CN111471671A - 一种具有抑制产气荚膜梭菌活性的蛋白质及其相关生物材料与应用 - Google Patents
一种具有抑制产气荚膜梭菌活性的蛋白质及其相关生物材料与应用 Download PDFInfo
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- CN111471671A CN111471671A CN202010298999.8A CN202010298999A CN111471671A CN 111471671 A CN111471671 A CN 111471671A CN 202010298999 A CN202010298999 A CN 202010298999A CN 111471671 A CN111471671 A CN 111471671A
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Abstract
本发明公开了具有抑制产气荚膜梭菌活性的蛋白质PlyH11及其相关生物材料与应用。蛋白质PlyH11是如下A1)、A2)或A3)的蛋白质:A1)氨基酸序列是序列表中序列1的蛋白质;A2)将A1)的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有90%以上的同一性且具有抑制产气荚膜梭菌活性的蛋白质;A3)在A1)或A2)的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。实验证明PlyH11粗酶液对随机选择的五株不同产气荚膜梭菌均具裂解活性。本发明的蛋白质PlyH11、其相关生物材料及其制备方法对于预防和治疗产气荚膜梭菌导致的感染具有重要意义。
Description
技术领域
本发明涉及生物技术领域中一种具有抑制产气荚膜梭菌活性的蛋白质及其相关生物材料与应用。
背景技术
抗生素作为饲料添加剂在养殖业中的广泛应用,对促进动物生长、预防疾病、保障动物健康等方面做出了重要贡献。然而,在全球健康养殖背景下,无抗养殖是大势所趋。根据欧洲经验,在禽类中禁止使用抗生素作为促生长剂之后,最显著的问题就是由产气荚膜梭菌(Clostridium perfringens)引起的坏死性肠炎的广泛流行(Van Immerseel F,DeBuck J,Pasmans F,Huyghebaert G,Haesebrouck F,Ducatelle R:Clostridiumperfringens in poultry:an emerging threat for animal and public health[J].Avian Pathol 2004,33(6):537-549.)。不仅直接导致养殖业的经济损失,同时病原菌可以通过食物链等途径传播至人体,导致人类食物中毒。研究表明,产气荚膜梭菌是导致人类死亡的七大食源病原菌之一(Uzal FA,Freedman JC,Shrestha A,Theoret JR,Garcia J,Awad MM,Adams V,Moore RJ,Rood JI,McClane BA:Towards an understanding of therole of Clostridiumperfringens toxins in human and animal disease[J].FutureMicrobiol 2014,9(3):361-377.)。有效减少产气荚膜梭菌造成的危害是畜牧养殖业面临的重要问题之一;同时,控制产气荚膜梭菌的流行也是防控食源病原菌的重要目标之一。
由于抗生素的大量使用,细菌耐药性不断增强。近年来,人们一直在探索对抗细菌感染的新策略。大量研究表明,噬菌体及其组分在替代抗生素预防和治疗细菌感染方面具有重要的潜在应用价值。噬菌体裂解酶(lysin),是噬菌体复制晚期合成的一类细胞壁水解酶,用于水解宿主菌的肽聚糖结构,从而释放子代噬菌体。跟据其催化结构域在细菌细胞壁的作用位点,噬菌体裂解酶可分为三个家族,分别为糖苷酶、酰胺酶和肽链内切酶( C.Engineering of Phage-Derived Lytic Enzymes:Improving TheirPotential as Antimicrobials[J].Antibiotics(Basel,Switzerland)2018,7(2),29.)。相比于噬菌体病毒颗粒的应用,噬菌体裂解酶具有很多自身的优点:可作为酶制剂应用、可标准化规模生产、不产生耐药性等。
