CN108752431B - 杂合抗菌肽Mel-MytB及其应用 - Google Patents
杂合抗菌肽Mel-MytB及其应用 Download PDFInfo
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- CN108752431B CN108752431B CN201810339292.XA CN201810339292A CN108752431B CN 108752431 B CN108752431 B CN 108752431B CN 201810339292 A CN201810339292 A CN 201810339292A CN 108752431 B CN108752431 B CN 108752431B
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Abstract
本发明公开了一种杂合抗菌肽Mel‑MytB及其应用，将杂合抗菌肽Mel‑MytB基因克隆到pPICZα‑A载体中，构建分泌型重组酵母表达载体pPICZα‑A‑MEM，转化P.pastoris受体菌X‑33。在醇氧化酶启动子调控下，分子量约3.0kD的Mel‑MytB杂合抗菌肽获得表达。抗菌特性研究表明，该表达产物具有广谱抗菌活性，对多数G‑菌及G+菌均有较好的抑菌活性；同原始抗菌肽相比，杂合后抗菌肽对常见致病菌的抑菌活性显著增强。具有热稳定性和酸稳定性。这些特点使得重组抗菌肽Mel‑MytB在疾病防治和动物饲料添加剂等方面具备较好应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种杂合抗菌肽Mel-MytB及其应用。
背景技术
抗菌肽是一类具有广谱抑菌活性的多肽类物质，部分抗菌肽还能激活免疫系统，参与细胞信号传导，调节细胞增殖，具有抗肿瘤、抗病毒、抗寄生虫功能。抗菌肽可通过破坏细菌细胞膜，造成胞内物外溢使细菌死亡，机理不同于抗生素，具有广谱的杀菌抑菌效果，同时抗菌肽可作为免疫细胞的趋化因子，刺激宿主细胞趋化因子的释放，激活免疫系统对抗原的识别和呈递，提高免疫细胞的分化增殖和应答水平。抗菌肽因其具有独特的杀菌机制和优良的理化性质成为安全绿色抗生素添加剂替代品的研究热点，抗菌肽在畜牧业生产中具有广阔的应用前景。其中将抗菌肽应用到畜牧养殖中的研究较多，人工设计杂合抗菌肽也是目前研究的热点之一。
然而，抗菌肽的研发中存在着诸多限制因素，由于天然生物资源中抗菌肽含量低，且提取分离纯化工艺繁琐，因此从天然资源中提取受到很大限制。而采用化学方法合成的成本仍然很高，目前仅满足于抗菌肽用量较少的基础实验研究。而生产抗生素替代品用于医疗以及畜牧养殖，对于生产成本的要求就比较高。通过微生物发酵和基因工程技术是规模化获得抗菌肽的有效途径，其可以规模化发酵生产，有效降低成本。
蜂毒肽(Melittin)亦称蜂毒溶血肽，是意大利蜂蜂毒的主要成分之一，不仅具有抗菌、消炎、抗关节炎等作用，对辐射还有预防和治疗作用。蜂毒肽的结构蜂毒肽由26个氨基酸残基组成，具有强碱性，溶于水，在水中存在2种形式，即在低浓度、低离子强度形成单体的自由结构，在高浓度、高离子强度则形成四聚体。蜂毒肽具有广谱杀菌作用，能抑制革兰氏阴性和革兰氏阳性病原微生物的发育，尤其对耐药菌株有明显抑杀作用，然而其溶血活性限制了其发展为高效抗菌药物的应用。贻贝素是富含半胱氨酸的阳离子抗菌肽，有5个同分异构体A、B、C、D和G1，主要对革兰氏阳性菌有抑菌活性，包括对一些海洋无脊椎动物的病原体，对革兰氏阴性菌和真菌的作用较弱。
