CN116425853B - 一种改造体抗菌肽及其应用 - Google Patents
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Abstract
本发明提供一种改造体抗菌肽，其氨基酸序列为SEQ ID NO:1。其编码基因的序列为SEQ ID NO:2。本发明所提供的改造体抗菌肽可用于制备抑制病原菌的制品。本发明所提供的改造体R‑Hep在大肠杆菌BL21中生产量显著提高，且其对革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌鳗弧菌的抑菌效果增强。
Description
技术领域
本发明属于生物技术领域，具体涉及一种改造体抗菌肽及其应用。
背景技术
病原性微生物的感染在全球范围内爆发，是导致养殖动物患病死亡的重要因素之一。特别对水生动物而言，在其生活环境中存在许多病原微生物(细菌、病毒、寄生虫等)。而在过去几十年中，水产养殖已经成为增长最快的食品生产部门，但水产养殖的病害问题已严重影响了该行业的发展。一直以来，人们大多选择用抗生素进行病原性微生物感染的治疗，然而抗生素的滥用所导致的细菌耐药性，对人类的食品安全、公共卫生带来了新的威胁。而寻找对病原体具有杀害作用且不会产生耐药性的抗生素替代品是水产领域病害防治的热点之一。
抗菌肽(宿主防御肽)是一类小分子多肽，大多对病原微生物(细菌、病毒、寄生虫等)有着广谱的抗性，能够快速抵挡病原体的入侵，是先天免疫中重要的组成部分。因此抗菌肽是目前具有较大潜力的抗生素替代品。
目前人工合成多肽成本较高，而体外通过基因工程方法表达生产抗菌肽具有表达时间短，成本低，操作步骤方便等优势，可应用在实际生产中。而目前原核表达所生产的鱼类天然抗菌肽表达量和抑菌活性相对不太理想。
发明内容
本发明提供一种改造体抗菌肽，即提供一种牙鲆(Paralichthys olivaceus)抗菌肽Hepcidin 2的改造体抗菌肽R-Hep及其制备方法和应用。
本发明首先提供一种改造体抗菌肽，其氨基酸序列如下：
IIFQGVQELEEAGGNDTIVAARQMMSMESWMESPVRQKRKISKI SMCRICCRCCKIKGCGICCKF(SEQ ID NO:1),
根据大肠杆菌密码子偏好进行密码子优化后的改造体抗菌肽的编码核酸片段序列如下：
ATTATTTTTCAGGGTGTACAAGAACTGGAAGAGGCAGGCGGT
AATGACACTATTGTAGCGGCTCGCCAGATGATGTCTATGGAATCTT
GGATGGAGTCTCCTGTTCGTCAGAAACGCAAAATCTCCAAAATCT
CCATGTGTCGCATCTGCTGTCGTTGTTGCAAGATCAAGGGTTGCGG
TATCTGCTGTAAATTC(SEQ ID NO:2)。
本发明再一个方面还提供一种重组表达载体，所述的重组表达载体中插入有用于编码上述改造体抗菌肽R-Hep的核酸片段，
作为一个实施例的具体记载，所述的表达载体为pET-32a载体。
本发明所提供的改造体抗菌肽可用于制备抑制病原菌的制品；
所述的病原菌，包含有金黄色葡萄球菌和哈维氏弧菌。
本发明所提供的改造体R-Hep在大肠杆菌BL21中生产量显著提高，且其对革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌哈维氏弧菌的抑菌效果增强。
附图说明：
图1为牙鲆Hepcidin 2重组蛋白(A)和改造体抗菌肽R-Hep重组蛋白(B)的SDSPAGE表达结果图；其中M为Maker；1为未诱导的全菌液；2为诱导后的全菌液；
图2为每升菌液纯化所得Hepcidin 2和改造体R-Hep重组蛋白的产量数据图；
图3为Hepcidin 2和改造体抗菌肽R-Hep重组蛋白的体外抑菌活性分析图。
具体实施方式
本发明旨在针对牙鲆(Paralichthys olivaceus)抗菌肽Hepcidin 2进行改造，以期提高重组表达效率和其抑菌活性，作为抗菌药物的有力备选。
下面结合实施例和附图对本发明进行详细的描述。
实施例1：抗菌肽的改造及重组表达
1、改造体抗菌肽及重组载体的构建
将Hepcidin 2抗菌肽N末端的24个氨基酸剪切去除，同时将部分氨基酸残基替换成Arg(R)，还将等电点相对较低的His(H)残基替换成了等电点较高的Lys(K)残基；将部分疏水性相对较差的Pro(P)残基替换为疏水性较强的Ile(I)残基，保留了其C端β折叠区域，这样获得的改造体抗菌肽R-Hep2序列如下：IIFQGVQELEEAGGNDTIVAARQMMSMESWMESPVRQKRKISKISMC RICCRCCKIKGCGICCKF(SEQ ID NO:1)。
根据大肠杆菌密码子偏好进行密码子优化后采用全基因合成方法合成改造体抗菌肽基因片段(SEQ ID NO:2)。
2、构建重组表达质粒