发明内容
本发明所要解决的技术问题是如何抑制产气荚膜梭菌。
为了解决以上技术问题,本发明提供了一种蛋白质,名称为PlyH11,是如下A1)、A2)或A3)的蛋白质:
A1)氨基酸序列是序列表中序列1第1-342位氨基酸残基的蛋白质;
A2)将A1)的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有90%以上的同一性且具有抑制产气荚膜梭菌活性的蛋白质;
A3)在A1)或A2)的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。
上述蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
上述蛋白质中,所述蛋白质标签(protein-tag)是指利用DNA体外重组技术,与目的蛋白质一起融合表达的一种多肽或者蛋白质,以便于目的蛋白质的表达、检测、示踪和/或纯化。所述蛋白质标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中,同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Perresidue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索以对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述蛋白质中,所述90%以上的同一性可为至少91%、92%、95%、96%、98%、99%或100%的同一性。
与PlyH11相关的生物材料也属于本发明的保护范围。
本发明所提供的与蛋白质PlyH11相关的生物材料,为下述B1)至B6)中的任一种:
B1)编码PlyH11的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体、或含有B1)所述表达盒的重组载体;
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物;
B5)含有B1)所述核酸分子的转基因动物细胞系、或含有B2)所述表达盒的转基因动物细胞系、或含有B3)所述重组载体的转基因动物细胞系;
B6)含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系、或含有B3)所述重组载体的转基因植物细胞系。
其中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA或hnRNA等。
上述生物材料中,B1)所述核酸分子为如下b1)或b2)所示的码基因:
b1)编码链的编码序列是序列表中序列3的第25-1053位核苷酸的cDNA分子或DNA分子;
b2)编码链的核苷酸是序列表中序列3的第25-1053位的cDNA分子或DNA分子。
其中,序列表中的序列3的第25-1053位由1029个核苷酸组成,编码序列表中的序列1所示的蛋白质。
上述生物材料中,B2)所述的含有编码PlyH11的核酸分子的表达盒(PlyH11基因表达盒),是指能够在宿主细胞中表达PlyH11的DNA,该DNA不但可包括启动PlyH11转录的启动子,还可包括终止PlyH11转录的终止子。进一步,所述表达盒还可包括增强子序列。
上述生物材料中,所述重组载体可为pPIC9-plyH11,pPIC9-plyH11是用核苷酸序列是序列3第1-1061位的DNA替换载体pPIC9的XhoI和NotI识别位点之间的片段(包括XhoI识别位点和NotI识别位点的小片段),保持pPIC9的其它核苷酸序列不变,得到的plyH11基因重组表达载体。
上述生物材料中,所述重组微生物可为E1)或E2):
E1)所述重组微生物为将所述的蛋白质的编码基因导入受体微生物,得到表达所述蛋白质的重组微生物,所述受体微生物可为C1)-C4)中的任一种:
C1)真核微生物;
C2)酵母;
C3)毕赤酵母属真菌;
C4)巴斯德毕赤酵母,如巴斯德毕赤酵母GS115;
E2)所述重组微生物为将所述pPIC9-plyH11导入巴斯德毕赤氏酵母GS115得到的分泌表达氨基酸序列是序列表的序列1的重组蛋白质(名称为PlyH11)的重组巴斯德毕赤酵母。
上述生物材料中,B4)至B6)可包括繁殖材料也可不包括繁殖材料。
为了解决以上技术问题,本发明提供了制备具有抑制产气荚膜梭菌活性的蛋白质的方法。
