CN115124610B - 类Crustin抗菌肽及编码基因的获取方法与应用 - Google Patents

类Crustin抗菌肽及编码基因的获取方法与应用 Download PDF

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CN115124610B
CN115124610B CN202210661973.4A CN202210661973A CN115124610B CN 115124610 B CN115124610 B CN 115124610B CN 202210661973 A CN202210661973 A CN 202210661973A CN 115124610 B CN115124610 B CN 115124610B
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李诗豪
李富花
胡婕
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Institute of Oceanology of CAS
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Abstract

本发明涉及由凡纳滨对虾基因组中多WAP编码基因转录并经选择性剪切产生的一组类Crustin抗菌肽的获取及其抗菌应用。该组成熟mRNA分子编码类Crustin抗菌肽LvTWD‑C1W1C3、LvTWD‑W2C4和LvTWD‑C3W2C4，分别由SEQ ID No.5、SEQ ID No.6和SEQ ID No.7所示的氨基酸序列组成。本发明利用表达载体pET32a(+)和表达菌株大肠杆菌BL21，对LvTWD‑C1W1C3、LvTWD‑W2C4和LvTWD‑C3W2C4分别进行原核重组表达，并获得了具有生物学活性的重组蛋白。对其生物学功能进行体外验证，证实重组蛋白能够对多种细菌产生有效的抑制作用。

Description

类Crustin抗菌肽及编码基因的获取方法与应用
技术领域
本发明涉及基因工程，尤其是涉及一组类Crustin抗菌肽LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的获取及其抗菌应用。
背景技术
对虾养殖是海水养殖的支柱产业之一，随着市场对优质水产品需求量的不断增长，虾类养殖在水产养殖中占据越来越重要的地位。然而，虾类病害的爆发性流行使对虾养殖遭受了巨大损失，多种细菌病毒引发的对虾疾病频发困扰着对虾养殖业的发展。目前抗生素、激素类等药物在水产养殖上，虽然对病害控制起到一定的作用，但也破坏了水环境及动物体内的微生态平衡，使水产动物失去了正常的菌群屏障及生物拮抗作用，同时抗生素的滥用也将导致细菌产生耐药性，变异出致病性更强、危害性更大的致病微生物。因此开发特异的生物制品药物来替代传统抗生素的任务十分紧迫。
对虾抗菌肽是一类基因编码的小分子多肽，在先天性免疫中起到了重要作用。它们一般带有正电荷，能够对某些细菌、真菌和病毒等具有较强的杀伤作用。同时，与传统抗生素相比，抗菌肽作为多肽抗生素具有无污染，针对性强，不易产生耐药性等特点，是解决对虾病害问题的重要突破口。
甲壳肽Crustin作为甲壳动物的一类抗菌肽，作为先天性体液免疫的重要效应分子，具有广谱的抗菌活性，并可以直接杀灭病原，在先天免疫反应中起着至关重要的作用，也是近年来抗菌肽类的研究热点之一。乳清蛋白(whey acidic protein，WAP)结构域是Crustin的关键结构域，在其抗菌活性发挥中具有重要作用。在前期的研究中，申请人所在项目组从凡纳滨对虾基因组数据中获取了34条完整的Crustin抗菌肽编码基因，鉴定获得部分新的Crustin类型，并通过功能研究揭示了它们抗菌活性的作用机制。随后，发明人经过数据挖掘，进一步从凡纳滨对虾基因组中获得一条编码多个WAP结构域的基因LvTWD，其转录本可经过选择性剪切产生多种形式的成熟mRNA分子，其中一组分子编码的氨基酸序列具有与Crustin类似的结构域组成，暗示其可能发挥抗菌肽作用。
发明内容
目的在于提供一组类Crustin抗菌肽的获取及其抗菌应用。
本发明技术方案如下：
一条来源于凡纳滨对虾基因组的多WAP编码基因LvTWD，具有序列表SEQ ID No.1中的碱基序列，其转录的前体mRNA经过选择性产生一组成熟mRNA序列LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4，分别具有序列表SEQ ID No.2、SEQ ID No.3和SEQ ID No.4中的碱基序列，编码的类Crustin抗菌肽序列分别具有列表SEQ ID No.5、SEQ ID No.6和SEQID No.7所示的氨基酸序列。
所述LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的重组表达：根据其序列信息，构建原核表达载体pET32a(+)，分别转化至大肠杆菌BL21菌株中，鉴定阳性克隆，并通过IPTG诱导表达，通过钴离子亲和层析柱，纯化出具有活性的重组抗菌蛋白，并检测其活性；具体为：
1.类Crustin抗菌肽编码DNA制备
根据LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的碱基序列信息，分别设计带有限制性内切酶酶切位点的PCR扩增引物，以cDNA为模板，经PCR扩增并进行产物胶回收，获得LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的成熟肽编码DNA。
2.重组表达载体及宿主菌的选择
确定使用pET32a(+)为原核表达载体，使用大肠杆菌BL21为表达宿主菌。
3.重组表达载体的构建
利用限制性内切酶份额分别酶切胶回收后的LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的PCR产物以及pET32a(+)表达载体质粒，并分别把不同抗菌肽DNA与酶切后的载体DNA进行连接，构建重组表达载体，将其转化到大肠杆菌Trans5α感受态细胞中，利用PCR技术检测阳性菌株。
4.转化与筛选
提取重组表达载体，并转化到大肠杆菌BL21感受态表达菌株中，挑选单克隆菌，进行PCR检测，筛选出阳性工程菌。
5.IPTG诱导表达
挑选阳性克隆，在LB液体培养基中过夜培养，培养液按照1:100稀释到新鲜LB液体培养基中，摇床培养至OD600约0.4～0.6，添加IPTG至终浓度为1mM进行诱导表达，SDS-PAGE检测重组蛋白的表达形式，三种重组蛋白均以可溶形式进行表达(图1、2、3)。
6.重组蛋白的分离纯化
选用TALON His-Tag钴离子纯化树脂(Clontech，美国)对重组表达蛋白进行纯化，纯化后在Tris-HCl溶液中进行透析，获得可溶重组蛋白。
所述的一组类Crustin抗菌肽重组蛋白对革兰氏阳性菌和阴性菌的生长均具有抑制作用。
本发明类Crustin抗菌肽来源于凡纳滨对虾基因组的多WAP编码基因LvTWD，由SEQID No.1所示的碱基序列组成，其编码的前体mRNA经选择性剪切后产生了一组可翻译成与Crustin抗菌肽结构域组成类似蛋白的成熟mRNA分子LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4，分别由SEQ ID No.2、SEQ ID No.3和SEQ ID No.4所示的碱基序列组成。该组成熟mRNA分子编码类Crustin抗菌肽LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4，分别由SEQ IDNo.5、SEQ ID No.6和SEQ ID No.7所示的氨基酸序列组成。本发明利用表达载体pET32a(+)和表达菌株大肠杆菌BL21，对LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4分别进行原核重组表达，并获得了具有生物学活性的重组蛋白。对其生物学功能进行体外验证，证实重组蛋白能够对多种细菌产生有效的抑制作用。
本发明有如下优点：
1.本发明中使用原核重组表达系统，获得可溶的重组表达蛋白，可以较为简单地打量制备目的活性蛋白。
2.利用本发明中使用的重组表达载体及宿主菌，能够成功表达出具有抗菌活性的重组蛋白。
3.本发明可开发有效的对虾细菌性疾病的防治制剂。
附图说明
图1重组蛋白LvTWD-C1W1C3纯化后的SDS-PAGE电泳图。泳道1未结合蛋白，泳道2为杂蛋白，泳道3为Wash Buffer洗脱液，泳道4为纯化后的蛋白，泳道M为蛋白Marker；
图2重组蛋白LvTWD-W2C4纯化后的SDS-PAGE电泳图。泳道1未结合蛋白，泳道2为杂蛋白，泳道3为Wash Buffer洗脱液，泳道4为纯化后的蛋白，泳道M为蛋白Marker；
图3重组蛋白LvTWD-C3W2C4纯化后的SDS-PAGE电泳图。泳道1未结合蛋白，泳道2为杂蛋白，泳道3为Wash Buffer洗脱液，泳道4为纯化后的蛋白，泳道M为蛋白Marker。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
实施例
一组类Crustin抗菌肽LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4及其编码基因，具有如下序列：
(1)SEQ ID No.1的信息(参见序列表)
(a)序列特征
*长度：11907碱基对
*类型：核酸
*链型：双链
*拓扑结构：线性
(b)分子类型：DNA
(c)假设：否
(d)反义：否
(e)最初来源：凡纳滨对虾(Litopenaeus vannamei)
序列描述：SEQ ID No.1
ACAAGTGAGGATCAAGATGCGCACCGGCACTCTCCTGCTGGTGGTGGCGGCTGCCCTCTCCGCTGCAGCACAGGGTGAGACTTAAAATAAAATCTAGATAAAAATCACGTTAACGATAGTACTAATATTGAAACAAAATATACAAATGAGTAAATATATAAAATCTAAATAAAAGTCACGTTAACGATAGTACTCATAATGAAGCAAAATAAACAAATCAGTAAATATTTAAAATCTAGAAAAAAATCACGTTAACAATAGTACTCATATTGAAACAAATAATGTAAATGAGTAAATACACGTAAAACAGTTGATCATGAGATAATATTAAGAATTTTGAAAATTACGAATAGAGGCTTGGAAGTGAGAATATGTGTGGAAAGTCAAGAATCAGAATACATACCAAAATAACGGTAAACAACATAGAATCCGAGGAAACTTTTGTTTAAGGAGGAAAGAATATGAATATAAGATATTAAAAATAGAGACTTTCGATAAAAAAAAAATGATTAATGGTTAAAAGCAAAATAAATGTGCTAGACATATAAGGTCATATAACACTATAGGAAGGTACAGCAAAAGGGTAGTGAAGTTAGAAACAATAGTAACAACAATAGTTAGTGAGTTAGTAGTTAGTTGCTATGCGTCAAATATGTAGAGCAGAGGTTCTCAACCTTTACTAGTTAGTGGCGCACTAAGGATATAGCCTGGACTTCGACGGCGCACAAACTATACAGTAATAATAATAATGATAAGTAATATTATTATATCTATTACCAATATCATTATTGTTGTTTTTATTATCATATATATGAGAATGAATTAATGAACACATGGGAATGAAATATATAATTCCAGTTGTAGATTAAACATACATGATTAATGGGACACTTGCGCTTGTCTGGATTTGCATATCCTTTCGATGTCATGAGGAAAAAATAATTGTGAAAATAAGGCGGCTAGAAGCAAGGCACAAATTCAAGACATAACTAGATATTCATTAAATTTTCGCGGCGACCTTGAGGGATGGTCGCGGCGCAGGTTGAGAACCACTGACCTAGAGTAATATTGAAAAGGTTAAAGATGGGTAAAGGAGTTGAGTGAATGGGTTACGGTGGGGTTAGGCAAGGGGATTAGATCAAGTGAAGGATATGTATAGTCTGAGGAAGGAGAACAGGTTGTCAAAGCAGAGGAGGATGCGTAGGAAAAGTCTCTTGGGAGGCAGAGAAGGGACGGGTTATACGGAGAAAGGATATGACGAAGGCCAAGTCTGTGAGAACGGAAACAGCGAATATTCTAATGCAGGAGAGCTATTGCTTAGTTAAAGGATATGACGAAGATCAGGTCTGTGTGAACGGAAACCGGGAATTATTACTTTGTTGATCTACGAGTGATAACGGTTACAGTTTGGAGAATTTGAAGGGAAAGGAACTAGGGGGTGAGAAAAGGAATTAGAGTGGGGGGGGGGGTAGTATCAGGAGGGGTATTAGGAGGTGGAGGATCTGGGGAAGGGTATAGTTGTGACATAAGGGGTTCACGTGTGTATCCAGGAGGGAATGGGAGGGATAGAGGGGATGGAGGGAGGGTTGATGTAGTTGGAACTGAGGGGGATGGGCTGTGTTGGGAGGGAGTAGGAGGAAGAGGTGTAGGAGGAATATGAGTAGGAGGAGGATGGATACCGGCAGTCACTTTGAGGGTGGAGGGAGCAGGAGTTGTGGAATTTTAGGGTGGGTGAGGGTTTTCGGTGTCATTTTGGGACTCGAGCAGATAGTTCTGAATGTCTTCAAGGGTTTCTGAAACGTAGTCGGCCGGGGAGTGTTGTGAGACAAAGGTTTTCTCATGAGGGGGGGGGGGGGGGAGAGAAACTGGTGTCTGAGGAGTGGAGGCAAAGGTAGGTGGAGGTGAGAGTGTGGTAGAAGTATGAGAAAAATCCCGTCGACGTGCTTCCTGTCTGGGTTCACGCAGAGTGAGACCAAGTCTGTATCTGAGAGTTGCCACCTCAGACTCAAACTTGTAGGTGGGGCAGCCCCTATAAAATACATTATGGGGGCTGCCGCAGTTAGCGCATGTGCGTGACTGCGCAGAGCAGATTGATCGGTTATGACCAGGTTGTGCACACAGAGGGCATCGGGCTGTGGGACGGCAGTGTTTGGCAGAATGTCCTTAACGCCAACAGTTTTAACATTGACGGGGAGGATGTTGATATGATCGGACAGGGAGGGATTCTCCACCAATGTAGACATTAAAGTGAAGGTCATGTCTACGGAAAGTAATTTTGGCAATGTTGGTGGGGTTCTTACGATGAATTTTAGGAGGAATGAAGTAGCACTGTACTGTTCGTACATCATCGTCCGCGGCTCTCACACCTTAGGAAGTAGGTAGTAGAAGGGGACGGAGAAGGGACAGAAACGAAAAAGCAGGAGGGAGAAAAGACCTTGCAAAATTAGTTGAGTCGAGGGCTGAGGCTCAAGGGAAGGGAGATCCCCAGCAGCATTGGGCCCCAGTCCTCGCCTTCCAAGCCCCCTCACGACAACAAAGGGTAAGGGATTGGGGGGGAGGGGGGTCGACAAAAAAAAAAAAAAAACGAAAATGTTGTACGAGAGGAGAGGGGAGAAGGGAGAGTGTAAATCTTGAACGGTTTTCGAGAGATCATATCATATCAATTTTTTTTTATTCTACCAGTTCCCCCATCCCCTCTCATTTTTGACCAATATCTACGTCATATTATCTCTATGCGTATCTATGCTAAAACATTATTCTCTTGACATTCATATTCACAATTTTCTTTTTTATTTTCCGTACAACTACAGAGGGCATCCCGACAGAAGGAAAACTTACACCAACAGGTAAGACTCATAATGATAATGAATAACATAAAAATGAATAAAACAGGATGTGATGATACAAAAAATAAACCCACTTATTAATAGGTTTAATAATTTGCATACAATTCTTGACATGTTTCAATTCATTATGATTAAATCTTAAAAGTATGACAATGGCACAAAAAAAAAAAAAAAATAATAATAAATAAATAAATATGGCGAACTACAATTTTGTGTTTGTTTGTTTTACAGAAGCTCCTCGAGAATTCCAGACTCTCCCCCCTAGCAGTAAGTCAATATACATCAATTAACAACTGATGATAATTATCCTTATTATGATTATCATCATTACTTTTATCCTTGTTATTATTATTATCATTATTGTTATTATCATTACTATTATCATCAGCATTGTTATTATCATTATTATTATTATCATCATCATTATCATCATCATTATCATCATCATTATCGTTATTATTATTTCTCATTATTTTATTATTATTATCATTTATCATTATATTATAGTTATTTGATTTTATTCCTATTATTATTATTCTATTCCTATCATTATCATCATAATTGTATACCTATTATTAGTAGTATTATCATTACTAATATCATTATCATTATCATAGTAATTAGCATCTTATTATCATCATACATCTGCCACACAATAGTAAATGGTCTAAGGGGATGTTATTTTCTCTAATTTATTCTCTAATTCTATTTGTTATACCGGATGTATTTATTTATTTAATTTAAGCATTTTTTTTATCTAATTTCATTTTGATTTACCGAATAAGATTTTTGTAGCCTCTTTTTATCTGATTTCATTTTCATTTAGCGAATAGATTTATCTTTTGTATTTTCTTTTCTCTAATTTTATTTTCATTTACTCAGCCGTCAAGCAGTAAGTCAGTGTACATAGTTTAATAAATTATTATTATCATTATCATCATTGTGCTATCATGGAATACGGTCTTAAACTTTGATCAAAAGTCTTAAGAGGTCTGAAAGTGAATAAAACATTTTTGTAAAATATTCCAACTCTCTTCTCTTCCTTTTTTTTCAATTTACTATATAATTATTGACATTTTTCTTCCTATATCATTTTTTTCAGATTGTCGCTACTATTGCGCTTACGAAGGAATTGCGTACTGCTGCGATGATGGCACTAGACCTAGTAAGTGAAAAAGAAATGTGTATATATATATGTATATCTGTCTATCTATCTATCAGTCCGTCTATTCATATATGTGTGTGTATATACATTATTTTCTGTTCTTTTTTAATATACATCAAAAGAACTTATCACTTATATAGTGGCGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCATAATCATCATTTCATCATAATAAAAAATAGCATAATTATTTATCTATCATCATCATCATCTATTATCATTATTACCATTATCATTATTACCATTATCATCATAATTATCAACACTTTGTCTCTTCCAGTTCCCCCAGATCACGATGAGAATGCAGGTACTTGCGTCGAAATTGAAGAACATATTTGTGAAAAGGATGCCATCTACTATAACGTCACTGACAGTGTAAGTCCCTCAGATTTAAAAAAAAGAAAGAAAGAAAGAAAAAAAAGAAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGGAAAGAAAGAAAAAAAAAAGAAAGAAAGAAAGAAAGAAAAGAAAAGAAGAAAGAAAGAAAGGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGGAAAAGGAAAGAAAGAAAAGTGTATCCGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTTGAAAGTCTTTGTTAGATTTGTGTTGTCTACACACAAATACCCGCACACATTCACATACACACATACACACGCACATAGATACCCACATACACAGATACCCGCACACACACACACAGATACCCACACATACATACAGATACCCCTCACACACATGCAAATACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACAGATACCCACACACACACACAGATACCCGCACACACACAGATACACACTACACACACAGATACCCGTACACACACACAGATACACACACAGATACCCGTACACACACACATACCCGCACACACACACATACCCGTACACACACACACACAGATACCCGCACACACACACATACTACACACAGGATACACACACACACATTTCTACACATTCTCCATCTATTGTTCTTCAGACTGGCGCGAACACGACGAAGCAGCTGTTCTCGGCTCGCCAACGGCCACGCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGCACAGGGTGAGGTCTCGAAAGTTTATTTATTTGTTTATTTATCTATTATTTATTCATTTTATTTATTTTTATTTTATTTATTCATTTATATTTATTTCATTTATTTAATTATTTATTTATTTTTATTCATTTATTTAATTATTTATTTATTATTTATTTATATTTACTTATTTATTTATTTATTTGTTTTTATTTCATTTATTTTTTTATTATGATTCATTTATTTTTTTAGAAGGACATGTGTAATAAAAGGATTGATGATCAAAGGAGACGAGGCAGAATAGGAGAGGATTCTGACGTGGGAATGAAGCACAGGGAAAAATTATTCTAGAATATTTTTTTAGAATTAGAATCAAAATTTCCTTTTTCTACTTCCTTTAACCTCTTCCTCTATTTTGATTATGCTCTCTACAAATATTTTGACTTTTTGTCGTGATGATAAACAACATTTTATTTTTTTATTTCTTTCTTAAATTTGCAGAAAAATATCCAGTCACAGATACGCCTCCAGTAACAGGTAAGACTAAAATAAGAGAAACTGAATATATGTATACTTATACAGTACATATATCACATTGTATATTTTCTACAATAATGAAAAACATATTTATATTCCATATATTAGTGGAATACATTAGTTAAATCGGAGAATAACTCAAATACATGCACTCGTGGAGGACTACATAAAAAAAAAATTTGACTTTGAACACAAAGCACTTAGCTATGTTGCCTTTTCTCTAAATTCATGGGTTATTTTTTTCGTCTCGCAAAATACACCGAGTTAGAAATAAACCCATTTTACTCTTATTTTCCTGATTTTTTATTCATTTACATATTTATTTATTTATTTTCCTGATGTTTTATTTATTTATATATTTATTTATTTATTTATTTATAGCCTGTCGGTACTACTGTGGCTACGCGGGGGTTAGCTACTGTTGCGACGGTGGACTCGCGCCCGGTCTGTTTCCTATTTTTCTTTCCTTCTTATTTTTCTTTCTTTCTTACTTTTTTTTCTTTCTTTCTTTTTTATTTTTCTTTCTTTCTTTCTGTCCTTCTTCCCTTTTTTTCCTCCCTCCCTCTCTCTCTCTCTGTCTCTCTGTCTCTCTCTGTCTGTCTGTCTCTGTCTCTCTGTCTGTCTGTCTGTCTGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACATATATAATGCATACACACGTATGTATGTACATAGATACACACGTGTATATGTATATATTTCTGTGTATGTGTGCATATATATATATGTTATACTCATTTCTGTTTGATTAGGAAAACAGAAAATTACATTTACTCTATTAGTGGAATAGACTAGACAGAAATATCAGAGAATGACTCAATTTCACCCTCTAATGGAATATTACATTGACAACTTTTGCTTTTGTTTGACAGATTTCCGCCGCCAATACCCAACTCAGCCGCCAAGCAGTAAGTCAATGTGTATAGTTTAGCAAATGATTATTATTATCATTATCATCTTTGCACTAGCCTGGAATACGGTCTCAAAGTTTGATCTAAATTCTTAGAGGTCTGTAAGTGGATTGAATATTTTTGTAAGATATTCCAACTCTCTCTTCTCTTACTTTTTATTCAATTTACTATATATCTATTGACATTTTTCTTCCTATATCTTTTTTTTTCAGATTGTCGCTACTTCTGTGGCTACGAGGGAGATGCTTACTGCTGTGACGATGGCACCAGACCCAGTAGGTGATGCTCGTCTTTGCAAAAAAGAAAGAAAGAAAGAGAAAGATAATAATAGAAAAATAAAATAAATAAATATCAATTAAAATTTAAAAATGATAATACAAAAATAGAATAAATAAATAAATATGAAAATAAAAAAGATCATAATACAAAATTAAGATAAATAAATAAATATGACAATAAAAATAAAATATGTTTTCTAAAAAGACCATTGCCTATTGCCGTACCTGTTCAGATAACTTTAGATCGTGTCTTCTCTATACATATATCCTTCTCTTTTGTTGTGTTGTCTCTGTCTCTTTCGTTTCCTCTCACTCCCTCTTGATTGCCCCTTTGAAAGTTATTATTCTCGTTGCCTCTTACAGTTCCCCAGGATCACGATGAGAATGCAGGTGTTTGTATCGAAATGAGCGAACACATCTGCGAGGATGACGCCATTTACTACAACACTACTGATGGGGTAAGTATAGTACTTATATGATACGTATGAAATTTAGACAACCAGAGTCTACTGAGGAAAAAATACTTGCAAATCTTACACACACTGCAACAGGAACAACGAAGGGAAGGACAAAAAAATACACGAATAGGCCCATATTCGTGTGTTTTCCTGTCCGTCATGAATTCCACACATACACACACATGTACACAAAGGGAGATTCAGGGAAAGACACGTTTATACCGAAGGTTTTTTTCACGTTATTGTTTCTTCATGCTCTGAAGAAGCGCTATGAAGGCCTTGGGTGTGATCTCAGGTTTCCGTTTTTTTCCTTTTTTTCATAACTTACTCATCATCAATCTTCTACACACTCACGCACATAAAAACCGTACAAACACACACTTAAAACACCCGCAAACATACACGCACAAAACACCCGCACACATACACACCCACAACATCCACCCACACACATATTTATACACATTCTCCATCTATTGTTCTTCAGACTGGCGCGAACACGACGAAGCAGCTGTTCTCGGGCTCGCCAACGGCCACGCCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGCACAGGGTGAGGCCTCGAGAAAGCTCATTAGAGGAAATAAGAGAGTTATTGGGGATGAAATTGAGGTTTCGGATAAAACAGGATTGTTTTAGGTGCCACCCCCCCCCCCCCTAGCACCACCAACCTCAACCCTCCTTCTCTGTCATATTGTAGGAAATGTATTTTGTTTTATTGTCAGTATGTTTGACTCCGTTAGTACCTACGTTTATTTTTTATTTATTTATTTACAGAGAATGCCCCGATAGAAAAGGCCCCTACGGCAGAAGGTGAGACTCGTAGAAATAAATAGAAAATAAGGAAACGGAAAAATTACGCATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAACACACACACACACACACACACACACACACACACACACCACACACATATACACTCACACAAACGTGCCTGTGAGTATATGTCACATCCGAACTTATTTTTGCTTGATAATGAATAACACTGCAGGAGTTACAACTGATAAAGTAATTTGTGAAAGAAACAAGTCTGCAATAAAAATAACGCCTTTGTTTCTTCCGACAGACGACCTTCTCGAGTTTCCGACTATCCCGCCGAACAGTAAGGCGTTCTATTATACATTATTTACAATACTTATTTCAACTTATAATCAAACAATGATATTCAGGATCAAAAAAAAAAAAAAAAAAAAATCAAAAATTTCTGAAAAACTCCAATTATGTGTATTTTCATTACTCTGTTGAATTAAATAGTCTAAAATCAGTGATTTAAATGCACAGTACAAAAAAAAAAAAGTTGCTATTCAAGATCGAAAGCAGAGTGGGATTAAATGGTTCAAGTAATAGAAAAAAAACTAAATTGACTGAATTAAAGTAATAATAATAATAATAATGATAATAATAACAATAATGATAATAAATAAAAGTTCAAGTATGCCAAAGGTTTCTCAATTTTCTTCAACAGCCTGTCGCTACTACTGCAGCTACGATAATACCGTCTACTGCTGTGACGATGGCACCAGACCTCGTAGGTGGTATTGTATTTGTATTAAGCCAGGGGAGGCCAGCCTTTTGTGGGATACGATCTATTCTATCTACAATTACCCTGATGCGATCTACCAGCCTAAGAGTCAAACATAAATAATTGTATGTTGTTCAGACATATCCGCCATTATGAATATAAATATAACTTATTCCCAGAAAAGAAAAGAAATATAATAATCTAATTATCATTCGTGCTCGGATATGATATTGAAGCTATTTACATGGCAACTTCTGAAGGCCGAATTATTAACAGGTACAGTAGTGTGATCGACTTAGAACAGGCTTGTGACCGACTGGTTGGCCAACCGTTGCGTTAACCCGTGCGTGCGTGCGTGTGTGTTCGTACGTACGTGTGTGTGTGTGTGTGCGTGCGACTGTTTGTGTGTGTGTGTGTGTGTCATCTTTTTAAGTTCCCATCTCCTTGAGCAACTGTTTTTCTCTCTCTCTTTTCAGTTCCTCCGGATCACTATGCCCACCAAGGAATGTGTGTAGCTAAGGAAGAACACATCTGCAAGAGTGACGGAATCTACTATAAGGTATGTCGCTAGATACAGTAACTGCAGGACATTCGGTGTAACTTTCTTTCTAAAGTCTAAAGATAAAAAGAAATATAACCATTTTGTGAGAGTATGACCATAGAAAGACAATGGTCAAACACACCAGTATGTATGTGTGGCTAATAGATAAATAGTTAATGAAATGAATAATGATAAAAAGACATAGATGTATACATGTATTTATACACACACACACACACACGTAAATGTATGTCTATGAATGTATACTCATACACACAAACCTCGTTACTCAGAGCGAGAAAGCCACGATCGAGAAATTGTTCTCGGGCTCGCCAGTGACCACGCCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAAGAAGCACCATTGCCTCAAGTATGTGCCGTATCCAAGCAGCTGCCGTTACTACTGCTTCTGGGACAGCGAGAGCTACTGCTGCGACGATGGTAGTTTACTAGGTGAGCTAGTCAACTGTCTATAGGATTTTTTTCTTATTTTGCCTTCATTCTTATTCTTATTTCTAATGAATATTTTGTATGTTTATTTTGGCGAGAATGCCAGTATGTATTATAACTCCATCTGTAATTTATCAACATTGTTATTATCTTCAACTGAATGAATCAAAATCCCACTGGAATCAATATGATAAAAAAATATATCAGAGAAACCTGAATAAGCAGTAATTAATAAGATAATCCAAAATATGACGAGATAACCTCCGATCTCTAGCGCCATTAACTAACACCCCCCCCTCCTCCCTCCCTCCCCCCTCCTCTCTCTCTCTCTCTCTCTCTCTCCAGAACCCAAGAACCATGACGACCACGAGGGACTCTGCCCCAAGATCGAGGATGAAGACTGCGACGGAGACAGGGAAGTCCCGATCAAGTTTGGGGTAAGTTCTCAAAACTGTTTTAGGGGATGTTTAATGCATGAACCTTCGGGTTTCTTTCGGTTGAACGAAGGCATTGTGCAACACACATACACACATATATATGTGTGTATATGTATATGTATACTTACATATACATACATGCATACATACATATATGAGTGAGAGAGTGATTAAGTGAATGGGTGCGCACCCGCGTATGTATATTATAGAAACATAAATTTACGATTCCCAACAGACTTTAATAAAACTTTCTCGTTACACAAAGTATGCCACATAACACCCACCTCTCCGTTCCAGGACAAAGGCTACAAGCAGAAAGCCGCCGCCTGTGCCTCCGACGGGTACTGCGCGCGCGACGAGAAGTGCTGCCCCAGCAAGTGTGCGCAGCGCCACGTCTGCCTCAAGGCCAACCGCATCGTGGAGGAGATCGACGGGAGTGGAGGAGCGCAGGAACTCGATTTTTAAGTTTCAAGTGCAACCGATGGGCGCCATGTTCAGGGAGGAGGAGGAGAAGGAGGAGCCAATAGAGGAGGAGGACGCGGAGAAGGAAGAAGTAGGGGAGGATGAGGATGAGGTGGAGGGGGAGGACGGGGAGAAGGAGGAAGTGGAGGAGTAGGTTGAGGAGGAGGATGGGGAGAAGAAAGTGGAGGAAGAGGACGGGGAGGAGAAAGTGGAGGAGGAGGACGGGGAAGAGAAAGTGGAGGAAGAGGAGGAGGAGGAGGAGGACGGGGAAGAGAAAGTGGAGGAAGAGGAGAAGGACGTGGAGCTGGAGGAAGAGAAAGAAGTGAGAATTGAATGGTGTTTAAGACGAAGACGGAGAGGATAGAAGATGAGAGGAAACGAATTGCAAACAGGAACGCAGCTCAGAAGCGAGTGTGGATTCGTTTGCAAAAATCTCTATAATGCGTAGATAGGAACAAGTTCTTGGTGATGCAGAGATGAAAAGCATAAATGTAACGCTTAGATAGAACTGAAATATGGTGACGCTGTCGGTTACATTTTTTATCTTTATGTAGATTCTTAGCGATGTAAATTCAGTGTAAAATTTGCACTTCTTGTGATTCATGTCATTAGTGCACACTGTTGAGCAAGTTTGCTTTTATGTTTGTTGAAGATTGTGAAATA
(2)SEQ ID No.2的信息
*长度：849碱基对
*类型：核酸
*链型：双链
*拓扑结构：线性
(b)分子类型：cDNA
(c)假设：否
(d)反义：否
(e)最初来源：凡纳滨对虾(Litopenaeus vannamei)
序列描述：SEQ ID No.2
CATACTCGAAAAGACAAGTGAGGATCAAGATGCGCACCGGCACTCTCCTGCTGGTGGTGGCGGCTACCCTCTCCGCTGCAGCACAGGAGGGCATCCCGACAGAAGGAAAACTTACACCAACAGAAGCTCCTCGAGAATTCCAGACTCTCCCCCCGAGCAATTGTCGCTACTATTGCGCTTACGAAGGAATTGCGTACTGCTGCGATGATGGCACTAGACCTATTCCCCCAGATCACGATGAGAATGCAGGTACTTGCGTCGAAATTGAAGAACATATTTGTGAAAAGGATGCCATCTACTATAACGTCACTGACAGTACTGGCGCGAACACGACGAAGCAGCTGTTCTCGGGCTCGCCAACGGTCACGCCCCCATGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGCACAGGAAAAATATCCAGTCACAGATACGCCTCCAGTAACAGATTTCCGCCGCCAATACCCAACTCAGCCGCCAAGCAATTGTCGCTACTTCTGTGGCTACGAGGGAGATGCTTACTGCTGTGACGATGGCACCAGACCCATTCCCCAGGATCACGATGAGAATGCAGGTGTTTGTATCGAAATGAGCGAACACATCTGCGAGGATGACGCCATTTACTACAACACTACTGATGGGACAGCGAGAGCTACTGCTGCGACGATGGTAGTTTACTAGAACCCAAGAACCATGACGACCACGAGGGACTCTGCCCCAAGATCGAGGATGAAGACTGCGACGGAGACAGGGAAGTCCCGATCAAGGCTGGGGACAAAGGCTACAAGCAGAAAGC
(3)SEQ ID No.3的信息
*长度：1366碱基对
*类型：核酸
*链型：双链
*拓扑结构：线性
(b)分子类型：cDNA
(c)假设：否
(d)反义：否
(e)最初来源：凡纳滨对虾(Litopenaeus vannamei)
序列描述：SEQ ID No.3
