CN103772494B - 一种丁酸梭菌分泌的抗菌肽及其制备方法和应用 - Google Patents
一种丁酸梭菌分泌的抗菌肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种丁酸梭菌分泌的抗菌肽及其氨基酸序列和应用。所述的抗菌肽氨基酸序列为序列表SEQ ID NO:1所示，分子量为2264.6道尔顿，等电点为8.64。其制备方法是：硫酸铵沉淀培养基中丁酸梭菌分泌的的抗菌肽，利用阳离子交换、分子筛层析和反向高效液相色谱法纯化抗菌肽。该抗菌肽具有显著的抗菌活性，且不会对猪的红细胞产生溶血作用，可用于开发制备饲用抗菌药物。
Description
技术领域
本发明属于生物技术，具体的，本发明涉及一种丁酸梭菌分泌的抗菌肽及其制备方法和应用。
背景技术
抗菌肽是机体先天免疫的重要组成成分，是许多生物体抵抗外来致病菌侵袭的第一道屏障。由于其独特且多样的生物学功能和作用机制引起了科学界的广泛关注和研究，一方面研究抗菌肽在先天免疫中发挥作用的生物学基础，一方面将其作为设计新型抗菌、抗感染制剂的分子模板。抗菌肽是自然进化的产物，和微生物一起已经存在了亿万年，尽管经过长期的共同进化，但抗菌肽并没有丢失其杀灭或抑制微生物的能力，或产生使微生物逃逸抗菌肽的能力，所以说抗菌肽是研发新型高效抗生素替代品的重要突破。
丁酸梭菌是一种益生菌，具有维持肠道微生态平衡、提高免疫力、抗癌、产生营养物质等功能。丁酸梭菌开发利用前景广阔，目前已应用于食品、医药、农牧、化工等诸多领域。但由于丁酸梭菌培养条件严格，需要在严格厌氧条件下才能生长，且分泌的成分复杂，难被分离纯化，因此目前尚无从丁酸梭菌中分离纯化抗菌肽的报道。
发明内容
本发明的目的在于提供一种新的具有广谱抗菌的丁酸梭菌分泌的抗菌肽及其制备方法和在饲料中的应用。
为了实现本发明，本发明提供如下技术方案：
一种丁酸梭菌分泌的抗菌肽，从丁酸梭菌分泌产物中分离达到的一种单链多肽，分子量是2264.6，等电点8.64，多肽的氨基酸序列如SEQ ID NO:1所示为：PSAWQITKCAGSIAWALGSGIF，该抗菌肽具有抗菌活性，且不会对猪的红细胞产生溶血作用。
所述的抗菌肽的制备方法，将丁酸梭菌置于严格厌氧条件培养24小时后离心，收集培养上清并用硫酸铵沉淀法富集所述的抗菌肽，经离子交换、分子筛层析和反相高效液相色谱纯化。
所述的离子交换为：将硫酸铵沉淀后得到的样品透析，上样于阳离子交换柱，用含0~0.5M NaCl的缓冲液梯度洗脱，收集洗脱的蛋白。
所述的分子筛层析为：将离子交换得到的活性成分透析，上样于分子筛层析柱，用缓冲液洗脱，收集第四个峰。
所述的反向高效液相色谱为：将分子筛层析得到的活性物质上样于C18柱，用含0.1%的乙腈从10~60%梯度洗脱，收集45min处峰。
所述的抗菌肽的应用，作为饲料的抗菌制剂。
本发明采用飞行质谱法测定分子量，使用ExPASy在线工具估算等电点，自动氨基酸测序仪测定氨基酸的序列结构。
下面用本发明丁酸梭菌分泌的抗菌肽的抗菌试验和安全性试验说明本发明的有益效果：地衣芽孢杆菌分泌的抗菌肽对革兰氏阳性菌和阴性菌均有较高的抗菌活性，对E. coli K 88的最小抑菌浓度为32μg/mL，对S. aureus ATCC25923的最小抑菌浓度为64 μg/mL；安全性高，抗菌肽浓度高达256 μg/mL对猪红细胞也无溶血作用。
附图说明
图1 阳离子交换色谱纯化丁酸梭菌分泌的抗菌肽SDS-PAGE图，（泳道1，蛋白marker；泳道2，丁酸梭菌分泌的总蛋白；泳道3，阳离子交换色谱纯化后得到的蛋白）；
图2 分子筛层析色谱纯化丁酸梭菌分泌的抗菌肽SDS-PAGE图，（泳道1，蛋白marker；泳道2，阳离子交换色谱纯化后得到的蛋白；泳道3，分子筛层析色谱纯化后得到的蛋白）；
图3丁酸梭菌分泌的抗菌肽对猪红细胞溶血率测定。
具体实施方式
以下结合附图和实施例对本发明做进一步的说明。
实施例1 阳离子交换纯化丁酸梭菌分泌的抗菌肽
1) 装柱
2) 10体积的低盐缓冲液，至紫外吸收值稳定
3) 将样品注射至进样柱内，调整进样流速为1 ml/min；
4) 10体积的低盐缓冲液，至紫外吸收值稳定
5) 0~0.5M NaCl氯化钠梯度洗脱