本发明所提供的制备具有抑制产气荚膜梭菌活性的蛋白质的方法,包括:使含有所述蛋白质的编码基因的DNA分子在生物中进行表达,得到具有抑制产气荚膜梭菌活性的蛋白质;所述生物为微生物、植物或非人动物。
上述方法中,所述微生物可为C1)-C4)中的任一种:
C1)真核微生物;
C2)酵母;
C3)毕赤酵母属真菌;
C4)巴斯德毕赤酵母。
上述方法中,使含有所述蛋白质的编码基因的DNA分子在生物中进行表达包括将所述的蛋白质的编码基因导入受体微生物,得到表达所述蛋白质的重组微生物,培养所述重组微生物,表达得到所述蛋白质。含有所述蛋白质的编码基因的DNA分子的编码链是序列表中序列3的第25-1053位。
上述方法中,所述受体微生物可为C1)-C4)中的任一种:
C1)真核微生物;
C2)酵母;
C3)毕赤酵母属真菌;
C4)巴斯德毕赤酵母,如巴斯德毕赤酵母GS115。
上述方法中,所述重组微生物可为将所述pPIC9-plyH11导入巴斯德毕赤氏酵母GS115得到的分泌表达氨基酸序列是序列表的序列1的重组蛋白质(名称为PlyH11)的重组巴斯德毕赤酵母。
上述方法中,在表达后包括收集分泌到胞外的蛋白质(分泌表达的蛋白质),得到具有抑制产气荚膜梭菌活性的蛋白质。
本发明还提供一种抗菌剂。
上述抗菌剂的活性成分含有所述蛋白质和/或生物材料,上述抗菌剂的活性成分还可含有其他生物成分或/和非生物成分,上述抗菌剂的其他活性成分本领域技术人员可根据抗菌效果确定。
上述抗菌剂中,所述抗菌剂可为抗产气荚膜梭菌的药剂。
上述蛋白质、或上述生物材料的下述P1-P2中的任一种应用也属于本发明的保护范围:
P1、所述蛋白质或所述生物材料在抗产气荚膜梭菌中的应用;
P2、所述蛋白质或所述生物材料在制备抗产气荚膜梭菌的产品中的应用。
所述产品可为药物、食品和/或保健品。
本发明优化蛋白质PlyH11编码基因的密码子利用毕赤酵母构建基因工程菌,对蛋白质PlyH11进行分泌表达,无需纯化蛋白质,直接检测发酵上清液,即具有较强杀灭产气荚膜梭菌的效果。实验证明,本发明PlyH11粗酶液对随机选择的五株不同产气荚膜梭菌均具有裂解活性。本发明所提供的蛋白质PlyH11、其相关生物材料及其制备方法对于预防和治疗产气荚膜梭菌导致的感染具有重要意义,在食品、卫生、畜牧业等领域具有广泛的应用前景。
附图说明
图1为本发明实施例1中重组表达载体pPIC9-plyH11以XhoI和NotI双酶切后产物的凝胶电泳图。其中,M为DNA分子量标准。左数第1泳道为pPIC9-plyH11以XhoI和NotI双酶切后产物。
图2为本发明实施例2中重组巴斯德毕赤氏酵母发酵液上清液的SDS-PAGE电泳图谱。其中,PlyH11(发酵液)即GS115/pPIC9-plyH11发酵上清液,对照为GS115发酵上清液,均经饱和硫酸铵沉淀,浓缩约20倍后上样;方框所示为PlyH11的分泌表达产物,可见两条带,较大条带为糖基化产物;其中,M为蛋白质分子量标准。
图3为本发明实施例3中重组巴斯德毕赤氏酵母发酵液上清液对五株产气荚膜梭菌的裂解活性。其中,处理为GS115/pPIC9-plyH11发酵上清液,对照为GS115发酵上清液;100为原液,101为10倍稀释液,102为100倍稀释液,103为1000倍稀释液;透明圈表示菌体被裂解。
图4为本发明实施例4中PlyH11粗酶液对产气荚膜梭菌ATCC13124和CP12两株菌株的杀菌动力学曲线。其中,control ATCC13124为ATCC13124菌液中不加入PlyH11粗酶液的对照处理,ATCC13124为ATCC13124菌液中加入PlyH11粗酶液的对照处理,control CP12为CP12菌液中不加入PlyH11粗酶液的对照处理,CP12为CP12菌液中加入PlyH11粗酶液的对照处理)
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中,pUC57载体(B300695)是生工生物工程(上海)股份有限公司产品;pPIC9载体(K171001)是Invitrogen公司产品。
下述实施例中,巴斯德毕赤氏酵母GS115(K171001)是Invitrogen公司产品。
下述实施例中,产气荚膜梭菌ATCC13124(BNCC185933)是ATCC产品,从北纳生物购买;产气荚膜梭菌CVCC2014(以下又称产气荚膜梭菌CP12)、CVCC2027(以下又称产气荚膜梭菌CP15)、CVCC52(以下又称产气荚膜梭菌CP36)、CVCC50(以下又称产气荚膜梭菌CP41)均是中国兽医微生物菌种保藏管理中心商品化菌株。
