CN104774842A - 一种具有抗菌活性的多肽Pt5e - Google Patents
一种具有抗菌活性的多肽Pt5e Download PDFInfo
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Abstract
本发明发现了一种新型具有中和内毒素作用的重组抗菌多肽的设计、重组表达和制备技术。该技术采用定点突变、表达载体构建、诱导表达和亲和层析，分离纯化重组多肽。高磷蛋白衍生抗菌多肽Pt5e具有广谱抗菌作用；中和内毒素作用；抑制炎症因子TNF-α分泌；并能显著提高内毒素血症小鼠成活率，且无细胞毒和溶血等副作用，是一种具有良好应用前景的治疗败血症的药物。应用该技术可以生产新型中和内毒素抗菌多肽，成本低，对环境无污染。
Description
技术领域
本发明属于基因工程药物技术领域。涉及一种具有中和内毒素作用的高磷蛋白衍生多肽。其技术涉及基因定点突变、重组表达、分离纯化、重组多肽的抗菌功能、中和内毒素功能以及细胞毒性分析技术。重组多肽在浓度1.2和1.8 μM时，可以完全抑制大肠杆菌和金黄色葡萄球菌生长。重组多肽Pt5e能抑制炎症因子TNF-α释放，结合并中和内毒素，保护内毒素血症小鼠，保护率达60%以上，且无细胞毒性、无溶血副作用。
背景技术
败血症是一种血液中毒病症，主要是由身体某些部位感染的细菌扩散至血液，细菌在血液中大量繁殖，产生内毒素(Lipoplysaccharide,LPS)，细菌内毒素主要通过两个方面发生作用：一方面为LPS在细菌周围形成一层保护屏障以逃避抗生素的作用；另一方面LPS作用于宿主细胞，诱导诸如TNF、IL等细胞因子的释放，从而使机体处于混乱状态，导致过度炎症，严重时引起人体多种器官功能受损、可导致脓毒性休克，使病人衰竭，最终死亡。即使在美国这样医疗水平高度发达的国家，每年仍有超过200,000人死于败血症。因此，败血症是常见而又严重的临床疾病，对患者生命威胁、危害极大。
败血症的治疗最主要在于阻断、遏制和消除内毒素作用。目前，治疗败血症主要依靠抗生素结合其他辅助方法来实现。迄今，临床上还缺乏一种可以特异性中和内毒素而没有副作用的药物。即使新近生产的抗内毒素抗体和血液净化方法等，临床上疗效也不甚明显。
抗菌肽由于具有广谱抗菌活性和结合内毒素、阻止炎症因子释放等抗菌作用，以及对正常细胞无损伤等作用。近年来已经成为理想的治疗败血症候选药物。例如，抗菌肽LL-37不但可以结合并中和内毒素，而且具有治疗败血症作用，能明显提高成活率。但是，LL-37具有溶血副作用，极大地限制了其在临床上的应用。但不容忽视的是抗菌肽已成为未来对抗内毒素的主要研究对象。我们发现，通过定点突变、重组表达的高磷蛋白（phosvitin）衍生抗菌多肽Pt5e，不但具有广谱抗菌作用、可以结合内毒素，而且能够抑制炎症因子释放、显著提高内毒素血症小鼠成活率，而无细胞毒性和溶血作用发生，因此是一种具有良好前景的治疗败血症的药物。
发明内容
本发明是发现了一种可用于治疗败血症的新型抗菌肽Pt5e，及其设计和制备技术。抗菌肽原始序列来源高磷蛋白基因，我们采用定点突变和重组表达制备高磷蛋白衍生抗菌多肽Pt5e，并中和内毒素功能以及细胞毒性分析，验证了Pt5e应用于败血症治疗的可行性。
本发明更详细的实施方法可参见实施例。
附图说明
图1：高磷蛋白与其C-端55个氨基酸组成多肽Pt5。示由Pt5经定点突变，获得重组表达突变体流程。
图2：重组表达多肽电泳(A)和免疫印迹(B)。M, 标准蛋白; Lane 1, 重组表达多肽Pt5；Lane 2, 重组表达多肽Pt5a；Lane 3, 重组表达多肽Pt5b；Lane 4, 重组表达多肽Pt5c；Lane 5, 重组表达多肽Pt5d；Lane 6, 重组表达多肽Pt5e；Lane 7, 重组表达多肽Pt5f。
图3：透射电镜结果。示PBS (A、C)或者Pt5e (B、D)与E. coli以及S. aureus在25°C共育1小时对细菌的影响。
具体实施方式
(1)定点突变以及重组蛋白表达与纯化
基于Pt5序列, 设计特定的系列引物制作6个突变体Pt5a、Pt5b、Pt5c、Pt5d、Pt5e和Pt5f。采用定点突变获得高磷蛋白衍生多肽Pt5e，构建表达载体导入大肠杆菌诱导表达，突变体在Escherichia coli，经亲和层析纯化，得电泳纯多肽。
多肽Pt5、Pt5a、Pt5b、Pt5c、Pt5d、Pt5e和Pt5f的DNA以及氨基酸序列见序列表，其中Pt5的核苷酸和氨基酸序列标识符为1； Pt5的纯氨基酸序列标识符为2；Pt5a的核苷酸和氨基酸序列标识符为3；Pt5a的纯氨基酸序列标识符为4；Pt5b的核苷酸和氨基酸序列标识符为5；Pt5b的纯氨基酸序列标识符为6；Pt5c的核苷酸和氨基酸序列标识符为7；Pt5c的纯氨基酸序列标识符为8；Pt5d的核苷酸和氨基酸序列标识符为9；Pt5d的纯氨基酸序列标识符为10；Pt5e的核苷酸和氨基酸序列标识符为11；Pt5e的纯氨基酸序列标识符为12；Pt5f的核苷酸和氨基酸序列标识符为13；Pt5f的纯氨基酸序列标识符为14。
（2）重组多肽具有广谱抗菌作用
先导实验采用微量稀释分析法（microdilution assay）分析6个重组多肽抗菌活性，发现其中Pt5e 抗E.coli和S. aureus活性最强，最小抑菌浓度分别为1.2和1.8 μM，比其母分子Pt5抑菌作用还强大。因此，我们选择Pt5e进行下面实验。分析Pt5e抗革蓝氏阴性细菌E. coli和V. anguillarum以及革蓝氏阳性细菌S. aureus、B. Subtilis和M. luteus作用，发现Pt5e可以显著抑制各种所测试细菌的生长，具有广谱抑菌活性。Pt5e抗菌活性具有热稳定性，其作用100度处理10分钟，不会受到影响。
