CN103641901B - 一种抗菌肽 - Google Patents

一种抗菌肽 Download PDF

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CN103641901B
CN103641901B CN201310631774.XA CN201310631774A CN103641901B CN 103641901 B CN103641901 B CN 103641901B CN 201310631774 A CN201310631774 A CN 201310631774A CN 103641901 B CN103641901 B CN 103641901B
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antibacterial peptide
melittin
thanatin
antibacterial
peptide
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CN103641901A (zh
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钱坤
周国庆
文良柱
沈子龙
葛海霞
孙来玉
肖莉
张海洋
徐秀芳
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Huzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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Abstract

本发明属于生物技术制药工业中基因工程生产药物的技术领域，具体为一种抗菌肽。该抗菌肽的一级结构为GLPLLISWIKRKRQQ-AGP-GSKKPVPIIYCNRRTGKCQRM。新设计的抗菌肽，同时具备死亡素和蜂毒素的优点，不但抗菌谱广，而且能抗金黄色葡萄球菌，同时避免了蜂毒素溶血性强的弱点。

Description

一种抗菌肽
技术领域
本发明属于生物技术制药工业中基因工程生产药物的技术领域，具体为一种抗菌肽。
背景技术
抗菌肽（antibacterial peptide）是一类分子量较小的多肽，它们大多数是由13-45个氨基酸组成，有些抗菌肽全部由L-氨基酸组成，有些抗菌肽分子中则同时包含了L-氨基酸和D-氨基酸，分子量一般为几千道尔顿，常带正电荷，1980年Hultmark等首先在天蚕(Hyatophoracecropia)蛹中分离得到一种杀菌肽,并将其命名为cecropin。抗菌肽具有抗菌、抗病毒、抗肿瘤等活性，水溶性好，耐高温，是一种比较理想的抗生素代用品，特别是其主要抑菌机制是破坏细胞膜的稳定性，难以产生耐药性，而且抗菌肽一般是小肽，可以被动物完全消化，不存在残留性的问题，所以，抗菌肽无论是作为饲料添加剂，兽药还是人药，都是抗生素的理想代用品。
死亡素（Thanatin）是昆虫抗菌肽中最广谱的抗菌肽，它对革兰氏阳性菌、革兰氏阴性菌和某些真菌有抗菌作用，但是对金黄色葡萄球菌没有抑制作用。
蜂毒素(Melittin)是意大利蜂(Apismellifera)毒液的主要活性成 分，由Habermanm等首先从蜂毒中分离出来的一种抗菌肽，具抗菌、消炎、抗辐射、抗关节炎、抗肿瘤、抗艾滋病以及对心血管等方面的作用特别是可以抗金黄色葡萄球菌，但是蜂毒素有很强的溶血性，限制了临床上的进一步使用。
发明内容
本发明的目的是设计一种新的抗菌肽，使其同时具备死亡素和蜂毒素的优点，避免各自缺陷，即设计的抗菌肽具有死亡素的抗菌广谱性和蜂毒素的抗金黄色葡萄球菌优势，克服了死亡素不抗金黄色葡萄球菌抗性和蜂毒素溶血性的缺陷。
实现上述发明的技术解决方案是：一种抗菌肽，该抗菌肽的一级结构为GLPLLISWIKRKRQQ-AGP-GSKKPVPIIYCNRRTGKCQRM。
本发明的主要有益效果有：新设计的抗菌肽，同时具备死亡素和蜂毒素的优点，不但抗菌谱广，而且能抗金黄色葡萄球菌，同时避免了蜂毒素溶血性强的弱点。
说明书附图 
图1为重组表达载体的构建过程。
具体实施方式
下面结合实施例对本发明做进一步说明。
实施例1：
蜂毒素一级结构：GIGAVLKVLTTGLPALISWIKRKRQQ，死亡素一级结 构：GSKKPVPIIYCNRRTGKCQRM，设计出新的抗菌肽：N-末端为蜂毒素的12-26号位氨基酸，并突变成GLPLLISWIKRKRQQ9，斜体氨基酸为突变氨基酸；C-末端为完整的死亡素，中间用AGP连接后，所设计的抗菌肽一级结构为GLPLLISWIKRKRQQ-AGP-GSKKPVPIIYCNRRTGKCQRM。（氨基酸简写规则：甘氨酸G，丝氨酸S，丙氨酸A，苏氨酸T，缬氨酸V，异亮氨酸I，亮氨酸L，酪氨酸Y，苯丙氨酸F，组氨酸H，脯氨酸P，天冬氨酸D，甲硫氨酸M，谷氨酸E，色氨酸W，赖氨酸K，半胱氨酸C，精氨酸R）
（1）重组表达载体的构建
构建过程见图1。
1）设计引物五段引物：
引物1：5-CTCGAGATGGGTCTTCCTCTTCTTATTTCTTGGATTAAGCGTAAGCGTCAACAAGCTGG
引物2：5－AAGCGTCAACAAGCTGGTCCTGGTTCTAAGAAGCCTGTTCCTATTATTTACTGCAACCG 
引物3：3－GGATAATAAATGACGTTGGCAGCATGACCATTCACGGTTGCATACATTATTCCTAGG 
引物4：5-ACGCGGATCCGACCCCGGTCTTCCTCTTCTT ATTTCTTGG
引物5：5-ACCGCTCGAGTTATTACATACGTTGGCACTTACC
2）PCR反应
按照表1的反应体系进行PCR反应，反应产物连接在PMD-18载体上（图1中的PMD-18MT）并保存于大肠杆菌DH5α中；提取重组 PMD-18MT质粒，按照表2的反应体系进行PCR反应，反应产物连接在PMD-18载体上（图1中的PMD-18MTE）并保存于大肠杆菌DH5α中。
表1 PCR反应体系1
表2 PCR反应体系2
3）重组载体的构建
提取PMD-18MTE质粒，XhoI和BamHI双酶切质粒过夜，琼脂糖 凝胶电泳酶切产物，染色后，切割目的条带，凝胶回收试剂盒纯化目的片段。
pET32-a质粒用XhoI和BamHI双酶切过夜，电泳，切割目的条带后，凝胶回收试剂盒纯化目的片段。
两种纯化的DNA片段，用连接酶22℃连接过夜，连接产物转入到大肠杆菌DH5α感受态细胞中。
（2）工程菌的表达与融合蛋白的分离纯化
1）工程菌的构建
提取质粒(图1中pET32-a-MT)，并转化到大肠杆菌BL21中，涂布的平板过夜培养后，挑取一个单菌落，用液体培养基过夜培养后，1％的接种量转接到新鲜的培养基中，振荡培养至对数中期，加入乳糖至终浓度1mg/ml，在0、2、4、6、8小时的分别取0.75ml的菌，离心后，加入样品缓冲液调节菌体密度OD600至0.5左右，沸水浴中加热15min，进行SDS-PAGE电泳，电泳结束后，考马斯亮蓝染色液染色过夜，再用脱色液脱色至背景清晰，电泳结果说明工程菌构建成功。
2）融合蛋白的表达与纯化
大规模培养工程菌，离心收集菌体，蒸馏水洗涤两次，采用间隙法超声法或者高压细胞法破碎菌体，菌体破碎后离心，收集上清液，加到His-Taq亲和柱中，用1倍柱体积各种浓度50mmol/L咪唑洗脱杂蛋白，再用100mmol/L的咪唑洗涤表达的融合蛋白，收集并浓缩融合蛋白。
（3）融合蛋白的酸水解与设计抗菌肽的纯化
上述步骤获得的浓缩融合蛋白加入盐酸至终浓度50±10mmol/L，于55±10℃水浴中水解35±10℃，超滤除盐酸后，加到His-Taq亲和柱中，用1倍柱体积各种浓度50mmol/L咪唑洗脱得到所设计的抗菌肽浓缩液。
（4）抑菌和溶血性实验
1）抑菌实验 
按照管碟法实验步骤，初步结果表明，所设计的抗菌肽浓缩液对大肠杆菌和金黄色葡萄球菌具有体外抑菌活性。
2）溶血实验 
取兔血10ml，加入洁净干燥的小烧杯中，并用玻璃棒搅拌除去纤维蛋白原，再加生理盐水10ml，摇匀、离心、倾去上清液，如此反复几次到上清液不呈红色为止；量取2.5ml血球放入试管，加生理盐水稀释成2％的混悬液，并加入纯化抗菌肽浓缩液，置37℃恒温箱中培养5小时，观察结果，试管内的液体没有发生颜色的变化。