实施例1、克隆裂解产气荚膜梭菌的蛋白质的编码基因
从实验室测序产气荚膜梭菌基因组中预测噬菌体裂解酶基因,其核苷酸序列如序列表的序列2第1-1029位所示;将上述基因经密码子优化后,得到基因plyH11,其核苷酸序列如序列表的序列3第25-1053位所示;将基因plyH11编码蛋白质命名为蛋白质PlyH11,其氨基酸序列如序列表的序列1第1-342位所示。
将基因plyH11交由上海生工公司进行基因合成,用序列表的序列3第1-1061位的DNA替换载体pPIC9的XhoI和NotI识别位点之间的片段(包括XhoI识别位点和NotI识别位点在内的小片段),保持pPIC9的其它序列不变,得到重组表达载体,该命名为pPIC9-plyH11。
为验证重组表达载体pPIC9-plyH11的正确性,使用限制性内切酶Xho I和Not I对其进行双酶切,酶切产物琼脂糖凝胶电泳,结果如图1所示:经双酶切后,出现1kb左右目的基因片段和8kb左右载体片段,表明重组表达载体构建成功。
实施例2、构建分泌表达蛋白质PlyH11的重组毕赤酵母工程菌并制备粗酶液
1、构建分泌表达蛋白质PlyH11的重组毕赤酵母工程菌
使用限制性内切酶Bgl II将实施例1得到的重组表达载体pPIC9-plyH11线性化,琼脂糖凝胶电泳后回收大片段(含有目的基因PlyH11及同源臂),将回收片段电击转化入巴斯德毕赤氏酵母GS115感受态细胞中,利用组氨酸营养缺陷平板进行筛选。随机挑取转化子进行PCR检测,确认阳性克隆,得到转入pPIC9-plyH11的重组巴斯德毕赤氏酵母,将其命名为GS115/pPIC9-plyH11。诱导表达重组巴斯德毕赤氏酵母GS115/pPIC9-plyH11,可得到蛋白质PlyH11,蛋白质PlyH11的氨基酸序列如序列表的序列1第1-342位所示的。
2、制备粗酶液
将上述构建得到的重组巴斯德毕赤氏酵母GS115/pPIC9-plyH11接种于100ml YPD培养基中,置于恒温摇床,30℃、250rpm/min培养18h得到液体培养物;按1%(v/v)接种量,将液体培养物接种于100ml BMGY培养基中,置于恒温摇床,30℃、250rpm/min培养24h;离心沉淀收集菌体,用BMMY培养基将菌体含量稀释至OD600nm=1(以BMMY培养基作为空白对照),置于30℃、250rpm/min诱导表达,每24h补加一次甲醇,使甲醇终浓度为0.5%(v/v),连续诱导表达96h。诱导表达结束后,4℃,6000rpm,离心10min,收集发酵上清液,经0.45μm滤膜除菌,获得的GS115/pPIC9-plyH11发酵上清液即为PlyH11粗酶液。以野生型巴斯德毕赤氏酵母GS115替代重组巴斯德毕赤氏酵母GS115/pPIC9-plyH11重复上述步骤得到GS115发酵上清液作为对照。SDS-PAGE检测蛋白质表达情况,将GS115/pPIC9-plyH11发酵上清液和GS115发酵上清液分别经饱和硫酸铵沉淀,浓缩约20倍后上样进行SDS-PAGE电泳,结果如图2所示,目的蛋白质PlyH11大小约为40KD。与对照相比,诱导表达后出现两条大小相近的条带,其中较大条带为蛋白质被糖基化修饰。
实施例3、PlyH11粗酶液对产气荚膜梭菌的裂解活性
选择5株产气荚膜梭菌菌株作为待测菌株,进行PlyH11粗酶液(即GS115/pPIC9-plyH11发酵上清液,制备方法见实施例2)的裂解活性分析。
5株产气荚膜梭菌菌株具体为产气荚膜梭菌ATCC13124、产气荚膜梭菌CP12(即产气荚膜梭菌CVCC2014)、产气荚膜梭菌CP15(即产气荚膜梭菌CVCC2027)、产气荚膜梭菌CP36(即产气荚膜梭菌CVCC52)、产气荚膜梭菌CP41(产气荚膜梭菌CVCC50)。
分析采用斑点试验方法:
将5株待测菌株:产气荚膜梭菌ATCC13124、产气荚膜梭菌CP12、产气荚膜梭菌CP15、产气荚膜梭菌CP36、产气荚膜梭菌CP41的单菌落分别接种于5ml液体硫乙醇酸盐培养基(FTG),37℃过夜培养备用,得到5种不同的待测菌株液体培养物。
制备双层培养基平板:制备20ml含2%琼脂的FTG培养基为下层培养基平板;制备5ml含0.7%琼脂的FTG培养基,每株待测菌株的处理同时混入500μl该待测菌株液体培养物作为上层培养基平板,一共得到5种菌株处理的双层培养基平板:ATCC13124双层培养基平板、CP12双层培养基平板、CP15双层培养基平板、CP36双层培养基平板、CP41双层培养基平板。