ACTCGAAAAGACAAGTGAGGATCAAGGTGCGCACCGGCACTCTCCTGCTGGTGGTGGCGGCTGCTCTCTCCGCTGCAGCACAGGAGGGCATCCCGACAGAAGGAAAACTTACACCAACAGATTGTCGCTACTATTGCGCTTACGAAGGAATTGCGTACTGCTGCGATGATGGCACTAGACCTATTCCCCCAGATCACGATGAGAATGCAGGTACTTGCGTCGAAATTGAAGAACATATTTGTGAAAAGGATGCCATCTACTATAACGTCACTGACAGTACTGGCGCGAACACGACGAGGCGGCTGTTCTCGGGCTCGCCAACGGACACGCCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGCACAGGAAAAATATCCAGTCACAGATACGCCTCCAGTAACAGATTTCCGCCGCCAATACCCAACTCAGCCGCCAAGCAATTGTCGCTACTTCTGTGGCTACGAGGGAGATGCTTACTGCTGTGACGATGGCACCAGACCCATTCCCCAGGATCACGATGAGAATGCAGGTGTTTGTATCGATATGAGCGAACACATCTGCGAGGATGACGCCATTTACTACAACACTACTGATGGGACTGGCGCGAACACGACGAAGCAGCTGTTCTCGGGCTCGCCAACGGCCACGCCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGAACAGGAGAATGCCCCGATAGAAAAGGCCCCTACGGCAGAAGACGACCTTCTCGAGTTTCCGACTATCCCGCCGAACACCTGTCGCTACTACTGCAGCTACGATAATACCGTCTACTGCTGTGACGATGGCACCAGACCTCGTAGGTGGTATTGTATTTGTATTAAGCCAGAGGAGGCCAGCCTTTTGTGGGATACGATCTATTCTATCTACAATTACCCTGATGCGATCTACCAGCCTAAGAGTCAAACATAAATAATTGTACGTTGTTCAGACATATCCGCCATTATGAATATAAATATAACTTATTCCCAGAAAAGAAAAGAAAATAATCTAATTATCATTCGTGCTTGGATGTGATATTGGAGCTATTTACATGGCAACTTCTGAAGGCCGAATTATTAGCAGGTACAGTAGTGTGACCGACTTAGAACAGGCTTGTGACGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGTGCCTCGCCGCCGCAGCACAGGAGAATGCCCCGATAGAAAAGGCCCCTACGGCAGAAG
(4)SEQ ID No.4的信息
*长度：872碱基对
*类型：核酸
*链型：双链
*拓扑结构：线性
(b)分子类型：cDNA
(c)假设：否
(d)反义：否
(e)最初来源：凡纳滨对虾(Litopenaeus vannamei)
序列描述：SEQ ID No.4
CATACTCGAAAAGACAAGTGAGGATCAAGATGCGCACCGGCACTCTCCTGCTGGTGGTGGCGGCTGCCCTCTCCGCTGCAGCACAGGAGGGCATCCCGACAGAAGGAAAACTTACACCAACAGAAGCTCCTCGAGAATTCCAGACTCTCCCCCCGAGCAATTGTCGCTACTTCTGTGGCTACGAGGGAGATGCTTACTGCTGTGACGATGGTACCAGACCCATTCCCCAGGATCACGATGAGAATGCAGGTGTTTGTATCGAAATGAGCGAACACATCTGCGAGGATGACGCCATTTACTACAACACTACTGATGGGACTGGCGCGAACACGACGAAGCAGCTGTTCTCGGGCTCGCCAGTGACCACGCCCCCCTGCGCCTCCGACGGCTACTGCGCCGAGGACGAGAAGTGCTGCCCGACGCCCTGCGCCAGGAAGCACCTGCGCCTCGCCGCCGCAGCACAGGAGAATGCCCCGATAGAAAAGGCCCCTACGGCAGAAGACGACCTTCTCGAGTTTCCGACTATCCCGCCGAACACCTGTCGCTACTACTGCAGCTACGATAATACCGTCTACTGCTGTGACGATGGCACCAGACCTCGTAGGTGGTATTGTATTTGTATTAAGCCAGGGGAGGCCAGCCTTTTGTGGGATACGATCTATTCTATCTACAATTACCCTGATGCGATCTACCAGCCTAAGAGTCAAACATAAATAATTGTACGTTGTTCAGACATATCCGCCATTATGAATATAAATATAACTTTTTCCCAGAAAAGAAAAGAAATATAATAATCTAATTATCATTAGTGCTCGGATATGATATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACCTGCCCTTTTT
(5)SEQ ID No.5的信息
(a)序列特征
*长度：234氨基酸残基
*类型：氨基酸
*链型：单链
*拓扑结构：线性
(b)分子类型：蛋白
序列描述：SEQ ID No.5
MRTGTLLLVVAATLSAAAQEGIPTEGKLTPTEAPREFQTLPPSNCRYYCAYEGIAYCCDDGTRPIPPDHDENAGTCVEIEEHICEKDAIYYNVTDSTGANTTKQLFSGSPTVTPPCASDGYCAEDEKCCPTPCARKHLCLAAAAQEKYPVTDTPPVTDFRRQYPTQPPSNCRYFCGYEGDAYCCDDGTRPIPQDHDENAGVCIEMSEHICEDDAIYYNTTDGTARATAATMVVY
(6)SEQ ID No.6的信息
(a)序列特征
*长度：149氨基酸残基
*类型：氨基酸
*链型：单链
*拓扑结构：线性
(b)分子类型：蛋白
序列描述：SEQ ID No.6
MSEHICEDDAIYYNTTDGTGANTTKQLFSGSPTATPPCASDGYCAEDEKCCPTPCARKHLCLAAAEQENAPIEKAPTAEDDLLEFPTIPPNTCRYYCSYDNTVYCCDDGTRPRRWYCICIKPEEASLLWDTIYSIYNYPDAIYQPKSQT
(7)SEQ ID No.7的信息
(a)序列特征
*长度：227氨基酸残基
*类型：氨基酸
*链型：单链
*拓扑结构：线性
(b)分子类型：蛋白
序列描述：SEQ ID No.7
MRTGTLLLVVAAALSAAAQEGIPTEGKLTPTEAPREFQTLPPSNCRYFCGYEGDAYCCDDGTRPIPQDHDENAGVCIEMSEHICEDDAIYYNTTDGTGANTTKQLFSGSPVTTPPCASDGYCAEDEKCCPTPCARKHLRLAAAAQENAPIEKAPTAEDDLLEFPTIPPNTCRYYCSYDNTVYCCDDGTRPRRWYCICIKPGEASLLWDTIYSIYNYPDAIYQPKSQT
一组类Crustin抗菌肽的获取及其抗菌应用：
针对凡纳滨对虾编码产生的LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4序列信息，设计PCR引物，分别扩增其成熟肽编码序列，利用大肠杆菌表达系统，获得了具有活性的重组抗菌蛋白。具体如下：
1)成熟肽编码序列扩增
根据LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的序列信息，分别设计两侧分别带有限制性内切酶BamHⅠ和Hind III酶切位点及15bp同源臂的PCR引物(表1)，使用PremixEx TaqTM Hot Start Version分别扩增其成熟肽编码序列，PCR反应体系及反应条件：
Figure BDA0003690878050000121
反应条件为：94℃预变性5min；94℃变性30s，50/52℃(此处对应温度见表1所示)退火30s，72℃延伸30s，35个循环；72℃延伸10min。取2μL PCR产物，用1％质量浓度的琼脂糖凝胶电泳检测目的条带，将大小正确的SteadyPure PCR反应液纯化试剂盒(艾科瑞，湖南)进行纯化。
表1不同剪接体引物序列
Figure BDA0003690878050000122
2)重组表达载体的构建
利用BamHⅠ和Hind III内切酶，双酶切pET32a(+)表达载体质粒，并利用In-Fusion连接酶(TaKaRa)，分别将目的基因片段连接于酶切后的表达载体pET32a(+)中，转化到大肠杆菌宿主菌Trans5α中，过夜培养，分别挑选单克隆用通用引物T7(F：TAATACGACTCACTATAGGG；R：GCTAGTTATTGCTCAGCGGT)进行PCR检测，并送样测序，确定重组质粒。
3)转化与筛选阳性菌
分别利用天根质粒提取试剂盒(DP106，天根)，提取重组质粒，分别转化至大肠杆菌宿主菌BL21中，涂布Amp+终浓度100μg/mL的LB培养平板，37℃过夜培养，并挑选单克隆菌株，测序并筛选出阳性克隆。
4)IPTG诱导表达
将测序成功的阳性克隆菌液分别接种于氨苄抗性(Amp+,100μg/mL)的LB固体培养基上，220rpm、37℃过夜活化，然后按体积比1:100接种于新鲜的氨苄抗性(Amp+,100μg/mL)的LB液体培养基中，220rpm、37℃继续培养至OD600为0.4-0.6时，每个菌株取2管菌液，一管加入终浓度为1mM的诱导剂异丙基-β-D-硫代半乳糖苷(isopropylthio-β-galactoside,IPTG)，另一管作为对照组不加诱导剂。在0h、2h、4h、6h取样，每个样品1mL。室温8000rpm离心2min沉淀菌体，加入1mL PBS缓冲液重悬，8000rpm离心2min去除培养基杂质，清洗2次；去除离心后的上清，用500μL PBS缓冲液重悬菌体；用超声波细胞破碎仪(新芝生物，宁波)进行超声破碎细胞；4℃，12000rpm离心5min，分离上清和沉淀，沉淀用200μL PBS缓冲液重悬；分别将上清和沉淀与30μL 4×SDS-PAGE上样缓冲液(P1015，Solarbio)充分混匀后100℃煮沸10min；4℃冷却5min后进行SDS-PAGE，用考马斯亮蓝染色，脱色后通过GelDocTM EZImager成像系统(BIO RAD，美国)检测蛋白的表达。
5)扩大培养纯化
将构建的菌株按体积比1:100接种量接种至5mL氨苄抗性(Amp+,100μg/mL)的LB培养基，37℃，220rpm过夜活化；将过夜活化的菌液按体积比1:100接种量转接至新鲜的200mLAmp+抗性(Amp+,100μg/mL)的LB培养基，37℃，220rpm培养3h至菌液OD值为0.4-0.6，加入终浓度1mM的IPTG诱导培养4h；预冷离心机，将菌液用无RNA酶的50mL离心管收集，4℃，8000rpm离心5min，弃去培养基；用15mL的Buffer A(50mM Na3PO4、300mM NaCl，pH 7.4，于水中)重悬菌体；使用超声波细胞破碎仪破碎菌体，工作/暂停时间设置为1s/4s超声破碎10min；4℃，8000rpm离心30min，分离上清和沉淀，因目的蛋白为可溶性表达，所以收集上清；将溶解于Buffer A中的目的蛋白使用TALON His-Tag钴离子纯化树脂(Clontech，美国)用亲和层析的方法纯化目的蛋白。
6)抑菌试验
最小抑菌浓度(MIC)的测定：利用最小抑菌浓度的测定方法来测定对革兰氏阳性菌：金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)和藤黄微球菌(Micrococcus luteus)；革兰氏阴性菌：副溶血弧菌(Vibrioparahaemolyticus)、哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(Vibrio alginolyticus)和大肠杆菌(Escherichia coli)的抑菌活性。副溶血弧菌(Vibrio parahaemolyticus)、哈维氏弧菌(Vibrio harveyi)和溶藻弧菌(Vibrio alginolyticus)的实验条件为：在TSB培养基中28℃培养，测定560nm下光密度。其它细菌的实验条件为：在LB培养基中37℃培养，测定600nm下光密度。即：将上述细菌分别在液体LB(或TSB)培养基中进行培养，过夜培养后的菌液按体积比1:100接种于新鲜的液体LB(或TSB)培养基中，分别在37℃或28℃培养至600nm(或560nm)下光密度(OD600或OD560)值达到相应于约1×108CFU/ml，用新鲜液体LB(或TSB)培养基将菌液稀释并在96微孔板中每孔加入150μl，使菌的终浓度至约1×106CFU/ml，并在96微孔板中加入重组表达蛋白使其终浓度分别为：24μM、12μM、6μM、3μM、1.5μM、0.75μM、0.375μM、0μM，过夜培养，在酶标仪中测定各孔中溶液的OD600(或OD560)。根据OD值检测目的蛋白对细菌的抑菌活性，当实验组的细菌生长浓度达到对照组生长浓度的90％时，被认为是最小抑制浓度(Liu,N.；Zhang,R.；Fan,Z X.；Zhao,X F.；Wang,X W.；Wang,JX.Characterization of a type-I crustin with broad-spectrum antimicrobialactivity from red swamp crayfish Procambarus clarkii.Dev.Comp.Immunol.2016,61,145–153)。
