CN115572325A - 一种脊尾白虾抗菌肽及其重组蛋白的制备方法与应用 - Google Patents
一种脊尾白虾抗菌肽及其重组蛋白的制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种脊尾白虾抗菌肽及其重组蛋白的制备方法与应用，包含具有与SEQ ID NO：2所示氨基酸序列至少95％同源性的氨基酸序列；所述脊尾白虾抗菌肽的基因EcCrustin，包含具有与SEQ ID NO：1所示核苷酸序列至少95％同源性的核苷酸序列；本发明的EcCrustin的核酸序列长378bp，开放阅读框架编码125个氨基酸，推测蛋白分子量大小为13.9KDa，重组蛋白表达载体pET‑22b‑Crustin转化到大肠杆菌BL21可以获得稳定的表达EcCrustin重组蛋白的菌株；采用本发明制备的表达菌株以及其表达的重组抗菌肽蛋白具有良好的抗菌效果，可应用于水产动物抗菌药物、疫苗或饲料添加剂。
Description
技术领域
本发明涉及生物技术领域，具体为一种脊尾白虾抗菌肽及其重组蛋白的制备方法与应用。
背景技术
渔业作为现代农业重要组成部分，是保障优质蛋白供给和食品安全的重要物质基础。近年来，随着高密度、工厂化、集约化养殖模式的建立及推广，病害频发已成为制约水产健康养殖的重要因素之一。例如各种细菌性疾病的爆发会引起大量水产养殖动物死亡，从而给水产养殖业带来巨大经济损失。目前行业内主要采用抗生素类药物来预防和治疗水产动物的细菌性疾病，虽然抗生素具有强效的杀菌作用，但其在抑制或杀灭病原微生物的同时会抑制某些有益微生物，破坏水产动物体内外生态平衡。长期滥用抗生素还会致使病菌产生耐药性，违背了绿色健康养殖理念。因此亟待开发可用于水产养殖的绿色、健康、安全的新型渔药，以减少或替代抗生素类药物的使用。
抗菌肽(Antimicrobial peptides)来源于动物自身，作为体液免疫中一种重要的免疫分子，在先天性免疫防御中有着非常重要的作用，它不仅可以抑制或杀灭多种病原体，也可以激发机体的其他抗病免疫相关反应，具有广谱杀菌、热稳定性好、不易使细菌产生耐药性等特点，是最有可能替代抗生素成为新一代相对安全的抗菌药物。
甲壳动物中的抗菌肽主要包括三种：penaeidins，Crustin(甲壳肽)和ALF(抗脂多糖因子)，上述三种抗菌肽都是阳离子抗菌肽，具有抗菌或抗病毒活性。另外在甲壳动物中还发现了许多抗菌功能的小肽，比如对虾血蓝蛋白断裂片段等等。
目前已经有许多甲壳动物的抗菌肽基因被发现并公布在NCBI(美国国立生物技术信息中心)上，但是本发明涉及到的抗菌肽基因EcCrustin为本发明人首次发现且命名，在NCBI或者相关文献中没有关于其重组蛋白应用的报道。
发明内容
本发明的目的是针对现有技术的缺陷，提供一种脊尾白虾抗菌肽及其重组蛋白的制备方法与应用，以解决上述背景技术提出的问题。
为实现上述目的，本发明提供如下技术方案：一种脊尾白虾抗菌肽，具有与SEQ IDNO：2所示氨基酸序列至少95％同源性的氨基酸序列；所述脊尾白虾抗菌肽的基因EcCrustin，包含具有与SEQ ID NO：1所示核苷酸序列至少95％同源性的核苷酸序列。
作为本发明的一种优选技术方案，将基因EcCrustin表达序列连接到原核表达载体pET-22b中，得到表达载体Pet-22b-EcCrustin。
一种脊尾白虾抗菌肽重组蛋白的制备方法，具体步骤如下：将脊尾白虾抗菌肽的基因EcCrustin转化到表达载体BL21中进行蛋白表达、纯化，获得凡纳滨对虾抗菌肽重组蛋白。
一种脊尾白虾抗菌肽的应用，脊尾白虾抗菌肽在抗菌药物、疫苗、饲料添加剂中的应用。
一种脊尾白虾抗菌肽抗菌效果的检测方法，利用分子生物学手段敲低EcCrustin基因，检测其抑菌效果。