将按照实施例3的方法制备的GS115/pPIC9-plyH11发酵上清液(PlyH11粗酶液)进行梯度稀释,取GS115/pPIC9-plyH11发酵上清液(即100)及10倍(即101)、100倍(即102)、1000倍(即103)稀释液各5μl分别滴到5种双层培养基平板的上层培养基平板表面,待液滴吸干后,将平板倒置,于37℃,厌氧过夜培养。以GS115发酵上清液(制备方法见实施例2)替换GS115/pPIC9-plyH11发酵上清液作为对照。
结果如图3所示,PlyH11粗酶液对五株不同产气荚膜梭菌均具有明显的裂解活性;对菌株CP12的裂解活性最强,1000倍稀释后仍有模糊斑点。
实施例4、PlyH11粗酶液对产气荚膜梭菌的杀菌动力学曲线
选择产气荚膜梭菌ATCC13124及产气荚膜梭菌CP12进行PlyH11粗酶液杀菌动力学曲线测定。PlyH11粗酶液(即GS115/pPIC9-plyH11发酵上清液,制备方法见实施例2)。设置三次重复实验,数据表示为平均值±标准差。
将待测菌株产气荚膜梭菌ATCC13124、产气荚膜梭菌CP12的单菌落分别接种于5ml液体硫乙醇酸盐培养基(FTG),37℃过夜培养备用,得到ATCC13124待测菌株液体培养物和CP12待测菌株液体培养物。
将ATCC13124待测菌株液体培养物和CP12待测菌株液体培养物分别离心收集菌体,用PBS重悬,分别得到ATCC13124菌液和CP12菌液。分别吸取100μl ATCC13124菌液和CP12菌液,各自加入100μl的PlyH11粗酶液(菌液和粗酶液混合后的初始OD600nm值约为1.0),迅速置于酶标仪中测定OD600nm吸光值。同时分别吸取100μl ATCC13124菌液和CP12菌液,各自加入100μl的野生型巴斯德毕赤氏酵母GS115发酵上清液,迅速置于酶标仪中测定OD600nm吸光值,作为对照。动力学参数为:恒温37℃,每10分钟测定一次吸光值,持续测定90min。
其中,PlyH11粗酶液的制备方法如下:将实施例2构建得到的重组巴斯德毕赤氏酵母GS115/pPIC9-plyH11接种于100ml YPD培养基中,置于恒温摇床,30℃、250rpm/min培养18h得到液体培养物;按1%(v/v)接种量,将液体培养物接种于100ml BMGY培养基中,置于恒温摇床,30℃、250rpm/min培养24h;离心沉淀收集菌体,用BMMY培养基将菌体含量稀释至OD600nm=1(以BMMY培养基作为空白对照),置于30℃、250rpm/min诱导表达,每24h补加一次甲醇,使甲醇终浓度为0.5%(v/v),连续诱导表达96h。诱导表达结束后,4℃,6000rpm,离心10min,收集发酵上清液,经0.45μm滤膜除菌,获得的GS115/pPIC9-plyH11发酵上清液即为PlyH11粗酶液。
结果如图4所示,PlyH11粗酶液在前10分钟对两株待测菌株均具有较高的杀菌活性。总体上对菌株ATCC13124的杀菌速度相对较慢;对菌株CP12的杀菌速度相对较快,10min内可使CP12的OD600nm值降低近70%,说明PlyH11对菌株CP12具有快速的杀菌活性,这与实施例3的斑点试验结果较为一致。可能由于残存菌体碎片的存在,10min后CP12的OD600nm值不再降低。
上述结果说明基因plyH11表达的蛋白质PlyH11能裂解产气荚膜梭菌,可用于预防和治疗产气荚膜梭菌导致的感染,在食品、卫生、畜牧业等领域具有广泛的应用前景。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
<110> 中国农业大学
<120> 一种具有抑制产气荚膜梭菌活性的蛋白质及其相关生物材料与应用
<130> GNCSY200869
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 342
<212> PRT
<213> 产气荚膜梭菌(Clostridium perfringens)
<400> 1
Met Thr Lys Leu Arg Gly Ile Asp Val Ser Glu His Gln Gly Val Val
1 5 10 15
Asn Trp Glu Gln Val Lys Asn Asn Val Asp Phe Val Met Leu Arg Ala
20 25 30
Gly Tyr Gly Arg Asn Asn Ile Asp Lys Gln Phe Ile Arg Asn Ile Glu
35 40 45