（3）Pt5e破坏细菌细胞壁
采用流式细胞仪和电子显微镜分析Pt5e 对E. coli和S. aureus细胞影响。结果表明：Pt5e可以造成E. coli和S. aureus细胞壁损伤，使细胞壁变薄甚至造成细胞壁破坏，失去完整性。Pt5e还可以造成细胞质流出细胞外。
（4）Pt5e结合LPS和LTA
免疫酶标法（ELISA）分析重组表达多肽Pt5e与LPS和LTA的结合。结果表明：Pt5e可以与革蓝氏阴性细菌表面保守分子LPS结合，也可以与革蓝氏阳性细菌表面保守分子LTA结合。
（5）Pt5e抑制LPS-处理的RAW264.7细胞分泌TNF-α
LPS处理大鼠巨噬细胞RAW264.7会分泌炎症因子TNF-α。我们发现：Pt5e可以显著抑制LPS处理的大鼠巨噬细胞RAW264.7分泌TNF-α，这证明Pt5e具有中和内毒素的功能。
（6）Pt5e对RAW264.7细胞无毒性
采用MTT法测试重组表达多肽Pt5e对RAW264.7细胞成活率影响。结果表明：Pt5e对大鼠RAW264.7生长没有影响，对大鼠RAW264.7细胞无毒性。
（7）Pt5e对人血红细胞没有破坏作用
溶血作用分析表明：重组表达多肽Pt5e对人人血红细胞无毒，不会造成血红细胞溶解，没有溶血作用。
（8）Pt5e保护内毒素血症小鼠
采用内毒素血症小鼠模型研究重组表达多肽Pt5e对其保护作用。结果表明：Pt5e可以保护体内注射LPS诱导的内毒素血症小鼠，使其死亡率显著降低，保护率可达60%以上。
表1：定点突变所用的引物。
| Primer | Sequence (5′–3′) | Sequence information |
| Pt5a (sense) | CCATCATT GTG CCTTTCAGGA | Mutation primer (E11V) |
| Pt5a (antisense) | TGGCAGTCTTAGACTAACGAG | Mutation primer (E11V) |
| Pt5b (sense) | GTAC AGG GCACACCATAGC | Mutation primer (L22R) |
| Pt5b (antisense) | CGATCTTTGTGGAATTTCCTG | Mutation primer (L22R) |
| Pt5c (sense) | CATTTGTTCAAAGCTAGC | Mutation primer (K45I) |
| Pt5c (antisense) | CAG ATA CAGAATAGATTCC | Mutation primer (K45I) |
| Pt5d (sense) | TTTCTGCATTTGTTCAAAGC | Mutation primer (R48L) |
| Pt5d (antisense) | CAGAAT CTA TTCCTTGGA | Mutation primer (R48L) |
| Pt5e (sense) | AGCACTTCCACTGCTAGTATCC | Mutation primer (E41V;Q42I) |
| Pt5e (antisense) | GCAGCTAGCTTT GTAATA ATGCAG | Mutation primer (E41V;Q42I) |
| Pt5f (sense) | CATTTGTTCAAAGCTAGC | Mutation primer (K45I;R48L) |
| Pt5f (antisense) | CAG ATA CAGAAT CTA TTCC | Mutation primer (K45I;R48L) |
表2：Pt5，LL-37及重组多肽对E.coli和S. aureus的最小抗菌活性。
| E.coli | S. aureus | |
| Pt5 | 2.4 | 1.8 |
| Pt5a | 2.4 | 1.8 |
| Pt5b | >26 | >26 |
| Pt5c | >26 | >26 |
| Pt5d | 1.8 | 1.8 |
| Pt5e | 1.8 | 1.2 |
| Pt5f | >26 | >26 |
| LL-37 | 7.2 | 3.6 |
表3：Pt5对革蓝氏阴性细菌E. coli和V. anguillarum以及革蓝氏阳性细菌S. aureus、B. Subtilis和M. luteus的抗菌作用。
| Pt5e抑菌浓度 | |
| E. coli | 1.8 |
| V. anguillarum | 1.2 |
| S. aureus | 1.2 |
| B. Subtilis | 1.2 |
| M. luteus | 1.2 |
表4：加热处理的组表达多肽Pt5e抑制细菌E. coli、V. Anguilarum、S. aureus、B. Subtilis和M. Luteus的最小抑菌浓度。
| Pt5e抑菌浓度 | 50度处理10分钟, Pt5e抑菌浓度 | 100度处理10分钟, Pt5e抑菌浓度 | |
| E. coli | 1.8 | 1.7 | 1.6 |
| V. anguillarum | 1.2 | 1.0 | 0.6 |
| S. aureus | 1.2 | 1.1 | 0.8 |