6) 收集样品，用Bradford试剂盒测定蛋白浓度，并用低盐缓冲液将蛋白浓度调整为1mg/ml，取5 μg样品用于NuPAGE分析，如图1所示，其余保存于﹣80℃超低温冰箱。
实施例2 分子筛层析纯化丁酸梭菌分泌的抗菌肽
1) 装柱
2) 10体积的缓冲液平衡，至紫外吸收值稳定
3) 将样品注射至进样柱内，调整进样流速为0.1 ml/min；
4）缓冲液洗脱，收集第四个峰
5) 收集样品，用Bradford试剂盒测定蛋白浓度。用琼脂糖孔穴扩散法检测每个峰的抗菌活性，用低盐缓冲液将蛋白浓度调整为1mg/ml，取5 μg样品用于NuPAGE分析，如图2所示，其余保存于﹣80℃超低温冰箱。
实施例3 丁酸梭菌分泌的抗菌肽对猪红细胞溶血率测定
1）将抗菌肽在96孔板上进行系列稀释，终浓度为256、128、64、32、16、8、4 μg/mL，每个浓度10uL，共七个浓度，同时设置三个重复；
2）90uL/孔的红细胞悬液加入到稀释好的多肽孔中，设置阳性对照孔为1%红细胞悬液中加入10uL 10%的Triton X-100（终浓度为1%）；阴性对照孔位1%红细胞悬液中加入10uL的1×PBS；
3）将96孔板置于37℃，5% CO2环境中培养18-24h；
4）培养结束后，将96孔板在离心机无制动停机状态下1000-1500rpm离心20min；
小心吸取上清至一新平底透明96孔培养板中，用酶标仪测定各孔在414nm和546nm下的吸光度；软件分析得到OD414nm、OD546nm和Delta OD值；通过下列公式计算抗菌肽对猪红细胞的溶血率，结果如图3所示：丁酸梭菌分泌的抗菌肽对猪红细胞无溶血作用。
实施例4 丁酸梭菌分泌的抗菌肽抗菌活性的测定
抗菌活性的测定采用最小抑菌浓度（MIC）法，方法如下：先向无菌96孔圆底板中加入90μL制备好的菌悬液，再逐一加入10μL相应浓度的肽稀释液或抗生素稀释液，待测肽的终浓度分别为256、128、64、32、16、8、4、2、1、0.5、0.25 μg/mL；待测抗生素的终浓度分别为64、32、16、8、4、2、1、0.5、0.25 、0.125、0.0625 μg/mL；另外设阳性对照孔为不加抗菌物质，只加100μL菌悬液，阴性对照孔加100μL MH肉汤培养基；将96孔板置于37℃生化培养箱中保湿静置培养18-24小时；肠道益生菌培养板置于厌氧培养箱中厌氧培养24-48h；每种抗菌物质做三次重复；培养完后观察各孔底部是否有细菌沉淀产生，无肉眼可见细菌沉淀的最小浓度可判定为抗菌物质的MIC。结果如下表1所示：
表1微量肉汤稀释法测定重组抗菌肽CBF的最小抑菌浓度
| 菌种 | 最小抑菌浓度 (μg/ml) |
| 革兰氏阴性菌 | |
| E. coli ATCC25922 | 32 |
| E. coli K 88 | 32 |
| E. coli K 12 | 64 |
| P. aeruginosa CMCC27853 | 64 |
| 革兰氏阳性菌 | |
| S. aureus ATCC25923 | 64 |
| S. epidermidis ATCC12228 | 64 |
SEQUENCE LISTING
<110> 浙江大学
<120> 一种丁酸梭菌分泌的抗菌肽及其制备方法和应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> PRT
<213> Clostridium butyricum
<400> 1
Pro Ser Ala Trp Gln Ile Thr Lys Cys Ala Gly Ser Ile Ala Trp Ala
1 5 10 15
Leu Gly Ser Gly Ile Phe
20
Claims (1)
1.一种丁酸梭菌分泌的抗菌肽，其特征是，从丁酸梭菌分泌产物中分离得到的一种单链多肽，分子量是2264.6，等电点8.64，多肽的氨基酸序列如SEQ ID NO:1所示为：PSAWQITKCAGSIAWALGSGIF，该抗菌肽具有抗菌活性，对E. coli K 88的最小抑菌浓度为32 μg/mL，S. aureus最小抑菌浓度为64 μg/ml，且对猪的红细胞不会产生溶血作用。
2. 一种根据权利要求1所述的抗菌肽的应用，作为饲料的抗菌制剂。
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