Glu Cys Asn Arg Leu Gly Ile Pro Val Gly Ile Tyr Trp Phe Ser Tyr
50 55 60
Ala Trp Asn Val Glu Met Ala Lys Asn Glu Ala Lys Tyr Ala Leu Glu
65 70 75 80
Ala Ile Lys Gly Tyr Lys Val Asp Tyr Pro Ile Ser Phe Asp Leu Glu
85 90 95
Tyr Asp Thr Leu Asn Tyr Ala Lys Lys Asn Gly Val Ile Ile Asn Lys
100 105 110
Arg Leu Ala Thr Asp Met Ile Asn Ala Phe Cys Ser Val Ile Glu Gln
115 120 125
Asn Gly Tyr Lys Ala Met Asn Tyr Ala Asn Pro Asp Phe Ile Asn Ser
130 135 140
Lys Phe Tyr Asn Asn Glu Val Lys Tyr Pro Leu Trp Leu Ala Trp Tyr
145 150 155 160
Asn Val Asp Glu Asp Lys Ala Lys Val Tyr Asn Pro Ser Met Trp Gln
165 170 175
Tyr Ser Glu Ser Gly Ser Ile Ser Gly Ile Gly Thr Asn Ser Val Asp
180 185 190
Met Asn Tyr Cys Tyr Glu Glu Phe Leu Lys Ser Asn Phe Val Leu Glu
195 200 205
Asn Ala Thr Thr Lys Asn Val Thr Thr His Leu Asn Ile Arg Ala Lys
210 215 220
Gly Asn Ile Asn Ser Asp Ile Ile Gly Ser Ile Pro Val Gly Glu Lys
225 230 235 240
Phe Phe Lys Lys Trp Val Asp Lys Asp Tyr Leu Gly Trp Tyr Val Val
245 250 255
Glu Tyr Lys Gly Ile Thr Gly Tyr Val Asn Ala Asp Tyr Val Glu Leu
260 265 270
Leu Gln Met Ala Thr Thr Asn Asn Val Ser Ser Val Leu Asn Val Arg
275 280 285
Glu Lys Gly Thr Val Asp Ser Lys Ile Leu Thr Ile Ile Asn Pro Gly
290 295 300
Glu Lys Phe Arg Ile Asp Trp Val Asp Glu Asp Tyr Ile Gly Trp Tyr
305 310 315 320
Arg Ile Thr Thr Ser Ser Gly Ala Ile Gly Phe Val Lys Ser Asp Phe
325 330 335
Val Lys Lys Leu Pro Lys
340
<210> 2
<211> 1029
<212> DNA
<213> 产气荚膜梭菌(Clostridium perfringens)
<400> 2
atgacaaagt taagaggaat agatgtatca gaacatcaag gcgttgtaaa ttgggagcaa 60
gtaaaaaata atgttgactt tgtaatgcta cgtgctggat atggtagaaa taatattgat 120
aagcaattta tcagaaatat agaagagtgt aatagacttg gaataccagt agggatttat 180
tggttctcat atgcttggaa tgtggaaatg gctaaaaatg aagctaaata tgcactagag 240
gctattaaag gttataaagt agattatcct ataagctttg acttagaata cgatacacta 300
aattatgcta aaaagaatgg ggttataatc aataaaagac ttgctacaga tatgataaat 360
gctttttgtt ctgtaataga gcagaatgga tataaagcta tgaactatgc taatccagac 420