| B. Subtilis | 1.2 | 1.1 | 1.0 |
| M. luteus | 1.2 | 1.1 | 0.6 |
表5：流式细胞仪分析结果。示重组表达多肽Pt5e对E. coli和S. aureus细胞膜完整性的破坏作用。
表6：免疫酶标分析。示重组表达多肽Pt5e与LPS和LTA和结合结果。
表7：重组表达多肽Pt5e对LPS-诱导的RAW264.7细胞的TNF-α分泌作用影响。
表8：重组表达多肽Pt5e对RAW264.7细胞成活影响。
| Pt5e浓度(μM) | 成活百分率(%) |
| 0 | 100 |
| 10 | 100±7 |
| 40 | 94±4 |
| 80 | 100±3 |
表9：重组表达多肽Pt5e的溶血作用分析。
表10：重组表达多肽Pt5e对内毒素血症小鼠保护作用。
序列表
<110> 张士璀 胡立立 宿烽
<120> 一种具有抗菌活性的多肽Pt5e
<160> 14
<210> 1
<211> 165
<212> DNA
<213> 斑马鱼（Danio rerio）
<400> 1
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag aaa cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 2
<211> 55
<212> PRT
<213> 斑马鱼（Danio rerio）
<400> 2
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 3
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 3
tct cgt atg tct aag act gcc acc atc att gtg cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Val Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag aaa cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 4
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 4
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Val Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 5
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 5
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac agg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Arg Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag aaa cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 6
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 6
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac agg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Arg Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag aaa cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 7
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 7
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gta ata atg cag aaa cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Val Ile Met Gln Lys Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 8
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 8
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Val Ile Met Gln Lys Gln Asn Arg
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 9
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 9
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag ata cag aat aga 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Ile Gln Asn Arg
