CN117384267A - 一种脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用 - Google Patents
一种脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用 Download PDFInfo
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Abstract
本发明提供了一种脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用，属于生物技术领域。本发明的脊尾白虾抗菌肽的氨基酸序列如SEQ ID NO：2所示，编码所述脊尾白虾抗菌肽的基因EcCrustin的核苷酸序列如SEQ ID NO：1所示。基因EcCrustin的核酸序列长378bp，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‑EcCrustin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，尤其涉及一种脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用。
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(Antimicrobialpeptides)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。但是目前抗菌肽存在着抗菌谱狭窄等问题。
发明内容
有鉴于此，为了克服现有抗菌肽抗菌谱狭窄的缺陷，本发明的目的在于提供一种脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种脊尾白虾抗菌肽，所述脊尾白虾抗菌肽的氨基酸序列如SEQ IDNO：2所示。
本发明还提供了编码所述脊尾白虾抗菌肽的基因EcCrustin，所述基因EcCrustin的核苷酸序列如SEQ ID NO：1所示。
本发明还提供了所述脊尾白虾抗菌肽的表达载体，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。
本发明还提供了一种能够表达所述脊尾白虾抗菌肽的重组菌，包含权利要求3所述的表达载体Pet-22b-EcCrustin。
本发明还提供了所述的脊尾白虾抗菌肽在制备抗菌药物、疫苗、饲料添加剂中的应用。
通过采用上述技术方案，本发明具有如下有益效果：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-EcCrustin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广谱抗菌效果，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Pet-22b-EcCrustin重组质粒图谱。
图2为EcCrustin重组载体构建SDS-PAGE电泳及WesternBlot实验结果图，其中：
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Westernblot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后的尾白虾抗菌肽。
图3为本发明病原物刺激后，EcCrustin在血淋巴中的表达变化(A：脂多糖、B：副溶血弧菌、C：磷壁酸、D：金黄色葡萄球菌)。
图4为EcCrustin基因的干扰效率验证图。
图5为在敲低EcCrustin后脊尾白虾在副溶血弧菌刺激下的存活率图(V.p：副溶血弧菌；Saline：生理盐水)。
图6为EcCrustin重组蛋白对不同菌株的最小抑菌浓度。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1EcCrustin基因的克隆、重组载体构建及蛋白纯化
本实施例中，编码脊尾白虾抗菌肽的EcCrustin基因序列如SEQ ID NO：1所示：
atgattcgtctatgcttcttggcggttggcatcctgtttgccgttgcacaagcgcaggtaataggtggccacttgggtacctgtccccctccaaagcaacaagtacaacagtgtaaaaacttctgtaaactggagttgccgggaaccagtggacaatactattgctgtgatcaacaacttgcgccacatggtcaccaaggctcctgccccacggtctccctcctgcccaacgagatcgaagtgatgtgtgaccccaacgactccaacagaccctacagtctgaactgcaagagtgatgacgattgcttcgagtgggagaagtgttgctacactccactcacgcagcagcgcatctgcagaatagcgatctacaattag
脊尾白虾抗菌肽的氨基酸序列如SEQ ID NO：2所示：
MIRLCFLAVGILFAVAQAQVIGGHLGTCPPPKQQVQQCKNFCKLELPGTSGQYYCCDQQLAPHGHQGSCPTVSLLPNEIEVMCDPNDSNRPYSLNCKSDDDCFEWEKCCYTPLTQQRICRIAIYN
1.EcCrustin基因的克隆
选取健康的脊尾白虾(Exopalaemon carinicauda)作为实验材料，实验用脊尾白虾来自连云港市佳信水产有限公司，取样时将脊尾白虾置于冰上，吸干残留水分后用一次性注射器于脊尾白虾围心腔插入，小心抽取血淋巴组织，离心收集沉淀，加入适量Trizol试剂，混匀后于-80℃箱中保存备用。采集血淋巴组织后将虾壳分离，迅速分离脊尾白虾胃、鳃、肠道、肌肉及肝胰腺组织，加入Trizol试剂提取样品中的总RNA，使用琼脂糖凝胶电泳检测RNA完整性。RNA样品检测合格后按照反转录试剂盒(HiScriptⅢRT SuperMix for qPCR,Vazyme)说明书进行反转录合成cDNA,于-20℃冰箱保存备用。