ttcataaata gtaaatttta taacaatgaa gtaaaatatc ctttatggct tgcttggtat 480
aatgtagatg aggataaggc aaaagtttat aatccttcta tgtggcaata tagtgaaagt 540
ggttctatct ctggaattgg tactaattct gtagatatga actattgtta tgaagagttt 600
ttaaaatcaa attttgtatt agaaaatgct actacaaaaa atgttacaac acaccttaat 660
attagagcta aaggaaatat taactctgac ataattggat ctataccagt aggagaaaag 720
ttttttaaaa aatgggtaga taaagattat ttaggttggt atgtagtaga atataaaggt 780
attacaggat atgttaacgc tgattatgta gaattactac aaatggcaac tactaataat 840
gttagctctg ttcttaatgt tagagaaaaa ggtacagtag attctaagat attaacaata 900
attaatccag gagagaaatt taggattgat tgggtagatg aagattatat tggttggtat 960
agaattacta catcaagtgg agcaattggg tttgttaaat cagattttgt taagaaactt 1020
ccaaaataa 1029
<210> 3
<211> 1061
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctcgagaaaa gagaggctga agctatgact aagttgagag gtattgacgt ttccgagcac 60
caaggtgttg ttaactggga gcaagttaag aacaacgttg acttcgttat gttgagagct 120
ggttacggta gaaacaacat tgacaagcaa ttcattagaa acattgagga gtgtaacaga 180
ttgggtattc cagttggtat ttactggttc tcctacgctt ggaacgttga gatggctaag 240
aacgaggcta agtacgcttt ggaggctatt aagggttaca aggttgacta cccaatttcc 300
ttcgacttgg agtacgacac tttgaactac gctaagaaga acggtgttat tattaacaag 360
agattggcta ctgacatgat taacgctttc tgttccgtta ttgagcaaaa cggttacaag 420
gctatgaact acgctaaccc agacttcatt aactccaagt tctacaacaa cgaggttaag 480
tacccattgt ggttggcttg gtacaacgtt gacgaggaca aggctaaggt ttacaaccca 540
tccatgtggc aatactccga gtccggttcc atttccggta ttggtactaa ctccgttgac 600
atgaactact gttacgagga gttcttgaag tccaacttcg ttttggagaa cgctactact 660
aagaacgtta ctactcactt gaacattaga gctaagggta acattaactc cgacattatt 720
ggttccattc cagttggtga gaagttcttc aagaagtggg ttgacaagga ctacttgggt 780
tggtacgttg ttgagtacaa gggtattact ggttacgtta acgctgacta cgttgagttg 840
ttgcaaatgg ctactactaa caacgtttcc tccgttttga acgttagaga gaagggtact 900
gttgactcca agattttgac tattattaac ccaggtgaga agttcagaat tgactgggtt 960