结果表明重组蛋白LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4对革兰氏阳性菌和革兰氏阴性菌均具有广谱的抑制活性，具体抑制活性见表2。
表2重组蛋白LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4的最小抑菌浓度MIC的测定
Figure BDA0003690878050000141
Figure BDA0003690878050000151
该组类Crusitn抗菌肽的成功表达与重组蛋白生物活性的鉴定，在新型抗菌药物研发上具有重要的应用前景。
序列表
<110> 中国科学院海洋研究所
<120> 类Crustin抗菌肽及编码基因的获取方法与应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11907
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
acaagtgagg atcaagatgc gcaccggcac tctcctgctg gtggtggcgg ctgccctctc 60
cgctgcagca cagggtgaga cttaaaataa aatctagata aaaatcacgt taacgatagt 120
actaatattg aaacaaaata tacaaatgag taaatatata aaatctaaat aaaagtcacg 180
ttaacgatag tactcataat gaagcaaaat aaacaaatca gtaaatattt aaaatctaga 240
aaaaaatcac gttaacaata gtactcatat tgaaacaaat aatgtaaatg agtaaataca 300
cgtaaaacag ttgatcatga gataatatta agaattttga aaattacgaa tagaggcttg 360
gaagtgagaa tatgtgtgga aagtcaagaa tcagaataca taccaaaata acggtaaaca 420
acatagaatc cgaggaaact tttgtttaag gaggaaagaa tatgaatata agatattaaa 480
aatagagact ttcgataaaa aaaaaatgat taatggttaa aagcaaaata aatgtgctag 540
acatataagg tcatataaca ctataggaag gtacagcaaa agggtagtga agttagaaac 600
aatagtaaca acaatagtta gtgagttagt agttagttgc tatgcgtcaa atatgtagag 660
cagaggttct caacctttac tagttagtgg cgcactaagg atatagcctg gacttcgacg 720
gcgcacaaac tatacagtaa taataataat gataagtaat attattatat ctattaccaa 780
tatcattatt gttgttttta ttatcatata tatgagaatg aattaatgaa cacatgggaa 840
tgaaatatat aattccagtt gtagattaaa catacatgat taatgggaca cttgcgcttg 900
tctggatttg catatccttt cgatgtcatg aggaaaaaat aattgtgaaa ataaggcggc 960
tagaagcaag gcacaaattc aagacataac tagatattca ttaaattttc gcggcgacct 1020
tgagggatgg tcgcggcgca ggttgagaac cactgaccta gagtaatatt gaaaaggtta 1080
aagatgggta aaggagttga gtgaatgggt tacggtgggg ttaggcaagg ggattagatc 1140
aagtgaagga tatgtatagt ctgaggaagg agaacaggtt gtcaaagcag aggaggatgc 1200
gtaggaaaag tctcttggga ggcagagaag ggacgggtta tacggagaaa ggatatgacg 1260
aaggccaagt ctgtgagaac ggaaacagcg aatattctaa tgcaggagag ctattgctta 1320
gttaaaggat atgacgaaga tcaggtctgt gtgaacggaa accgggaatt attactttgt 1380
tgatctacga gtgataacgg ttacagtttg gagaatttga agggaaagga actagggggt 1440
gagaaaagga attagagtgg gggggggggt agtatcagga ggggtattag gaggtggagg 1500
atctggggaa gggtatagtt gtgacataag gggttcacgt gtgtatccag gagggaatgg 1560
gagggataga ggggatggag ggagggttga tgtagttgga actgaggggg atgggctgtg 1620
ttgggaggga gtaggaggaa gaggtgtagg aggaatatga gtaggaggag gatggatacc 1680
ggcagtcact ttgagggtgg agggagcagg agttgtggaa ttttagggtg ggtgagggtt 1740
ttcggtgtca ttttgggact cgagcagata gttctgaatg tcttcaaggg tttctgaaac 1800
gtagtcggcc ggggagtgtt gtgagacaaa ggttttctca tgaggggggg ggggggggag 1860
agaaactggt gtctgaggag tggaggcaaa ggtaggtgga ggtgagagtg tggtagaagt 1920
atgagaaaaa tcccgtcgac gtgcttcctg tctgggttca cgcagagtga gaccaagtct 1980
gtatctgaga gttgccacct cagactcaaa cttgtaggtg gggcagcccc tataaaatac 2040
attatggggg ctgccgcagt tagcgcatgt gcgtgactgc gcagagcaga ttgatcggtt 2100
atgaccaggt tgtgcacaca gagggcatcg ggctgtggga cggcagtgtt tggcagaatg 2160
tccttaacgc caacagtttt aacattgacg gggaggatgt tgatatgatc ggacagggag 2220
ggattctcca ccaatgtaga cattaaagtg aaggtcatgt ctacggaaag taattttggc 2280
aatgttggtg gggttcttac gatgaatttt aggaggaatg aagtagcact gtactgttcg 2340
tacatcatcg tccgcggctc tcacacctta ggaagtaggt agtagaaggg gacggagaag 2400
ggacagaaac gaaaaagcag gagggagaaa agaccttgca aaattagttg agtcgagggc 2460
tgaggctcaa gggaagggag atccccagca gcattgggcc ccagtcctcg ccttccaagc 2520
cccctcacga caacaaaggg taagggattg ggggggaggg gggtcgacaa aaaaaaaaaa 2580
aaaacgaaaa tgttgtacga gaggagaggg gagaagggag agtgtaaatc ttgaacggtt 2640
ttcgagagat catatcatat caattttttt ttattctacc agttccccca tcccctctca 2700
tttttgacca atatctacgt catattatct ctatgcgtat ctatgctaaa acattattct 2760
cttgacattc atattcacaa ttttcttttt tattttccgt acaactacag agggcatccc 2820
gacagaagga aaacttacac caacaggtaa gactcataat gataatgaat aacataaaaa 2880
tgaataaaac aggatgtgat gatacaaaaa ataaacccac ttattaatag gtttaataat 2940
ttgcatacaa ttcttgacat gtttcaattc attatgatta aatcttaaaa gtatgacaat 3000
ggcacaaaaa aaaaaaaaaa ataataataa ataaataaat atggcgaact acaattttgt 3060
gtttgtttgt tttacagaag ctcctcgaga attccagact ctccccccta gcagtaagtc 3120
aatatacatc aattaacaac tgatgataat tatccttatt atgattatca tcattacttt 3180
tatccttgtt attattatta tcattattgt tattatcatt actattatca tcagcattgt 3240
tattatcatt attattatta tcatcatcat tatcatcatc attatcatca tcattatcgt 3300
tattattatt tctcattatt ttattattat tatcatttat cattatatta tagttatttg 3360
attttattcc tattattatt attctattcc tatcattatc atcataattg tatacctatt 3420
attagtagta ttatcattac taatatcatt atcattatca tagtaattag catcttatta 3480
tcatcataca tctgccacac aatagtaaat ggtctaaggg gatgttattt tctctaattt 3540
attctctaat tctatttgtt ataccggatg tatttattta tttaatttaa gcattttttt 3600
tatctaattt cattttgatt taccgaataa gatttttgta gcctcttttt atctgatttc 3660
attttcattt agcgaataga tttatctttt gtattttctt ttctctaatt ttattttcat 3720
ttactcagcc gtcaagcagt aagtcagtgt acatagttta ataaattatt attatcatta 3780
tcatcattgt gctatcatgg aatacggtct taaactttga tcaaaagtct taagaggtct 3840
gaaagtgaat aaaacatttt tgtaaaatat tccaactctc ttctcttcct tttttttcaa 3900
tttactatat aattattgac atttttcttc ctatatcatt tttttcagat tgtcgctact 3960
attgcgctta cgaaggaatt gcgtactgct gcgatgatgg cactagacct agtaagtgaa 4020
aaagaaatgt gtatatatat atgtatatct gtctatctat ctatcagtcc gtctattcat 4080
atatgtgtgt gtatatacat tattttctgt tcttttttaa tatacatcaa aagaacttat 4140
cacttatata gtggcgtctc tctctctctc tctctctctc tctctctctc tctctctctc 4200
tctctctctc cataatcatc atttcatcat aataaaaaat agcataatta tttatctatc 4260