Claims (1)

1.一种抗菌肽，其特征在于：该抗菌肽的一级结构为GLPLLISWIKRKRQQ-AGP-GSKKPVPIIYCNRRTGKCQRM。
CN201310631774.XA 2014-01-07 2014-01-07 一种抗菌肽 Expired - Fee Related CN103641901B (zh)

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CN104223065A (zh) * 2014-08-30 2014-12-24 北京安和亿泰生物工程技术有限公司 一种食用肉虫的繁殖培育方法
CN104327168B (zh) * 2014-09-29 2017-11-14 广西中医药大学 一种小分子多肽及其应用
CN105622763B (zh) * 2016-03-08 2019-03-12 浙江理工大学 抗菌肽融合蛋白及其制备方法和应用
CN110498848B (zh) * 2019-09-10 2020-05-12 中国医学科学院基础医学研究所 一种蜂毒肽变体及其应用
CN110903404A (zh) * 2019-12-30 2020-03-24 佛山科学技术学院 一种蜂毒素-死亡素重组多肽及其应用

Non-Patent Citations (2)

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文良柱.蜂毒肽与死亡素杂合肽(MT)在大肠杆菌中融合表达的研究.《食品与生物技术学报》.2006,第25卷(第6期),第45-48页. *
重组死亡素-蜂毒素杂合肽表达·分离纯化的研究;张莉;《安徽农业科学》;20101231;第38卷(第28期);15513-15514 *

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