随着抗生素残留，耐药菌株不断出现，人们意识到有必要开发一类新的抗生素。抗菌肽有着许多抗生素无法比拟的优点，具有广谱的生物学活性，杀菌迅速，不受传统抗生素耐药菌株影响，与典型的抗生素具有协同作用，中和内毒素等，在医药、农业、食品工业等领域具有广泛的应用前景。改造已有抗菌肽和设计新抗菌肽分子是提高其活性的有效途径。采用基因工程技术生产抗菌肽具有活性高、产量大、成本低廉、生产速度快、易于规模化生产等优点。目前，抗菌肽的抗菌活性较抗生素还存在一定的差距，且很多抗菌肽还存在着毒性强、稳定性差、成本高等诸多问题。因此，对抗菌肽进行分子设计、结构改造和修饰、降低其溶血活性、提高其抑菌效果及广泛性、提高其热稳定性、酸碱稳定性等成为抗菌肽研发的重要内容之一。
发明内容
本发明的目的在于提供一种杂合抗菌肽Mel-MytB及其应用
本发明所采取的技术方案是：
杂合抗菌肽Mel-MytB，其氨基酸序列如SEQ ID NO:1所示。
编码上述所述杂合抗菌肽Mel-MytB的基因序列，如SEQ ID NO:2所示。
上述所述杂合抗菌肽Mel-MytB在制备饲料添加剂中的应用。
上述所述杂合抗菌肽Mel-MytB在制备抑菌产品中的应用。
进一步的，上述所述抑菌产品包括防腐剂、保健品、药。
进一步的，上述所述抑菌产品为能够抑制大肠杆菌、沙门氏菌、金黄色葡萄球菌、副溶血弧菌或/和创伤弧菌的抑菌产品。
能够表达上述所述杂合抗菌肽Mel-MytB的菌株。
含有上述所述杂合抗菌肽Mel-MytB基因序列的菌株。
本发明的有益效果是：
(1)本发明杂合肽Mel-MytB是一小分子肽，具有热稳定性，可以在100℃的高温保持45min，具有较好的热稳定性。可以满足饲料制粒的高温要求。
(2)革兰氏阳性和革兰氏阴性细菌混合感染所致疾病的治疗，一直是兽医领域面临的难题。本发明中，重组杂合肽Mel-MytB对大肠杆菌、沙门氏菌、金黄色葡萄球菌具有较好的抑菌活性，对水产养殖中常见的副溶血弧菌、创伤弧菌也具有较好抑菌效应，但是改造后的杂合抗菌肽对芽孢杆菌类益生菌的抑制效果较弱，这对维持动物胃肠道菌群平衡及健康方面具有重要的意义。
(3)本发明杂合肽具有广谱抗菌活性，对多数革兰氏阳性菌及革兰氏阴性菌均有不同程度的抑菌活性，同原始抗菌肽相比，杂合后抗菌肽对常见致病菌的抑菌活性显著增强。抑菌特别是同时又具有热稳定性和酸稳定性的特点，使其在食品防腐、疾病防治和动物饲料添加剂等方面显示很好的应用前景。
附图说明
图1为重组表达载体质粒的PCR鉴定；
图2为重组抗菌肽的抑菌活性测定；1，氨苄青霉素；2，阴性对照(ddH2O)；3、4、5、6、7，酵母蛋白；A，金黄色葡萄球菌；B，大肠杆菌；C，鸡沙门氏菌；
图3为重组Mel-MytB的Tricine-SDS-PAGE电泳检测；M，低分子量Marker；1，2，3，4重组酵母菌株诱导后72h，48h，24h和0h样品；
图4为重组杂合肽Mel-MytB的酸碱稳定性。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1
1材料与方法
1.1材料
1.1.1质粒及菌株：
毕赤酵母X-33(His-Mut+)、表达载体pPICZα-A购于Invitrogen公司；Escherichiacoli ATCC25922、Staphylococcus aureus ATCC25923、Staphylococcus aureusATCC6538、Salmonella enterica ATCC13076、Salmonella typhimurium ATCC14028、Vibrio parahaemolyticus ATCC17802、Vibrio vulnificus ATCC27562、Bacillussubtilis ATCC6633E为本实验室保存。