本发明的有益效果是：本发明的EcCrustin的核酸序列长378bp，开放阅读框架编码125个氨基酸，推测蛋白分子量大小为13.9KDa，重组蛋白表达载体pET-22b-Crustin转化到大肠杆菌BL21可以获得稳定的表达EcCrustin重组蛋白的菌株；采用本发明制备的表达菌株以及其表达的重组抗菌肽蛋白具有良好的抗菌效果，可应用于水产动物抗菌药物、疫苗或饲料添加剂。
附图说明
图1为本发明的Pet-22b-EcCrustin重组质粒图谱；
图2为本发明EcCrustin重组载体构建SDS-PAGE电泳及WesternBlot实验结果图；
A：泳道M为蛋白marker，泳道1为未添加IPTG，泳道2-4加入0.2mM、0.4mM和0.6mM浓度IPTG；
B：泳道M为蛋白marker，泳道3为Western blot结果；
C：泳道M：Protein Marker；泳道1：破碎后沉淀；泳道2：破碎后上清；泳道3：流出液；泳道4：清洗样；泳道5：洗脱样成功表达目的蛋白并进行纯化。
图3为本发明病原物刺激后EcCrustin在血淋巴中的表达模式图；
A：脂多糖、B：副溶血弧菌、C：磷壁酸、D：金黄色葡萄球菌；
在外界病原物刺激后，EcCrustin在血淋巴中均呈现上调趋势，说明在病原物入侵时EcCrustin在机体免疫中发挥了一定的作用。
图4为本发明EcCrustin基因的干扰效率验证图；
通过注射5μL siRNA后对EcCrustin基因进行定量检测，观察表达量变化趋势确定干扰效率，在6小时后表达量有显著下降的趋势，干扰效率约为50％。
图5为本发明在敲低EcCrustin后脊尾白虾在副溶血弧菌刺激下的存活率图；siRNA：5μL siRNA；V.p：副溶血弧菌；Saline：生理盐水；
在敲低EcCrustin后，相较于未被干扰虾，受副溶血弧菌刺激的脊尾白虾后存活率显著下降；
图6为本发明EcCrustin重组蛋白最小抑菌浓度图；
EcCrustin重组蛋白复性后在体外对多种革兰氏阳性菌和革兰氏阴性菌有明显的抑制功能。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易被本领域人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
请参阅图1-6，本发明提供一种技术方案：一种脊尾白虾抗菌肽，具有与SEQ IDNO：2所示氨基酸序列至少95％同源性的氨基酸序列；所述脊尾白虾抗菌肽的基因EcCrustin，包含具有与SEQ ID NO：1所示核苷酸序列至少95％同源性的核苷酸序列。
一种脊尾白虾抗菌肽重组蛋白的制备方法，具体步骤如下：将脊尾白虾抗菌肽的基因EcCrustin转化到表达载体BL21中进行蛋白表达、纯化，获得凡纳滨对虾抗菌肽重组蛋白。
一种脊尾白虾抗菌肽的应用，脊尾白虾抗菌肽在抗菌药物、疫苗、饲料添加剂中的应用。
一种脊尾白虾抗菌肽抗菌效果的检测方法，利用分子生物学手段敲低EcCrustin基因，检测其抑菌效果。
实施例1：
1、EcCrustin基因的克隆；
选取健康的脊尾白虾(Exopalaemon carinicauda)作为实验材料，根据脊尾白虾转录组数据设计引物，提取脊尾白虾胃、鳃、肠道、肌肉、肝胰腺和血淋巴细胞组织样品中的RNA，逆转录合成cDNA并作为模板利用合成的引物进行PCR扩增，1％琼脂糖凝胶电泳检测，胶回收纯化目的产物,与克隆载体连接后转化到大肠杆菌中，挑取10个阳性克隆扩大培养并进行测序验证，对验证的序列进行分析。
2、重组载体构建及蛋白纯化；
