CN101168564A - 人源性抗菌肽及其衍生物的用途 - Google Patents
人源性抗菌肽及其衍生物的用途 Download PDFInfo
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- CN101168564A CN101168564A CNA2007100663029A CN200710066302A CN101168564A CN 101168564 A CN101168564 A CN 101168564A CN A2007100663029 A CNA2007100663029 A CN A2007100663029A CN 200710066302 A CN200710066302 A CN 200710066302A CN 101168564 A CN101168564 A CN 101168564A
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- antimicrobial peptide
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- antimicrobial
- peptide
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Abstract
本发明涉及人源性抗菌肽及其衍生物的用途，所述人源性抗菌肽具有选自序列表SEQ ID NO：1所示的氨基酸序列，还包括此氨基酸序列多肽中个别氨基酸的取代、环化、L－型氨基酸变为D－型氨基酸、缺失或加入而得到的功能等同物。与其它来源碱性抗菌多肽相比，该人源性抗菌肽具有结构简单、人工合成方便、应用于人体无抗原性的有益特点。本发明的人源性抗菌肽及其衍生物具有显著的抑制细菌以及真菌生长的作用，可以应用于制备抗微生物感染制剂。
Description
技术领域
本发明涉及人源性抗菌肽及其衍生物的用途，具体涉及人精液凝固蛋白降解小肽SgI-29及其衍生物的氨基酸序列、核苷酸编码序列以及它们的抗菌用途。
背景技术
感染性疾病一直是威胁人类生命的主要原因之一，上个世纪30年代抗生素被发现并广泛地应用于临床治疗，拯救了无数患者的生命。由于传统抗生素的大量使用导致了耐药病原微生物的迅速增长。因此现在亟待开发出新型抗生素来对付世界范围内日益严重的病原微生物耐药性。
抗菌肽(又名肽类抗生素)在自然界分布广泛，是生物体产生的抵御外源性感染的内源性物质，历经数百万年而很少产生耐受性，具有作用迅速、强大、广谱等特点。因而当其发现之时，就引起了药学界及高科技生物产业界的瞩目(Koczulla AR，Bals R，2003.Antimicrobialpeptides：current status and therapeutic potential.Drugs 63，389-406)。
抗菌肽广泛地分布在两栖类、昆虫、植物和哺乳动物中。研究发现抗菌肽在两栖类动物皮肤中含量尤其丰富，30多年来人们已经从自然界发现了近千个抗菌肽(可在http://www.bbcm.univ.trieste.it/～tossi/pag1.htm检索到)，其中有800多个来自两栖类皮肤(主要是蛙类)(Apponyi MA，et al.，2004.Host-defence peptides of Australian anurans：structure，mechanism of action and evolutionary significance.Peptides 25，1035-1054)。
