CN114437187B - 一种细菌素Bacin A4及其应用 - Google Patents
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Abstract
本发明属于医药应用领域，具体涉及一种细菌素Bacin A4及其应用。所述细菌素Bacin A4具有如SEQ ID NO.1所示的氨基酸序列，可以采用多肽固相合成法或液相多肽合成法合成，也可以将所述细菌素Bacin A4的编码基因在工程菌中表达得到。其结构简单，安全低毒，合成较容易，能高效杀灭金黄色葡萄球菌，可用于抗菌药物、饲料、食品等领域，具有广阔的应用前景。
Description
技术领域
本发明属于医药应用领域，具体涉及一种细菌素Bacin A4及其应用。
背景技术
金黄色葡萄球菌是全球院内感染的首要病原菌，可引起皮肤和软组织感染、菌血症、骨髓炎、脓毒性关节炎、肺炎、心内膜炎等疾病。临床分离的80％以上金葡菌对为耐甲氧西林金葡菌(MRSA)，因其多重耐药性，抗MRSA感染日益成为抗感染治疗的热点和难点，因此迫切需要新的抗MRSA药物。细菌素是由细菌通过核糖体合成机制产生的一类具有抗菌活性的蛋白质或多肽类物质。目前，一些芽胞杆菌产生的细菌素，如在蜡样芽胞杆菌As1.1846中报道的羊毛硫细菌素cerecin对耐甲氧西林金葡菌(MRSA)具有显著的抑菌活性。研究表明芽胞杆菌中依然存在大量未鉴定的新颖细菌素，这些新颖细菌素可被鉴定、开发做新型抗MRSA药物，具有重要开发应用前景。
发明内容
本发明的第一个目的，提供一种细菌素Bacin A4，具有如SEQ ID NO.1所示的氨基酸序列。本发明的细菌素Bacin A4可以采用多肽固相合成法或液相多肽合成法合成，也可以将所述细菌素Bacin A4的编码基因在工程菌中表达得到。
本发明的第二个目的，提供所述的细菌素Bacin A4在制备抑菌剂中的应用，该抑制剂抑制金黄色葡萄球菌。
本发明的第三个目的，提供所述的细菌素Bacin A4在制备用于治疗金黄色葡萄球菌染性疾病的药物中的应用。
本发明的第四个目的，提供一种用于治疗金黄色葡萄球菌染性疾病的药物，包含所述细菌素Bacin A4、一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
本发明的第五个目的，提供一种用于抑制金黄色葡萄球菌的抑菌剂，其包含所述细菌素Bacin A4。
本发明的第六个目的，提供一种含有所述抑菌剂的饲料。
本发明的第七个目的，提供含有所述抑菌剂的食品。
本发明具有如下有益效果：
本发明所述的细菌素Bacin A4结构简单，安全低毒，合成较容易，能高效杀灭金黄色葡萄球菌。本发明的细菌素Bacin A4可用于抗菌药物、饲料、食品等领域，具有广阔的应用前景。
附图说明
图1为实施例3中细菌素Bacin A4的溶血活性图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，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。
实施例1：细菌素Bacin A4的获得
本发明提出的细菌素来源于我们构建的芽胞杆菌XIN-TL12B(acillus sp. XIN-TL2，该菌的保藏编号为CCTCC NO：M 2021718，保藏信息及生物材料样品保藏证明参见本申请人同日提交的另一专利申请，申请号为 202210122032.3)的多肽序列数据库，其氨基酸序列(SEQ ID NO.1所示)如下： MVTFLRIVAQLGARAARWAWANKDRVLGWIRDGIAIDWIINKINDMVN，如序列表SEQ ID NO.所1示。通过在线BlastP比对分析(Protein BLAST:search proteindatabases using a protein query(nih.gov))，该细菌素与已鉴定、报道的细菌素的氨基酸序列均无同源性，说明它是一种新颖细菌素，属发明人首次研究、鉴定、报道。根据所示的氨基酸序列，经吉尔生化(上海)有限公司合成得到对应的抗菌多肽，其纯度>95％。
实施例2：细菌素Bacin A4抗金黄色葡萄球菌活性的测定
以生理盐水为稀释液，通过二倍稀释法配置系列梯度的细菌素溶液(0.5， 1，2，4，8μM)。利用琼脂扩散法检测不同浓度下的Bacin A4对金黄色葡萄球菌的最低抑菌浓度(MIC)。在未凝固的琼脂培养基中加入适量的指示菌(细菌个数约为5×105cfu/mL)，混匀，倒平板。选取金黄色葡萄球菌ATCC 6538、金黄色葡萄球菌ATCC 43300、金黄色葡萄球菌ATCC 12600为指示菌。待其凝固后用孔径为6mm的打孔器打孔。每孔中加入约50μL样品后将平板先放置于4℃， 2h左右，让待测样品充分扩散，而后放置于30℃下培养12h，观察抑菌效果 (观察有无透明的抑菌圈出现)。经测定Bacin A4对金黄色葡萄球菌ATCC 6538、金黄色葡萄球菌ATCC 43300、金黄色葡萄球菌ATCC 12600的最小抑菌浓度分别为0.5μM，1μM，1μM(表1)。