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 10
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 10
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Ile Gln Asn Arg
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 11
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 11
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 48
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag aaa cag aat cta 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Leu
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 12
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 12
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Lys Gln Asn Leu
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 13
<211> 165
<212> DNA
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 13
tct cgt atg tct aag act gcc acc atc att gag cct ttc agg aaa ttc 68
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
cac aaa gat cgg tac ttg gca cac cat agc gcc aca aag gat act agc 96
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
agt gga agt gct gca gct agc ttt gaa caa atg cag ata cag aat cta 144
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Ile Gln Asn Leu
35 40 45
ttc ctt gga aat gat att cca 165
Phe Leu Gly Asn Asp Ile Pro
50 55
<210> 14
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> 根据疏水性和极性而设计，以用作寻找比多肽Pt5具有更强抑菌活性的多肽。
<400> 14
Ser Arg Met Ser Lys Thr Ala Thr Ile Ile Glu Pro Phe Arg Lys Phe
1 5 10 15
His Lys Asp Arg Tyr Leu Ala His His Ser Ala Thr Lys Asp Thr Ser
20 25 30
Ser Gly Ser Ala Ala Ala Ser Phe Glu Gln Met Gln Ile Gln Asn Leu
35 40 45
Phe Leu Gly Asn Asp Ile Pro
50 55。
Claims (2)
1.一种具有抗菌活性的高磷蛋白衍生多肽Pt5e，其特征在于编码的DNA核苷酸序列为：
Tctcgtatgtctaagactgccaccatcattgagcctttcaggaaattccacaaagatcggtacttggcacaccatagcgccacaaaggatactagcagtggaagtgctgcagctagctttgaacaaatgcagaaacagaatctattccttggaaatgatattcca；
其氨基酸序列为：
SRMSKTATIIEPFRKFHKDRYLAHHSATKDTSSGSAAASFEQMQKQNLFLGNDIP；
所述的高磷蛋白衍生多肽Pt5e，具体是利用基因定点突变，构建表达载体导入大肠杆菌诱导表达；通过亲和层析纯化重组表达多肽。
2.权利要求1所述高磷蛋白衍生多肽Pt5e，其特征在于其具有广谱抗菌作用可用于治疗败血症。
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| CN112724222A (zh) * | 2021-02-22 | 2021-04-30 | 青岛森盛生物医药科技有限公司 | 一种鱼源抗菌肽 |
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| CN112724222A (zh) * | 2021-02-22 | 2021-04-30 | 青岛森盛生物医药科技有限公司 | 一种鱼源抗菌肽 |
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