自从抗生素发明以来，人类在控制和治疗微生物感染中取得了较大的成就，但随着目前抗生素的持续使用，目前微生物抗药性已成为微生物感染控制中的重大问题，以至于某些微生物细菌已没有控制杀灭的一线物质。如在临床药物治疗中，万古霉素抗性的葡萄球菌、肠球菌以及其他革兰氏阴性感染疾病目前均是世界范围内的临床难题。三类主要引起脑膜炎的细菌在临床上也出现了强的抗药性，抗青霉素、氯霉素脑膜炎双球菌，肺炎球菌，对抗新头孢菌素抗生素的肺炎球菌也广泛出现。因此新一类抗微生物物质的发现和研制已成为当务之急和国际上的热点。与目前广泛使用的抗生素相比，多肽抗生素具有很多优点：如在最小作用浓度时，快速而广谱的杀灭微生物(包括目前临床抗药菌)，对真菌也有抑制作用，抗性菌株生成性小，对局部感染和全身感染都有效，正成为新一类抗生素，其研制目前受到广泛重视(Brogden KA，2005.Antimicrobial peptides：pore formers or metabolic inhibitors in bacteria？Nat.Rev.Microbiol.3，238-250)。
人源性抗菌肽一直是研究的热点之一，它没有抗原性，因此更易作为治疗药物使用(DeSmet K，Contreras R，2005.Human antimicrobial peptides：defensins，cathelicidins and histatins.Biotechnol.Lett.27，1337-1347)。
发明内容
本发明的一个目的是提供人源性抗菌肽及其衍生物，具有结构简单、人工合成方便、应用于人体无抗原性的有益特点，本发明的另一个目的是提供人源性抗菌肽及其衍生物的用途，其具有具有显著的抑制细菌以及真菌生长的作用。
本发明的目的是这样实现的：
本发明涉及一种人源性抗菌肽，是从健康男性液化精液分离得到的，其氨基酸序列如下(SEQ ID NO：1)：
组氨酸-天冬酰胺-赖氨酸-谷氨酰胺-谷氨酸-甘氨酸-精氨酸-天冬氨酸-组氨酸-天冬氨酸-赖氨酸-丝氨酸-赖氨酸-甘氨酸-组氨酸-苯丙氨酸-组氨酸-精氨酸-缬氨酸-缬氨酸-异亮氨酸-组氨酸-组氨酸-赖氨酸-甘氨酸-甘氨酸-赖氨酸-丙氨酸-组氨酸(His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val IleHis His Lys Gly Gly Lys Ala His)。
本发明的人源性抗菌肽包括上述多肽中个别氨基酸的取代、环化、L-型氨基酸变为D-型氨基酸、缺失或加入而得到的功能等同物的多肽。
即衍生自上述人源性抗菌肽的分离的人源性抗菌肽氨基酸残基替代且具有抗菌活性的抗菌肽，所述抗菌肽具有选自序列表SEQ ID NO：2所示的氨基酸序列：His Asn Lys Gln Glu GlyLys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His。
还可以是衍生自上述人源性抗菌肽的多个氨基酸缺失且具有抗菌活性的抗菌肽，所述抗菌肽具有选自序列表SEQ ID NO：3所示的氨基酸序列：Lys Gln Glu Gly Arg Asp His Asp LysSer Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys。
本发明提供的人源性抗菌肽，其制备方法可以是固相化学法，也可以将抗菌肽的编码基因克隆到载体上，然后在宿主细胞中表达后获得。其中表达载体可以是质粒或病毒中的一种。宿主细胞可以是原核细胞，包括大肠杆菌或枯草芽孢杆菌等，宿主细胞也可以是真核细胞，包括酵母细胞、植物细胞、昆虫细胞或哺乳动物细胞等。制备的抗菌肽可通过质谱鉴定。
本发明的人源性抗菌肽的分离方法，分离抗菌肽具有SEQ ID NO：1所示的氨基酸序列。
本发明的人源性抗菌肽及其衍生物的制备方法，采用固相化学法合成。
一种分离的核苷酸序列或其互补序列，所述核苷酸序列编码上面所述的抗菌肽。
一种构建物，所述的构建物含有具有选自序列表SEQ ID NO：3所示的氨基酸序列的核苷酸序列。
一种细胞，所述的细胞含有上面所述的肽、上面所述核苷酸序列或上面所述的构建物。
制备上面所述抗菌肽的方法，该方法包括：
(1)在适合上面所述的抗菌肽表达的条件下，培养上面所述的细胞；
(2)分离出上面所述的抗菌肽。
本发明的人源性抗菌肽及其衍生物的用途，所述的抗菌肽及其衍生物在制备抗微生物感染的药物中的应用。
为了深入研究本发明的这种人源性抗菌肽及其衍生物的结构与功能关系，利用全自动多肽合成仪合成一组多肽，以进行研究。
下面以制备的具有下列氨基酸序列的SgI-29，SgI-29R7K和SgI-25抗菌肽为例来进行叙述：
SgI-29(SEQ ID NO：1)：