表1细菌素Bacin A4对金黄色葡萄球菌最小抑菌浓度
实施例3：细菌素Bacin A4溶血活性的测定
采集健康人新鲜血液3mL(添加抗凝血剂)，3000g离心5min，弃血浆收集红细胞。将收集的红细胞用生理盐水洗涤三遍，并按2％(V/V)的比例重悬血细胞。在无菌的96孔细胞培养板中加入100μL红细胞重悬液，再分别加入不同浓度的细菌素溶液，每个细菌素浓度设置3个平行。以0.1％Tritonx-100作为完全溶血的阳性对照，以生理盐组作为不溶血的阴性对照。37℃孵育2h，3000g离心10min。将离心后的上清转移到新的无菌96孔细胞培养板中，并利用酶标仪检测570nm处的光吸收值，计算细菌素的溶血活性。
细菌素BacinA1的溶血活性如图1所示，该细菌素在100μM的浓度下仅表现出1.8％的溶血活性，说明其毒副作用非常低。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 淮北师范大学
<120> 一种细菌素Bacin A4及其应用
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<170> SIPOSequenceListing 1.0
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Met Val Thr Phe Leu Arg Ile Val Ala Gln Leu Gly Ala Arg Ala Ala
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Arg Trp Ala Trp Ala Asn Lys Asp Arg Val Leu Gly Trp Ile Arg Asp
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Gly Ile Ala Ile Asp Trp Ile Ile Asn Lys Ile Asn Asp Met Val Asn
35 40 45
Claims (5)
1.一种细菌素Bacin A4，其特征在于，所述细菌素Bacin A4的氨基酸序列如SEQ IDNO.1所示。
2.权利要求1所述的细菌素Bacin A4在制备抑菌剂中的应用，其特征在于，该抑制剂抑制金黄色葡萄球菌。
3.权利要求1所述的细菌素Bacin A4在制备用于治疗金黄色葡萄球菌感染性疾病的药物中的应用。
4.一种用于治疗金黄色葡萄球菌感染性疾病的药物，其特征在于，包含权利要求1所述细菌素Bacin A4、一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
5.一种用于抑制金黄色葡萄球菌的抑菌剂，其特征在于，包含权利要求1所述细菌素Bacin A4。
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| JP2003339377A (ja) * | 2002-05-23 | 2003-12-02 | Prima Meat Packers Ltd | 新規バクテリオシン |
| CN103214583A (zh) * | 2013-04-15 | 2013-07-24 | 中国农业大学 | 细菌素lacticin Q的分泌表达方法 |
| CN111212569A (zh) * | 2017-08-31 | 2020-05-29 | 辛格隆股份公司 | 用于制备细菌素和抗微生物肽的方法和组合物 |
| CN112513251A (zh) * | 2018-06-06 | 2021-03-16 | 辛格隆股份公司 | 改造的抗微生物肽 |
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| JP2003339377A (ja) * | 2002-05-23 | 2003-12-02 | Prima Meat Packers Ltd | 新規バクテリオシン |
| CN103214583A (zh) * | 2013-04-15 | 2013-07-24 | 中国农业大学 | 细菌素lacticin Q的分泌表达方法 |
| CN111212569A (zh) * | 2017-08-31 | 2020-05-29 | 辛格隆股份公司 | 用于制备细菌素和抗微生物肽的方法和组合物 |
| CN112513251A (zh) * | 2018-06-06 | 2021-03-16 | 辛格隆股份公司 | 改造的抗微生物肽 |
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| 枯草芽孢杆菌所产细菌素的理化性质及抑菌谱的研究;徐海燕;张志焱;曹银生;李美丽;谷巍;;饲料博览(第04期);29-32 * |
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