通过限制性酶切位点将合成后的牙鲆抗菌肽Hepcidin 2及改造体抗菌肽R-Hep序列分别插入到表达载体pET-32a中。构建了重组表达质粒：pET-32a-Hepcidin 2和pET-32a-R-Hep。
3、蛋白的诱导表达
1)小量诱导表达
将菌液以1：100的比例接种至3mL LB培养液内培养。在37℃，220rpm/min条件下培养至指数生长期(OD600＝0.6)时，加入终浓度为1mM的IPTG。16℃，180rpm/min培养12h。吸取1mL诱导后菌液与1mL未加诱导剂的菌液一起6000g离心5mins。倒掉上清并用100μL PBS重悬菌体，于通风橱内加入100μL 2×上样缓冲液，沸水中煮15mins。利用SDS-PAGE检测诱导情况。将成功诱导表达的阳性菌株送北京擎科生物科技有限公司青岛分公司测序。
2)大量诱导表达
将测序正确的成功诱导表达菌株以1：100的比例加入1L锥形瓶内进行扩大培养，37℃，220rpm/min培养4h至指数生长期(OD600＝0.6)，加入终浓度为1mM的IPTG，于16℃，180rpm/min诱导12h。6000g，离心10mins收集菌体。加入PBS重悬细胞，于-80℃反复冻融三次后置于超声破碎仪内破碎菌体。200W破碎40mins后，12000g离心10mins。收集上清液加入2×上样缓冲液，沸水中煮10mins后利用SDS-PAGE检测诱导情况(图1)。
3)重组蛋白纯化：
将诱导后的大量菌体离心收集，用PBS重悬后置于超声破碎仪内破碎，直至菌液澄清透亮(破碎过程中在冰浴进行)。在4℃条件下，12000g离心15mins，收集上清并用0.22μM过滤器过滤。使用HisTrap HP亲和层析柱纯化重组蛋白Hepcidin2(rHep2)和rR-Hep。首先用超纯水洗管、洗柱，再用Binding Buffer平衡亲和层析柱，平衡后用注射器缓慢注入5mL过滤后的蛋白溶液，待蛋白与纯化柱充分结合后继续用Binding Buffer清洗掉杂蛋白，最后用Elusion Buffer(500mM咪唑)洗脱收集重组蛋白。吸取部分洗脱液加入2×上样缓冲液进行SDS-PAGE电泳检测纯化后结果。将收集的重组蛋白液体在PBS缓冲液中于4℃下透析12h，连续透析3次。将透析后的重组蛋白溶液于-80℃冻存6h后放入冷冻干燥机中冻干成粉末状重组蛋白。将冻干后的重组蛋白用无菌PBS重悬，通过BCA法测定1L大肠杆菌所产生的重组蛋白rHep2和rR-Hep的产量，如图2显示，经过改造和密码子优化后的rR-Hep产量提高了38％。
实施例2：重组蛋白rR-Hep抑菌活性检测
通过微量二倍稀释法对重组蛋白rR-Hep的抑菌活性进行检测。用LB培养基将金黄色葡萄球菌和哈维氏弧菌培养至指数生长期，用MH培养基稀释菌液，调整浓度为107CFU/mL。用MH培养基梯度稀释重组蛋白rR-Hep和rHep2(1.95-1000μg/mL)，并依次加入到96孔细胞培养板中，每孔90μL。
将调整好浓度的病原菌分别加入各孔内，每孔90μL。以90μL无菌PBS+90μL MH培养基作为阴性对照组；以90μL MH培养基+90μL细菌悬液作为阳性对照组。28℃下培养24h后，使用酶标仪对每孔在600nm处的吸光值进行测定。并以导致100％抑制细菌生长的蛋白最低浓度和观察到细菌生长的蛋白最高浓度之间的范围作为最小抑菌浓度(MIC)。
结果如图3所示，改造体抗菌肽rR-Hep的抑菌能力强于rHep2。rR-Hep对革兰氏阴性菌哈维氏弧菌的最小抑菌浓度由62.5μg/mL减少到了31.25μg/m；对革兰氏阳性菌金黄色葡萄球菌的最小抑菌浓度减少到了15.63μg/mL。
综上，本发明所提供的抗菌肽rR-Hep相比于改造前的Hepcidin2在大肠杆菌中重组表达量明显提高，其对金黄色葡萄球菌和哈维氏弧菌的抑菌活性也得到了明显的加强，有潜力成为抗生素替代品并应用在生产中。
Claims (9)
1.一种改造体抗菌肽，其特征在于，所述的改造体抗菌肽的氨基酸序列为SEQ ID NO:1。
2.一种核酸片段，其特征在于，所述的核酸片段用于编码权利要求1所述的改造体抗菌肽。
3.如权利要求2所述的核酸片段，其特征在于，所述的核酸片段的序列为SEQ ID NO:2。
4.一种重组表达载体，其特征在于，所述的重组表达载体中插入有用于编码权利要求1所述的改造体抗菌肽的核酸片段。
5.如权利要求4所述的重组表达载体，其特征在于，所述的核酸片段为权利要求2或3所述的核酸片段。
6.如权利要求4所述的重组表达载体，其特征在于，所述的表达载体为pET-32a载体。
7.一种重组工程菌，其特征在于，所述的重组工程菌中包含有权利要求4所述的重组表达载体。
8.权利要求1所述的改造体抗菌肽在制备抑制金黄色葡萄球菌或哈维氏弧菌的制品中的应用。
9.一种用于抑制金黄色葡萄球菌或哈维氏弧菌的制品，其特征在于，所述的制品中包含有药理有效浓度的权利要求1所述的改造体抗菌肽。
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