,經序列稀釋以測量活菌數。
請參照第3圖,未添加噬菌體溶菌蛋白(第C0組)於培養6小時後,其Km18鮑氏不動桿菌菌量由106CFU/ml增長至108CFU/ml,而第C1~C5組則皆有抑菌之效果,第C1組之菌量為107CFU/ml,第C2組之菌量為105CFU/ml,而第C5組甚至可以溶裂Km18鮑氏不動桿菌至僅剩104CFU/ml之活菌量。
(三)噬菌體溶菌蛋白之耐熱、耐酸鹼活性分析
各取5ng該噬菌體溶菌蛋白為D0~D6組進行耐熱活性分析,以D0組於室溫(25℃)下作為對照,另將D1~D6組分別以室溫40℃、50℃、60℃、70℃、80℃及100℃之水浴槽進行熱處理30分鐘,續進行SDS-PAGE分離及膠體內溶菌活性分析,其結果如第4圖所示。第D0~D2組皆有溶菌斑表現,意即,本發明之噬菌體溶菌蛋白於25~50℃之範圍下熱處理30分鐘,仍能保有溶裂Km18鮑氏不動桿菌之能力。
另取5mg/ml之該噬菌體溶菌蛋白各1μl為E1~E12組進行耐酸鹼活性分析,將E1~E12組分別加入pH值為2、3、4、5、6、7、8、9、10、11、12、13且經滅菌之磷酸鹽緩衝生理鹽水(phosphate buffer saline,
簡稱PBS)溶液99μl,於室溫下靜置30分鐘,續進行SDS-PAGE分離及膠體內溶菌活性分析,其結果如第5圖所示。第E2~E11組皆有溶菌斑表現,意即,本發明之噬菌體溶菌蛋白於pH值3~12之範圍下,仍能保有溶裂Km18鮑氏不動桿菌之能力。
經上述實驗可以證實,本發明之噬菌體溶菌蛋白確實可以被表現及純化,且透過該第一、第二胜肽片段之功能區,該噬菌體溶菌蛋白可以辨識並穿過鮑氏不動桿菌之外膜,進而可以直接溶裂未經前處理之鮑氏不動桿菌,故而該噬菌體溶菌蛋白可以作為藥劑使用,達成治療鮑氏不動桿菌之功效。
本發明之噬菌體溶菌蛋白係可以耐受25~50℃及pH值3~12之環境,仍保有溶裂鮑氏不動桿菌之能力,故而該噬菌體溶菌蛋白可以製成殺菌劑,用於環境、器具等,達成殺菌之功效。
雖然本發明已利用上述較佳實施例揭示,然其並非用以限定本發明,任何熟習此技藝者在不脫離本發明之精神和範圍之內,相對上述實施例進行各種更動與修改仍屬本發明所保護之技術範疇,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。
財團法人食品工業發展研究所、103年12月27日、BCRC940658
(無)
<110> 義守大學
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Claims (6)
- 一種經分離之噬菌體溶菌蛋白,係包含:一第一胜肽片段,具有如SEQ ID NO:4所示之胺基酸序列;及一第二胜肽片段,具有如SEQ ID NO:5所示之胺基酸序列。
- 如申請專利範圍第1項所述經分離之噬菌體溶菌蛋白,係具有如SEQ ID NO:6所示之序列。
- 如申請專利範圍第1或2項所述經分離之噬菌體溶菌蛋白,其中,該經分離之噬菌體溶菌蛋白係由一大腸桿菌所表現。
- 如申請專利範圍第3項所述經分離之噬菌體溶菌蛋白,其中,該大腸桿菌係藉由表現一質體以獲得該經分離之噬菌體溶菌蛋白,該質體係包含如SEQ ID NO:1及2所示之核苷酸序列。
- 如申請專利範圍第4項所述經分離之噬菌體溶菌蛋白,其中,該質體係包含如SEQ ID NO:3所示之核苷酸序列。
- 如申請專利範圍第5項所述經分離之噬菌體溶菌蛋白,其中,包含該質體之該大腸桿菌係寄存於財團法人食品工業發展研究所,寄存編號BCRC940658。
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US14/700,872 US20160230161A1 (en) | 2015-02-10 | 2015-04-30 | Purified lysin protein |
US15/203,638 US20160312203A1 (en) | 2015-02-10 | 2016-07-06 | Method of manufacturing a lysin protein capable of directly lysing acinetobacter baumannii |
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CN116478966B (zh) * | 2022-08-01 | 2023-11-21 | 中南大学湘雅三医院 | 新型鲍曼不动杆菌噬菌体内溶酶蛋白及其制备和应用 |
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CN116478966B (zh) * | 2022-08-01 | 2023-11-21 | 中南大学湘雅三医院 | 新型鲍曼不动杆菌噬菌体内溶酶蛋白及其制备和应用 |
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