1.1.2主要试剂
限制性内切酶XholⅠ、XbalⅠ、SacⅠ、T4连接酶(购自Takara(大连)有限公司)；Zeocin、Tricine、琼脂糖凝胶回收试剂盒、质粒提取试剂盒、DNA分子质量Marker、小分子质量蛋白Maker(购自上海生工生物工程技术服务有限公司)；pMD18-T由本实验室保藏；其他试剂为国产分析纯；多核苷酸及DNA测序由华大基因完成。
1.2杂合抗菌肽Mel-MytB基因的克隆
选用酵母偏爱密码子设计Mel-MytB杂合肽基因，其基因序列为GGTGCTGTTTTGAAGGTTTTGACTACTGGTTTGCCAAGAAGATGTGGTTACTACGTTTCTGTTTTGTACAGAGGTAGATGT(SEQ ID NO2)，其编码的氨基酸序列为GAVLKVLTTGLPRRCGYYVSVLYRGRC(SEQ ID NO 1)。在Mel-MytB杂合肽N端加上α信号肽Kex2裂解位点(-Glu-Lys-Arg-)。化学合成具有酵母密码子偏爱性编码基因，并在编码基因5'和3'端分别引入XholⅠ、XbalⅠ酶切位点。应用SOE-PCR的方法(两次PCR)合成所需要的目的基因。反应体系(25μL):10×PCR Buffer(Mg2+free)2.5μL，MgCl2(25mmol/L)1.5μL，dNTP(10mmol/L)0.5μL，上下游引物(100pmol/L)各0.5μL，TaKaRa ExTaqTM 0.25μL，灭菌超纯水19.25μL。混匀，瞬间离心。PCR反应条件：94℃预变性2min，进入PCR循环：94℃30s，退火温度从65℃降至50℃，每循环1min，每一个循环降低0.5℃，72℃1min，共30个循环后温度降至50℃，再在最适退火温度55℃的条件下进行15个循环；最后72℃延伸6min。取产物7.5μL，2％琼脂糖凝胶电泳，凝胶成像系统下观察并记录拍照。
1.3重组酵母表达载体的构建和Mel-MytB的诱导表达
经SOE合成好的基因经XhoⅠ、XbaⅠ双酶切，定向插入酵母表达载体pPICZα-A中，构建Mel-MytB重组质粒，缺失酶切位点鉴定阳性的质粒命名为pPICZα-A-MEM，送深圳华大基因测序。将测序正确的重组质粒转化酵母菌X-33，重组酵母的电击转化、筛选按Invitrogen公司酵母表达操作手册进行，略有改进。将筛选到的阳性酵母菌接种于20mL YPD培养基，30℃，300r/min培养至A600达到3～5，离心收集菌体转接于50mL BMMY培养基中，进行诱导表达。30℃，300r/min培养72h，期间每24h补加终浓度为0.5％(V/V)的甲醇。诱导后，12000r/min离心15min收集培养液上清，进行抑菌活性测定。
1.4重组杂合肽Mel-MytB抗菌活性初步测定
采用琼脂孔穴扩散法来初步检测表达产物的抗菌活性及抗菌谱。供试菌株包括大肠杆菌、金黄色葡萄球菌、鸡沙门氏菌。使用平板稀释法将上述各处于对数生长期的菌株悬浮液稀释为108cfu/mL，将其按1％接种到适宜的固体培养基中，混匀后倒置平板。待平板冷凝后，用高压灭菌处理的打孔器打孔，向每孔中分别滴加20μL液体。37℃恒温培养过夜后，观察杂合抗菌肽的抑菌情况。以空载体上清作为阴性对照。
1.5重组杂合肽Mel-MytB最小抑菌浓度(MIC)测定