atcatcatca tctattatca ttattaccat tatcattatt accattatca tcataattat 4320
caacactttg tctcttccag ttcccccaga tcacgatgag aatgcaggta cttgcgtcga 4380
aattgaagaa catatttgtg aaaaggatgc catctactat aacgtcactg acagtgtaag 4440
tccctcagat ttaaaaaaaa gaaagaaaga aagaaaaaaa agaaaggaag gaaggaagga 4500
aggaaggaag gaagaaagaa agaaagaaag aaagaaagaa agaaaggaaa gaaagaaaaa 4560
aaaaagaaag aaagaaagaa agaaaagaaa agaagaaaga aagaaaggaa agaaagaaag 4620
aaagaaagaa agaaagaaag gaaaaggaaa gaaagaaaag tgtatccgtg tttttttttt 4680
tttttttttt tttttttttt tttttacttg aaagtctttg ttagatttgt gttgtctaca 4740
cacaaatacc cgcacacatt cacatacaca catacacacg cacatagata cccacataca 4800
cagatacccg cacacacaca cacagatacc cacacataca tacagatacc cctcacacac 4860
atgcaaatac acacacacac acacacacac acacacacac acacacacac acacacacac 4920
acacacacac acacacacac acacacacag atacccacac acacacacag atacccgcac 4980
acacacagat acacactaca cacacagata cccgtacaca cacacagata cacacacaga 5040
tacccgtaca cacacacata cccgcacaca cacacatacc cgtacacaca cacacacaga 5100
tacccgcaca cacacacata ctacacacag gatacacaca cacacatttc tacacattct 5160
ccatctattg ttcttcagac tggcgcgaac acgacgaagc agctgttctc ggctcgccaa 5220
cggccacgcc ccctgcgcct ccgacggcta ctgcgccgag gacgagaagt gctgcccgac 5280
gccctgcgcc aggaagcacc tgtgcctcgc cgccgcagca cagggtgagg tctcgaaagt 5340
ttatttattt gtttatttat ctattattta ttcattttat ttatttttat tttatttatt 5400
catttatatt tatttcattt atttaattat ttatttattt ttattcattt atttaattat 5460
ttatttatta tttatttata tttacttatt tatttattta tttgttttta tttcatttat 5520
ttttttatta tgattcattt atttttttag aaggacatgt gtaataaaag gattgatgat 5580
caaaggagac gaggcagaat aggagaggat tctgacgtgg gaatgaagca cagggaaaaa 5640
ttattctaga atattttttt agaattagaa tcaaaatttc ctttttctac ttcctttaac 5700
ctcttcctct attttgatta tgctctctac aaatattttg actttttgtc gtgatgataa 5760
acaacatttt atttttttat ttctttctta aatttgcaga aaaatatcca gtcacagata 5820
cgcctccagt aacaggtaag actaaaataa gagaaactga atatatgtat acttatacag 5880
tacatatatc acattgtata ttttctacaa taatgaaaaa catatttata ttccatatat 5940
tagtggaata cattagttaa atcggagaat aactcaaata catgcactcg tggaggacta 6000
cataaaaaaa aaatttgact ttgaacacaa agcacttagc tatgttgcct tttctctaaa 6060
ttcatgggtt atttttttcg tctcgcaaaa tacaccgagt tagaaataaa cccattttac 6120
tcttattttc ctgatttttt attcatttac atatttattt atttattttc ctgatgtttt 6180
atttatttat atatttattt atttatttat ttatagcctg tcggtactac tgtggctacg 6240
cgggggttag ctactgttgc gacggtggac tcgcgcccgg tctgtttcct atttttcttt 6300
ccttcttatt tttctttctt tcttactttt ttttctttct ttctttttta tttttctttc 6360
tttctttctg tccttcttcc ctttttttcc tccctccctc tctctctctc tgtctctctg 6420
tctctctctg tctgtctgtc tctgtctctc tgtctgtctg tctgtctgtc tctctctctc 6480
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc 6540
tctctctctc tctctctctc tctctctctc tctctctcac acacacacac acacatatat 6600
aatgcataca cacgtatgta tgtacataga tacacacgtg tatatgtata tatttctgtg 6660
tatgtgtgca tatatatata tgttatactc atttctgttt gattaggaaa acagaaaatt 6720
acatttactc tattagtgga atagactaga cagaaatatc agagaatgac tcaatttcac 6780
cctctaatgg aatattacat tgacaacttt tgcttttgtt tgacagattt ccgccgccaa 6840
tacccaactc agccgccaag cagtaagtca atgtgtatag tttagcaaat gattattatt 6900
atcattatca tctttgcact agcctggaat acggtctcaa agtttgatct aaattcttag 6960
aggtctgtaa gtggattgaa tatttttgta agatattcca actctctctt ctcttacttt 7020
ttattcaatt tactatatat ctattgacat ttttcttcct atatcttttt ttttcagatt 7080
gtcgctactt ctgtggctac gagggagatg cttactgctg tgacgatggc accagaccca 7140
gtaggtgatg ctcgtctttg caaaaaagaa agaaagaaag agaaagataa taatagaaaa 7200
ataaaataaa taaatatcaa ttaaaattta aaaatgataa tacaaaaata gaataaataa 7260
ataaatatga aaataaaaaa gatcataata caaaattaag ataaataaat aaatatgaca 7320
ataaaaataa aatatgtttt ctaaaaagac cattgcctat tgccgtacct gttcagataa 7380
ctttagatcg tgtcttctct atacatatat ccttctcttt tgttgtgttg tctctgtctc 7440
tttcgtttcc tctcactccc tcttgattgc ccctttgaaa gttattattc tcgttgcctc 7500
ttacagttcc ccaggatcac gatgagaatg caggtgtttg tatcgaaatg agcgaacaca 7560
tctgcgagga tgacgccatt tactacaaca ctactgatgg ggtaagtata gtacttatat 7620
gatacgtatg aaatttagac aaccagagtc tactgaggaa aaaatacttg caaatcttac 7680
acacactgca acaggaacaa cgaagggaag gacaaaaaaa tacacgaata ggcccatatt 7740
cgtgtgtttt cctgtccgtc atgaattcca cacatacaca cacatgtaca caaagggaga 7800
ttcagggaaa gacacgttta taccgaaggt ttttttcacg ttattgtttc ttcatgctct 7860
gaagaagcgc tatgaaggcc ttgggtgtga tctcaggttt ccgttttttt cctttttttc 7920
ataacttact catcatcaat cttctacaca ctcacgcaca taaaaaccgt acaaacacac 7980
acttaaaaca cccgcaaaca tacacgcaca aaacacccgc acacatacac acccacaaca 8040
tccacccaca cacatattta tacacattct ccatctattg ttcttcagac tggcgcgaac 8100
acgacgaagc agctgttctc gggctcgcca acggccacgc ccccctgcgc ctccgacggc 8160
tactgcgccg aggacgagaa gtgctgcccg acgccctgcg ccaggaagca cctgtgcctc 8220
gccgccgcag cacagggtga ggcctcgaga aagctcatta gaggaaataa gagagttatt 8280
ggggatgaaa ttgaggtttc ggataaaaca ggattgtttt aggtgccacc cccccccccc 8340
ctagcaccac caacctcaac cctccttctc tgtcatattg taggaaatgt attttgtttt 8400
attgtcagta tgtttgactc cgttagtacc tacgtttatt ttttatttat ttatttacag 8460
agaatgcccc gatagaaaag gcccctacgg cagaaggtga gactcgtaga aataaataga 8520
aaataaggaa acggaaaaat tacgcatata tatatatata tatatatata tatatatata 8580
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 8640
taacacacac acacacacac acacacacac acacacacac cacacacata tacactcaca 8700
caaacgtgcc tgtgagtata tgtcacatcc gaacttattt ttgcttgata atgaataaca 8760
ctgcaggagt tacaactgat aaagtaattt gtgaaagaaa caagtctgca ataaaaataa 8820
cgcctttgtt tcttccgaca gacgaccttc tcgagtttcc gactatcccg ccgaacagta 8880
aggcgttcta ttatacatta tttacaatac ttatttcaac ttataatcaa acaatgatat 8940
tcaggatcaa aaaaaaaaaa aaaaaaaaat caaaaatttc tgaaaaactc caattatgtg 9000
tattttcatt actctgttga attaaatagt ctaaaatcag tgatttaaat gcacagtaca 9060
aaaaaaaaaa agttgctatt caagatcgaa agcagagtgg gattaaatgg ttcaagtaat 9120