gacgaggact acattggttg gtacagaatt actacttcct ccggtgctat tggtttcgtt 1020
aagtccgact tcgttaagaa gttgccaaag taagcggccg c 1061
Claims (10)
1.蛋白质,其特征在于:是如下A1)、A2)或A3)的蛋白质:
A1)氨基酸序列是序列表中序列1第1-342位氨基酸残基的蛋白质;
A2)将A1)的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有90%以上的同一性且具有抑制产气荚膜梭菌活性的蛋白质;
A3)在A1)或A2)的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。
2.与权利要求1所述蛋白质相关的生物材料,其特征在于:所述生物材料为下述B1)至B6)中的任一种:
B1)编码PlyH11的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体、或含有B1)所述表达盒的重组载体;
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物;
B5)含有B1)所述核酸分子的转基因动物细胞系、或含有B2)所述表达盒的转基因动物细胞系、或含有B3)所述重组载体的转基因动物细胞系;
B6)含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系、或含有B3)所述重组载体的转基因植物细胞系。
3.根据权利要求2所述的相关生物材料,其特征在于:B1)所述核酸分子为如下b1)或b2)所示的码基因:
b1)编码链的编码序列是序列表中序3的第25-1053位核苷酸的cDNA分子或DNA分子;
b2)编码链的核苷酸是序列表中序列3的第25-1053位的cDNA分子或DNA分子。
4.根据权利要求2或3所述的相关生物材料,其特征在于:B3)所述重组载体为pPIC9-plyH11,pPIC9-plyH11是用核苷酸序列是序列3第1-1061位的DNA替换载体pPIC9的XhoI和NotI识别位点之间的片段,保持pPIC9的其它核苷酸序列不变,得到的plyH11基因重组表达载体。
5.根据权利要求2或3所述的相关生物材料,其特征在于:B4)所述重组微生物为将权利要求1所述的蛋白质的编码基因导入受体微生物,得到表达权利要求1所述蛋白质的重组微生物;所述受体微生物为C1)-C4)中的任一种:
C1)真核微生物;
C2)酵母;
C3)毕赤酵母属真菌;
C4)巴斯德毕赤酵母。
6.一种制备权利要求1所述蛋白质的方法,其特征在于,使含有权利要求1所述蛋白质的编码基因的DNA分子在生物中进行表达,得到权利要求1所述蛋白质;所述生物为微生物、植物或非人动物。
7.根据权利要求6所述的方法,其特征在于,使含有权利要求1所述蛋白质的编码基因的DNA分子在生物中进行表达包括将权利要求1所述的蛋白质的编码基因导入受体微生物,得到表达权利要求1所述蛋白质的重组微生物,培养所述重组微生物,表达得到权利要求1所述蛋白质。
8.根据权利要求6或7所述的方法,其特征在于,所述DNA分子的编码链的编码序列是序列表中序列3的第25-1053位。
9.一种抗菌剂,其特征在于:所述抗菌剂为抗产气荚膜梭菌的药剂,所述药剂的活性成分含有权利要求1所述的蛋白质和/或权利要求2所述的生物材料。
10.权利要求1所述的蛋白质或权利要求2所述的生物材料的应用,其特征在于:所述应用为下述P1-P2中的任一种:
P1、权利要求1所述的蛋白质或权利要求2所述的生物材料在抗产气荚膜梭菌中的应用;
P2、权利要求1所述的蛋白质或权利要求2所述的生物材料在制备抗产气荚膜梭菌的产品中的应用。
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CN116042593A (zh) * | 2023-01-16 | 2023-05-02 | 山东省农业科学院畜牧兽医研究所 | 产气荚膜梭菌噬菌体裂解酶及其在制备抗产气荚膜梭菌感染药物中的应用 |
CN116042593B (zh) * | 2023-01-16 | 2023-07-14 | 山东省农业科学院畜牧兽医研究所 | 产气荚膜梭菌噬菌体裂解酶及其在制备抗产气荚膜梭菌感染药物中的应用 |
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