采用微量肉汤稀释法测定重组Mel-MytB抑菌活性。0.02％乙酸溶液(含0.4％牛血清白蛋白)稀释重组杂合肽，得到2倍梯度稀释抗菌肽贮存液(2560，1280，…，20和10μg/mL)，存放于聚丙烯离心管中。测试菌接种于MHA培养基中过夜培养，将测试菌过夜培养物稀释至2×105～7×105cfu/mL，分别取100μL加到96孔细胞培养板每行1～10号孔，将按梯度稀释抗菌肽10μL对应加到1～9号孔，10号孔不加抗菌肽，作为阳性对照，11号孔加入100μL新鲜MHB培养基作为空白对照，37℃培养18至24h后，用酶标仪测490nm吸光值。吸光度比10号孔低50％以上孔内浓度即定义为对该测试菌最小抑菌浓度MIC。试验在无菌条件下操作，重复3次。
1.6重组Mel-MytB的表达优化
为提高重组酵母的表达效率，对Mel-MytB重组酵母菌株的摇瓶发酵条件进行了优化，包括其诱导起始时种菌的OD值、诱导时间、诱导温度、培养基的pH值控制、甲醇浓度及营养成分的追加等多种参数。
1.7表达产物的SDS-PAGE分析
重组杂合肽Mel-MytB的分子量约为3.0kD，理论上，分子量低于15kD的多肽在常规Tris-甘氨酸电泳系统中难以得到理想的电泳分辨率。因此，我们使用Tricine(三羟甲基氨基甘氨酸)代替甘氨酸作为终止离子，并增加电泳缓冲液的摩尔浓度，使重组Mel-MytB多肽得到了较好的分辨率及带型。
1.8重组杂合肽Mel-MytB的热稳定性和酸碱稳定性检测
将杂合抗菌肽Mel-MytB分别煮沸10min、1h、2h、3h、4h，再以金黄色葡萄球菌为指示剂进行抑菌试验。实验同时，配置pH 2～12的缓冲液。取20μL表达上清，分别加入20μL不同pH值缓冲液，以不加抗菌肽的不同pH值缓冲液为对照，做抑菌实验。
2结果
2.1序列验证
根据测序核苷酸序列推导出氨基酸序列，杂合肽Mel-MytB编码的氨基酸序列为：GAVLKVLTTGLPRRCGYYVSVLYRGRC(SEQ ID NO 1)，已正确插入表达载体的Kex2蛋白酶裂解位点之后，使用了酵母偏好密码子。通过提取酵母阳性转化子的基因组DNA，并用通用引物进行PCR验证，可以看到一条特异、明亮的条带分别约位于580bp(图1)。与预期结果相符，证明构建的Mel-MytB已成功转入。
2.2重组杂合肽Mel-MytB初步抗菌活性测定结果
试验结果显示，Mel-MytB杂合抗菌肽对E.coli ATCC25922具有良好的杀菌活性。20μL浓缩10倍的表达上清对E.coli DH5α的抑菌直径可达到14.5mm，与10μL Amp(25μg/mL)抑菌圈相当，而小于10μL Amp(50μg/mL)相应的抑菌直径(17.5mm)(图2)。
2.3重组杂合肽Mel-MytB的表达优化参数和表达产物的Tricine-SDS-PAGE
通过对重组酵母菌发酵条件的优化，得到一组发酵参数：BMMY发酵培养基pH 6.0，表达后期BMMY培养基pH控制在5.0～6.0，25℃培养72h，其间每6h添加2mL/L的甲醇。在诱导72h时其表达量达到高峰。在Tris-Tricine电泳系统中，杂合肽Mel-MytB大小为3.0kD(图3)。
2.4重组杂合肽Mel-MytB的抑菌效价
重组杂合肽Mel-MytB的热稳定性和酸稳定性结果显示，重组杂合肽Mel-MytB煮沸40min以内抑菌圈没有变化。煮沸40min～1h的杂合肽Mel-MytB抑菌圈出现轻微下降。煮沸1h～2h，其抑菌直径出现显著下降，由最初的21mm下降至8mm。表明抗菌肽具有很强的热稳定性。在酸碱稳定性方面，结果显示，pH7～2下降时时，抑菌直径较原来出现轻微下降，pH8～11时，随着pH值增高，抑菌直径逐渐减小。说明此抗菌肽对酸具有较好的抵抗力，而当pH值高于10，则对其抗菌活性有显著的影响(图4)。
2.5重组杂合肽Mel-MytB的最小抑菌浓度(MIC)检测