agaaaaaaaa ctaaattgac tgaattaaag taataataat aataataatg ataataataa 9180
caataatgat aataaataaa agttcaagta tgccaaaggt ttctcaattt tcttcaacag 9240
cctgtcgcta ctactgcagc tacgataata ccgtctactg ctgtgacgat ggcaccagac 9300
ctcgtaggtg gtattgtatt tgtattaagc caggggaggc cagccttttg tgggatacga 9360
tctattctat ctacaattac cctgatgcga tctaccagcc taagagtcaa acataaataa 9420
ttgtatgttg ttcagacata tccgccatta tgaatataaa tataacttat tcccagaaaa 9480
gaaaagaaat ataataatct aattatcatt cgtgctcgga tatgatattg aagctattta 9540
catggcaact tctgaaggcc gaattattaa caggtacagt agtgtgatcg acttagaaca 9600
ggcttgtgac cgactggttg gccaaccgtt gcgttaaccc gtgcgtgcgt gcgtgtgtgt 9660
tcgtacgtac gtgtgtgtgt gtgtgtgcgt gcgactgttt gtgtgtgtgt gtgtgtgtca 9720
tctttttaag ttcccatctc cttgagcaac tgtttttctc tctctctttt cagttcctcc 9780
ggatcactat gcccaccaag gaatgtgtgt agctaaggaa gaacacatct gcaagagtga 9840
cggaatctac tataaggtat gtcgctagat acagtaactg caggacattc ggtgtaactt 9900
tctttctaaa gtctaaagat aaaaagaaat ataaccattt tgtgagagta tgaccataga 9960
aagacaatgg tcaaacacac cagtatgtat gtgtggctaa tagataaata gttaatgaaa 10020
tgaataatga taaaaagaca tagatgtata catgtattta tacacacaca cacacacacg 10080
taaatgtatg tctatgaatg tatactcata cacacaaacc tcgttactca gagcgagaaa 10140
gccacgatcg agaaattgtt ctcgggctcg ccagtgacca cgcccccctg cgcctccgac 10200
ggctactgcg ccgaggacga gaagtgctgc ccgacgccct gcgccaagaa gcaccattgc 10260
ctcaagtatg tgccgtatcc aagcagctgc cgttactact gcttctggga cagcgagagc 10320
tactgctgcg acgatggtag tttactaggt gagctagtca actgtctata ggattttttt 10380
cttattttgc cttcattctt attcttattt ctaatgaata ttttgtatgt ttattttggc 10440
gagaatgcca gtatgtatta taactccatc tgtaatttat caacattgtt attatcttca 10500
actgaatgaa tcaaaatccc actggaatca atatgataaa aaaatatatc agagaaacct 10560
gaataagcag taattaataa gataatccaa aatatgacga gataacctcc gatctctagc 10620
gccattaact aacacccccc cctcctccct ccctcccccc tcctctctct ctctctctct 10680
ctctctccag aacccaagaa ccatgacgac cacgagggac tctgccccaa gatcgaggat 10740
gaagactgcg acggagacag ggaagtcccg atcaagtttg gggtaagttc tcaaaactgt 10800
tttaggggat gtttaatgca tgaaccttcg ggtttctttc ggttgaacga aggcattgtg 10860
caacacacat acacacatat atatgtgtgt atatgtatat gtatacttac atatacatac 10920
atgcatacat acatatatga gtgagagagt gattaagtga atgggtgcgc acccgcgtat 10980
gtatattata gaaacataaa tttacgattc ccaacagact ttaataaaac tttctcgtta 11040
cacaaagtat gccacataac acccacctct ccgttccagg acaaaggcta caagcagaaa 11100
gccgccgcct gtgcctccga cgggtactgc gcgcgcgacg agaagtgctg ccccagcaag 11160
tgtgcgcagc gccacgtctg cctcaaggcc aaccgcatcg tggaggagat cgacgggagt 11220
ggaggagcgc aggaactcga tttttaagtt tcaagtgcaa ccgatgggcg ccatgttcag 11280
ggaggaggag gagaaggagg agccaataga ggaggaggac gcggagaagg aagaagtagg 11340
ggaggatgag gatgaggtgg agggggagga cggggagaag gaggaagtgg aggagtaggt 11400
tgaggaggag gatggggaga agaaagtgga ggaagaggac ggggaggaga aagtggagga 11460
ggaggacggg gaagagaaag tggaggaaga ggaggaggag gaggaggacg gggaagagaa 11520
agtggaggaa gaggagaagg acgtggagct ggaggaagag aaagaagtga gaattgaatg 11580
gtgtttaaga cgaagacgga gaggatagaa gatgagagga aacgaattgc aaacaggaac 11640
gcagctcaga agcgagtgtg gattcgtttg caaaaatctc tataatgcgt agataggaac 11700
aagttcttgg tgatgcagag atgaaaagca taaatgtaac gcttagatag aactgaaata 11760
tggtgacgct gtcggttaca ttttttatct ttatgtagat tcttagcgat gtaaattcag 11820
tgtaaaattt gcacttcttg tgattcatgt cattagtgca cactgttgag caagtttgct 11880
tttatgtttg ttgaagattg tgaaata 11907
<210> 2
<211> 849
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catactcgaa aagacaagtg aggatcaaga tgcgcaccgg cactctcctg ctggtggtgg 60
cggctaccct ctccgctgca gcacaggagg gcatcccgac agaaggaaaa cttacaccaa 120
cagaagctcc tcgagaattc cagactctcc ccccgagcaa ttgtcgctac tattgcgctt 180
acgaaggaat tgcgtactgc tgcgatgatg gcactagacc tattccccca gatcacgatg 240
agaatgcagg tacttgcgtc gaaattgaag aacatatttg tgaaaaggat gccatctact 300
ataacgtcac tgacagtact ggcgcgaaca cgacgaagca gctgttctcg ggctcgccaa 360
cggtcacgcc cccatgcgcc tccgacggct actgcgccga ggacgagaag tgctgcccga 420
cgccctgcgc caggaagcac ctgtgcctcg ccgccgcagc acaggaaaaa tatccagtca 480
cagatacgcc tccagtaaca gatttccgcc gccaataccc aactcagccg ccaagcaatt 540
gtcgctactt ctgtggctac gagggagatg cttactgctg tgacgatggc accagaccca 600
ttccccagga tcacgatgag aatgcaggtg tttgtatcga aatgagcgaa cacatctgcg 660
aggatgacgc catttactac aacactactg atgggacagc gagagctact gctgcgacga 720
tggtagttta ctagaaccca agaaccatga cgaccacgag ggactctgcc ccaagatcga 780
ggatgaagac tgcgacggag acagggaagt cccgatcaag gctggggaca aaggctacaa 840
gcagaaagc 849
<210> 3
<211> 1366
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
actcgaaaag acaagtgagg atcaaggtgc gcaccggcac tctcctgctg gtggtggcgg 60
ctgctctctc cgctgcagca caggagggca tcccgacaga aggaaaactt acaccaacag 120
attgtcgcta ctattgcgct tacgaaggaa ttgcgtactg ctgcgatgat ggcactagac 180
ctattccccc agatcacgat gagaatgcag gtacttgcgt cgaaattgaa gaacatattt 240
gtgaaaagga tgccatctac tataacgtca ctgacagtac tggcgcgaac acgacgaggc 300
ggctgttctc gggctcgcca acggacacgc ccccctgcgc ctccgacggc tactgcgccg 360
aggacgagaa gtgctgcccg acgccctgcg ccaggaagca cctgtgcctc gccgccgcag 420
cacaggaaaa atatccagtc acagatacgc ctccagtaac agatttccgc cgccaatacc 480
caactcagcc gccaagcaat tgtcgctact tctgtggcta cgagggagat gcttactgct 540
gtgacgatgg caccagaccc attccccagg atcacgatga gaatgcaggt gtttgtatcg 600
atatgagcga acacatctgc gaggatgacg ccatttacta caacactact gatgggactg 660
gcgcgaacac gacgaagcag ctgttctcgg gctcgccaac ggccacgccc ccctgcgcct 720
ccgacggcta ctgcgccgag gacgagaagt gctgcccgac gccctgcgcc aggaagcacc 780
tgtgcctcgc cgccgcagaa caggagaatg ccccgataga aaaggcccct acggcagaag 840
acgaccttct cgagtttccg actatcccgc cgaacacctg tcgctactac tgcagctacg 900
ataataccgt ctactgctgt gacgatggca ccagacctcg taggtggtat tgtatttgta 960
ttaagccaga ggaggccagc cttttgtggg atacgatcta ttctatctac aattaccctg 1020
atgcgatcta ccagcctaag agtcaaacat aaataattgt acgttgttca gacatatccg 1080