设计目的基因引物，分别命名为Cru-F1和Cru-R1，引物序列如表1所示，引物由上海生工生物工程有限公司合成。以脊尾白虾cDNA为模板进行PCR，扩增获得目的基因CDS序列，1％琼脂糖凝胶电泳检测，电泳检测后利用胶回收试剂盒(FastPure Gel DNAExtraction Mini kit,Vazyme)回收纯化目的基因片段,纯化回收的目的PCR产物与克隆载体(pEM-T质粒)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。
表1扩增目的基因的引物
| 名称 | 序列 | 序列编码 |
| Cru-F1 | ATTACATGATTCGTCTATGC | SEQ ID NO：3 |
| Cru-R1 | CGACGCCAGTTCCTCGACTC | SEQ ID NO：4 |
2.重组载体构建及蛋白纯化
设计带有NdeI和XhoI酶切位点的引物，如表2所示，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-EcCrustin，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 Ureabuffer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。
表2带有NdeI和XhoI酶切位点的的引物
| 名称 | 序列 | 序列编码 |
| Cru-F3 | CCCATATGATTACATGATTCGTCTATGC | SEQ ID NO：5 |
| Cru-R3 | CCCTCGAGCGACGCCAGTTCCTCGACTC | SEQ ID NO：6 |
注：下划线表示酶切位点
实施例2脊尾白虾抗菌肽的抑菌效果验证
1.不同病原物刺激后，EcCrustin基因的表达变化
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，具体操作为：将副溶血性弧菌和金黄色葡萄球菌培养至对数期，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。分别将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)于第二节腹肢肌肉注射，对照组注射等体积的生理盐水。注射后，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72h(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，结果如图3所示。图3显示，在副溶血弧菌(A)、金黄色葡萄球菌(B)、脂多糖(C)、磷壁酸(D)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。
2.RNA干扰及存活率试验
首先制备siRNA干扰试剂，包含四种siRNA干扰引物，siRNA干扰引物如表3所示，选取健康虾注射5μL siRNA干扰试剂，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10μL1.2×108CFU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。图4显示，6小时后，EcCrustin的相对表达量有显著下降的趋势，干扰效率约为50％；图5显示，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。
表3 siRNA干扰引物
3.体外抑菌试验
将EcCrustin重组蛋白进行稀释复性后，利用用复性蛋白测试其对杀鲑气单胞菌、大肠杆菌、幽门螺杆菌、铜绿假单胞菌、鮼弧菌、霍乱弧菌、哈维氏弧菌、拟态弧菌、副溶血弧菌、创伤弧菌、枯草芽孢杆菌和金黄色葡萄球菌的抗菌活性。分别将上述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，在黑暗条件下在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，检测结果如图6所示。图6显示，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。
由以上实施例可知，本发明提供了一种对多种革兰氏阳性菌和革兰氏阴性菌有抑菌效果的脊尾白虾抗菌肽、编码基因EcCrustin、重组菌及其应用。本发明提供的脊尾白虾抗菌肽具有良好的广谱抗菌效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种脊尾白虾抗菌肽，其特征在于，所述脊尾白虾抗菌肽的氨基酸序列如SEQ IDNO：2所示。
2.编码权利要求1所述脊尾白虾抗菌肽的基因EcCrustin，其特征在于，所述基因EcCrustin的核苷酸序列如SEQ ID NO：1所示。
3.权利要求1所述脊尾白虾抗菌肽的表达载体，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。
4.一种能够表达权利要求1所述脊尾白虾抗菌肽的重组菌，其特征在于，包含权利要求3所述的表达载体Pet-22b-EcCrustin。
5.权利要求1所述的脊尾白虾抗菌肽在制备抗菌药物、疫苗、饲料添加剂中的应用。
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