最小抑菌浓度是从定量的角度来分析抗菌肽对待供试菌株的抑菌能力。本实验对本发明杂合肽Mel-MytB同原始抗菌肽Melittin(简称Mel)、Mytilin-B(简称MytB)间的抑菌活性进行了比较，其中原始抗菌肽Mel的氨基酸序列为GLGAVLK VLTTGLPALISWIKRKRQQ(SEQID NO:3)，MytB的氨基酸序列为SCASRCKGHCRARRCGYYVSVLYRGRCYC KCLRC(SEQ ID NO:4)。
由表1可知，杂合肽Mel-MytB对大肠杆菌、沙门氏菌的MIC为5.1μg/ml、4.6μg/ml和7.6μg/ml，显著低于原始抗菌肽Mel和MytB；杂合肽Mel-MytB对2种金黄色葡萄球菌的MIC分别为2.2μg/ml和1.96μg/ml，显著低于原始抗菌肽Mel和MytB；对副溶血弧菌、创伤弧菌的MIC为9.4μg/ml、13.2μg/ml，同样显著低于原始抗菌肽Mel和MytB。此外，还检测了抗菌肽枯草芽孢杆菌的抑菌效应，MIC为59.4μg/ml。由表1的结果可知，抗菌肽对常见致病菌具有较好的抑菌活性，其抑菌活性相比原始抗菌肽Mel和MytB显著增强；同时本发明杂合肽Mel-MytB对供试的有益菌的抑菌作用相对较弱。
表1杂合抗菌肽的最小抑菌浓度(MIC)
目前，畜牧养殖行业普遍将添加剂在饲料制粒前进行混料加工，这对添加剂的耐热性提出了较高的要求。本发明杂合肽Mel-MytB是一小分子肽，具有热稳定性，可以在100℃的高温保持45min，具有较好的热稳定性。可以满足饲料制粒的高温要求。另外，革兰氏阳性和革兰氏阴性细菌混合感染所致疾病的治疗，一直是兽医领域面临的难题。本发明中，重组杂合肽Mel-MytB对大肠杆菌、沙门氏菌、金黄色葡萄球菌具有较好的抑菌活性，对水产养殖中常见的副溶血弧菌、创伤弧菌也具有较好抑菌效应，但是改造后的杂合抗菌肽对芽孢杆菌类益生菌的抑制效果较弱，这对维持动物胃肠道菌群平衡及健康方面具有重要的意义。
本发明成功构建基因工程菌P.pastoris X-33/pPICZα-A-MEM，杂合肽具有广谱抗菌活性，对多数革兰氏阳性菌及革兰氏阴性菌均有不同程度的抑菌活性，特别是同时又具有热稳定性和酸稳定性的特点，使其在食品防腐、疾病防治和动物饲料添加剂等方面显示很好的应用前景。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 广州格拉姆生物科技有限公司
<120> 杂合抗菌肽Mel-MytB及其应用
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Claims (10)
1.杂合抗菌肽Mel-MytB，其氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述杂合抗菌肽Mel-MytB的基因序列，如SEQ ID NO:2所示。
3.权利要求1所述杂合抗菌肽Mel-MytB在制备饲料、饲料添加剂中的应用。
4.权利要求1所述杂合抗菌肽Mel-MytB在制备抑菌产品中的应用。
5.根据权利要求4所述的应用，其特征在于：所述抑菌产品为饲料添加剂。
6.根据权利要求4所述的应用，其特征在于：饲料添加剂为防腐剂或药。
7.根据权利要求6所述的应用，其特征在于：药为兽药。
8.根据权利要求4所述的应用，其特征在于：所述抑菌产品为能够抑制大肠杆菌、沙门氏菌、金黄色葡萄球菌、副溶血弧菌或/和创伤弧菌的抑菌产品。
9.能够表达权利要求1所述杂合抗菌肽Mel-MytB的菌株。
10.含有权利要求2所述杂合抗菌肽Mel-MytB基因序列的菌株。
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