ccattatgaa tataaatata acttattccc agaaaagaaa agaaaataat ctaattatca 1140
ttcgtgcttg gatgtgatat tggagctatt tacatggcaa cttctgaagg ccgaattatt 1200
agcaggtaca gtagtgtgac cgacttagaa caggcttgtg acgcctccga cggctactgc 1260
gccgaggacg agaagtgctg cccgacgccc tgcgccagga agcacctgtg cctcgccgcc 1320
gcagcacagg agaatgcccc gatagaaaag gcccctacgg cagaag 1366
<210> 4
<211> 872
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catactcgaa aagacaagtg aggatcaaga tgcgcaccgg cactctcctg ctggtggtgg 60
cggctgccct ctccgctgca gcacaggagg gcatcccgac agaaggaaaa cttacaccaa 120
cagaagctcc tcgagaattc cagactctcc ccccgagcaa ttgtcgctac ttctgtggct 180
acgagggaga tgcttactgc tgtgacgatg gtaccagacc cattccccag gatcacgatg 240
agaatgcagg tgtttgtatc gaaatgagcg aacacatctg cgaggatgac gccatttact 300
acaacactac tgatgggact ggcgcgaaca cgacgaagca gctgttctcg ggctcgccag 360
tgaccacgcc cccctgcgcc tccgacggct actgcgccga ggacgagaag tgctgcccga 420
cgccctgcgc caggaagcac ctgcgcctcg ccgccgcagc acaggagaat gccccgatag 480
aaaaggcccc tacggcagaa gacgaccttc tcgagtttcc gactatcccg ccgaacacct 540
gtcgctacta ctgcagctac gataataccg tctactgctg tgacgatggc accagacctc 600
gtaggtggta ttgtatttgt attaagccag gggaggccag ccttttgtgg gatacgatct 660
attctatcta caattaccct gatgcgatct accagcctaa gagtcaaaca taaataattg 720
tacgttgttc agacatatcc gccattatga atataaatat aactttttcc cagaaaagaa 780
aagaaatata ataatctaat tatcattagt gctcggatat gatatctcta gaggatcccc 840
gggtaccgag ctcgaattca cctgcccttt tt 872
<210> 5
<211> 234
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Arg Thr Gly Thr Leu Leu Leu Val Val Ala Ala Thr Leu Ser Ala
1 5 10 15
Ala Ala Gln Glu Gly Ile Pro Thr Glu Gly Lys Leu Thr Pro Thr Glu
20 25 30
Ala Pro Arg Glu Phe Gln Thr Leu Pro Pro Ser Asn Cys Arg Tyr Tyr
35 40 45
Cys Ala Tyr Glu Gly Ile Ala Tyr Cys Cys Asp Asp Gly Thr Arg Pro
50 55 60
Ile Pro Pro Asp His Asp Glu Asn Ala Gly Thr Cys Val Glu Ile Glu
65 70 75 80
Glu His Ile Cys Glu Lys Asp Ala Ile Tyr Tyr Asn Val Thr Asp Ser
85 90 95
Thr Gly Ala Asn Thr Thr Lys Gln Leu Phe Ser Gly Ser Pro Thr Val
100 105 110
Thr Pro Pro Cys Ala Ser Asp Gly Tyr Cys Ala Glu Asp Glu Lys Cys
115 120 125
Cys Pro Thr Pro Cys Ala Arg Lys His Leu Cys Leu Ala Ala Ala Ala
130 135 140
Gln Glu Lys Tyr Pro Val Thr Asp Thr Pro Pro Val Thr Asp Phe Arg
145 150 155 160
Arg Gln Tyr Pro Thr Gln Pro Pro Ser Asn Cys Arg Tyr Phe Cys Gly
165 170 175
Tyr Glu Gly Asp Ala Tyr Cys Cys Asp Asp Gly Thr Arg Pro Ile Pro
180 185 190
Gln Asp His Asp Glu Asn Ala Gly Val Cys Ile Glu Met Ser Glu His
195 200 205
Ile Cys Glu Asp Asp Ala Ile Tyr Tyr Asn Thr Thr Asp Gly Thr Ala
210 215 220
Arg Ala Thr Ala Ala Thr Met Val Val Tyr
225 230
<210> 6
<211> 149
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser Glu His Ile Cys Glu Asp Asp Ala Ile Tyr Tyr Asn Thr Thr
1 5 10 15
Asp Gly Thr Gly Ala Asn Thr Thr Lys Gln Leu Phe Ser Gly Ser Pro
20 25 30
Thr Ala Thr Pro Pro Cys Ala Ser Asp Gly Tyr Cys Ala Glu Asp Glu
35 40 45
Lys Cys Cys Pro Thr Pro Cys Ala Arg Lys His Leu Cys Leu Ala Ala
50 55 60
Ala Glu Gln Glu Asn Ala Pro Ile Glu Lys Ala Pro Thr Ala Glu Asp
65 70 75 80
Asp Leu Leu Glu Phe Pro Thr Ile Pro Pro Asn Thr Cys Arg Tyr Tyr
85 90 95
Cys Ser Tyr Asp Asn Thr Val Tyr Cys Cys Asp Asp Gly Thr Arg Pro
100 105 110
Arg Arg Trp Tyr Cys Ile Cys Ile Lys Pro Glu Glu Ala Ser Leu Leu
115 120 125
Trp Asp Thr Ile Tyr Ser Ile Tyr Asn Tyr Pro Asp Ala Ile Tyr Gln
130 135 140
Pro Lys Ser Gln Thr
145
<210> 7
<211> 227
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Arg Thr Gly Thr Leu Leu Leu Val Val Ala Ala Ala Leu Ser Ala
1 5 10 15
Ala Ala Gln Glu Gly Ile Pro Thr Glu Gly Lys Leu Thr Pro Thr Glu
20 25 30
Ala Pro Arg Glu Phe Gln Thr Leu Pro Pro Ser Asn Cys Arg Tyr Phe
35 40 45
Cys Gly Tyr Glu Gly Asp Ala Tyr Cys Cys Asp Asp Gly Thr Arg Pro
50 55 60
Ile Pro Gln Asp His Asp Glu Asn Ala Gly Val Cys Ile Glu Met Ser
65 70 75 80
Glu His Ile Cys Glu Asp Asp Ala Ile Tyr Tyr Asn Thr Thr Asp Gly
85 90 95
Thr Gly Ala Asn Thr Thr Lys Gln Leu Phe Ser Gly Ser Pro Val Thr
100 105 110
Thr Pro Pro Cys Ala Ser Asp Gly Tyr Cys Ala Glu Asp Glu Lys Cys
115 120 125
Cys Pro Thr Pro Cys Ala Arg Lys His Leu Arg Leu Ala Ala Ala Ala
130 135 140
Gln Glu Asn Ala Pro Ile Glu Lys Ala Pro Thr Ala Glu Asp Asp Leu
145 150 155 160
Leu Glu Phe Pro Thr Ile Pro Pro Asn Thr Cys Arg Tyr Tyr Cys Ser
165 170 175
Tyr Asp Asn Thr Val Tyr Cys Cys Asp Asp Gly Thr Arg Pro Arg Arg
180 185 190
Trp Tyr Cys Ile Cys Ile Lys Pro Gly Glu Ala Ser Leu Leu Trp Asp
195 200 205
Thr Ile Tyr Ser Ile Tyr Asn Tyr Pro Asp Ala Ile Tyr Gln Pro Lys
210 215 220
Ser Gln Thr
225

Claims (7)

1.类Crustin抗菌肽，其特征在于：包括凡纳滨对虾类Crustin蛋白LvTWD-C1W1C3、LvTWD-W2C4或LvTWD-C3W2C4中的一种或二种或三种，它们分别为序列表SEQ ID No.5、SEQID No.6或SEQ ID No.7所示的氨基酸序列。
2. 一种编码权利要求1所述类Crustin抗菌肽的基因，其特征在于：包括mRNA序列LvTWD-C1W1C3、LvTWD-W2C4或LvTWD-C3W2C4中的一种或二种或三种，分别为序列表SEQ IDNo.2、SEQ ID No.3或SEQ ID No.4中的碱基序列，它们分别编码对应的类Crustin抗菌肽序列，分别为列表SEQ ID No.5、SEQ ID No.6和SEQ ID No.7所示的氨基酸序列。
3. 一组权利要求1所述的类Crustin抗菌肽的重组表达方法，其特征在于：分别根据LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4中的一种或二种或三种的类Crustin抗菌肽序列信息，确定其编码碱基序列信息，设计PCR引物，分别扩增其成熟肽编码序列，构建原核表达载体，利用大肠杆菌表达体系，获得有活性的重组抗菌蛋白SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中的一种或二种或三种。
4.一组权利要求1所述的类Crustin抗菌肽的获取方法，其特征在于：
其是由一条来源于凡纳滨对虾基因组的多WAP编码基因LvTWD，其为序列表SEQ IDNo.1中的碱基序列；其编码的前体mRNA在对虾体内经过选择性剪切产生一组成熟mRNA序列LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4。
5.权利要求1所述的类Crustin抗菌肽的应用，其特征在于：所述凡纳滨对虾类Crustin蛋白LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4中的一种或二种或三种在制备抑制细菌生长过程中或制备细菌的生长抑制剂药物中的应用，所述细菌为金黄色葡萄球菌、枯草芽孢杆菌、藤黄微球菌、副溶血弧菌、哈维氏弧菌、溶藻弧菌和大肠杆菌。
6. 细菌的生长抑制剂，其特征在于：以凡纳滨对虾类Crustin蛋白LvTWD-C1W1C3、LvTWD-W2C4和LvTWD-C3W2C4中的一种或二种或三种为活性成分，它们分别为序列表SEQ IDNo.5、SEQ ID No.6或SEQ ID No.7所示的氨基酸序列。
7.按照权利要求6所述的细菌的生长抑制剂，其特征在于：其中还添加有药剂学或药物学上可接受的载体或辅料。
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