通过设计带有NdeI和XhoI酶切位点的引物，以脊尾白虾cDNA为模板扩增目的基因的完整CDS序列，扩增产物经胶回收纯化后利用限制性内切酶进行双酶切，酶切产物纯化后利用T4连接酶与表达载体pET-22b(+)相连，获得重组载体pET-22b-Crustin，将重组载体转化至大肠杆菌BL21(DE3)中表达，挑选10个阳性克隆，进行测序验证，比对正确后扩大培养至OD值0.6-0.8，添加IPTG至终浓度0.6mM，37℃条件下诱导4个小时，离心收集菌体，PBS冲洗2次并重悬菌体，超声破碎。沉淀用Tris-8M Urea buffer溶解，10,000rpm离心10分钟收集上清液进行Ni柱亲和层析纯化。收集纯化蛋白进行SDS-PAGE和Westen Blot分析。洗脱蛋白复性至buffer(20mM Tris-HCL、50mM Nacl)，蛋白无沉淀析出，过滤除菌备用，如图2所示。
3、不同病原物刺激的体内表达模式；
通过对健康的脊尾白虾进行LPS、LTA、副溶血性弧菌和金黄色葡萄球菌刺激，副溶血性弧菌和金黄色葡萄球菌培养至对数，PBS洗涤菌体3次，然后用生理盐水将菌体浓度调节至1.2×108CFU mL-1。0.5mg/kg虾LPS(脂多糖)和LTA(脂磷壁酸)、10μL副溶血性弧菌(1.2×108CFU mL-1)和10μL金黄色葡萄球菌(1.2×108CFU mL-1)注射，对照组注射等体积的生理盐水。注射后，使用前面提到的方法在不同的时间间隔收集血淋巴细胞：0(空白对照)、3、6、12、24、48和72小时(每组3个平行样本)，然后提取总RNA并逆转录合成cDNA进行qRT-PCR检测，如图3所示。
4、RNA干扰及存活率试验；
首先制备siRNA干扰试剂，选取健康虾注射5μL siRNA，使用前面提到的方法在不同的时间间隔收集血淋巴细胞：0(空白对照)、3、6、12、24、48和72小时(每组3个平行样本)，随后使用qRT-PCR验证EcCrustin在血淋巴细胞中的干扰效率。在确认干扰效率后开展存活率试验，在对健康虾注射5μL siRNA后注射10μL 1.2×108CFU副溶血性弧菌来评估EcCrustin对病原体攻击虾的存活率的影响,统计虾的死亡数目并制图，如图4和图5所示。
5、体外抑菌试验；
将体外诱导表达蛋白进行稀释复性后，使用复性蛋白与多种菌株测试其抗菌活性。将菌株扩大培养至对数期培养物后进行稀释，统一浓度至1×106CFU，之后在96孔平底组织培养板中与不同浓度的蛋白混匀，在黑暗中在28℃下培养24小时。与阴性对照相比，没有引起可见生长的最低蛋白质浓度，并定义为MIC值，一式三份测定，如图6所示。
本发明的EcCrustin的核酸序列长378bp，开放阅读框架编码125个氨基酸，推测蛋白分子量大小为13.9KDa，重组蛋白表达载体pET-22b-Crustin转化到大肠杆菌BL21可以获得稳定的表达EcCrustin重组蛋白的菌株；采用本发明制备的表达菌株以及其表达的重组抗菌肽蛋白具有良好的抗菌效果，可应用于水产动物抗菌药物、疫苗或饲料添加剂。
以上实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
Claims (5)
1.一种脊尾白虾抗菌肽，其特征在于：包含具有与SEQ ID NO：2所示氨基酸序列至少95％同源性的氨基酸序列；所述脊尾白虾抗菌肽的基因EcCrustin，包含具有与SEQ ID NO：1所示核苷酸序列至少95％同源性的核苷酸序列。
2.根据权利要求1所述的一种脊尾白虾抗菌肽，其特征在于：将基因EcCrustin表达序列连接到原核表达载体pET-22b中，得到表达载体Pet-22b-EcCrustin。
3.根据权利要求1所述的一种脊尾白虾抗菌肽重组蛋白的制备方法，其特征在于：具体步骤如下：将脊尾白虾抗菌肽的基因EcCrustin转化到表达载体BL21中进行蛋白表达、纯化，获得凡纳滨对虾抗菌肽重组蛋白。
4.一种脊尾白虾抗菌肽的应用，其特征在于：脊尾白虾抗菌肽在抗菌药物、疫苗、饲料添加剂中的应用。
5.一种脊尾白虾抗菌肽抗菌效果的检测方法，其特征在于：利用分子生物学手段敲低EcCrustin基因，检测其抑菌效果。
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