His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile HisHis Lys Gly Gly Lys Ala His
SgI-29R7K(SEQ ID NO：2)：
His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile HisHis Lys Gly Gly Lys Ala His
SgI-25(SEQ ID NO：3)：
Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His LysGly Gly Lys
利用琼脂扩散实验检测多肽的抑菌活性。结果表明本发明合成抗菌肽有强的杀菌活性。
同样对多肽中氨基酸缺失衍生物的抗菌肽功能等同物也进行了制备与生物活性测定，结果表明这些多肽也具有较好的杀菌活性。
本发明的有益效果在于：人源性抗菌肽及其衍生物具有显著的抑制细菌以及真菌生长的作用。与其它来源碱性抗菌多肽相比，该人源性抗菌肽具有结构简单、人工合成方便、应用于人体无抗原性的有益特点。
附图说明
图1为本发明的人源性抗菌肽的SP-Sepharose离子交换层析图。
图2为本发明的人源性抗菌肽的G-25分子筛层析图。
图3为本发明的人源性抗菌肽的Mono-S离子交换层析图。
图4为本发明的人源性抗菌肽的HPLC反相C18柱层析图。
图5为本发明的人源性抗菌肽HPLC反相C18柱层析第III峰的质谱图。
图6为本发明的人源性抗菌肽SgI-29基因的核苷酸序列。
图7为本发明的人源性抗菌肽SgI-29的氨基酸序列。
图8为人工合成本发明的人源性抗菌肽SgI-29的质谱图。
具体实施方式
实施例1
1、人源性抗菌肽的分离纯化：
正常人禁欲3-7天后手淫取精液于干燥消毒量杯内，室温液化后，经精子微生物动静态计算机辅助自动分析系统(型号：CASAS-QH-III，北京清华同方产品)确定精子各项指标正常的样本于10000转/分离心10分钟分离精子与精液，小心收集上清液置-20℃备用。
第一步：SP-Sepharose离子交换：按上述方法获得的正常人精液(n＝40)上样于预先用50mM Na2HPO4-NaH2PO4，PH＝7.4缓冲液平衡的SP-Sepharose(Pharmacia产品) 阳离子交换柱(130mm长，直径20mm)。上样完毕后用0-0.5M NaCl进行线性洗脱。每管收集3ml。抑菌活性检测表明58-131管有很强的杀菌活性。合并58-131管冰冻干燥浓缩，见附图1。
第二步：分子筛Sephadex G-25层析：第一步冻干浓缩样品用Milli-Q超纯水溶解后，上样于预先用50mM Tris-HCl，pH8.8缓冲液平衡的AKTAHiPrepTM G-25脱盐柱(26/10，Pharmacia产品)，所用仪器为AKTAExplore 100(Amersham Biosciences产品)。收集第II峰并进行冰冻干燥浓缩，参见附图2。
第三步：Mono-S离子交换：第二步冻干浓缩样品用Milli-Q超纯水溶解后，上样于预先用50mM Tris-HCl，pH8.8缓冲液平衡的Mono-S阳离子交换柱(1ml，HR5/5，Pharmacia产品)，所用仪器为AKTAExplore 100(Amersham Biosciences产品)。收集第VII峰，见附图3。
第四步：反相高压液相层析：经Mono-S阳离子交换柱层析所得第VII峰以水(含0.1％三氟乙酸)：乙晴(含0.1％三氟乙酸)构成的洗脱系统进行梯度洗脱，收集第III峰即为含有抗菌活性的人精液凝固蛋白I衍生肽，见附图4。
含有抗菌活性的人精液凝固蛋白I衍生肽的鉴定：
第一步：反相高压液相层析第III峰的氨基酸序列测定：采用全自动氨基酸测序仪(型号：491，ABI公司产品)测定氨基酸序列结构。结果表明反相高压液相层析第III峰的氨基酸序列为：His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser。
第二步：快原子轰击质谱法(Fast atom bombardment mass spectrometry，FAB-MS)测定分子量：以甘油∶间硝基苄醇∶二甲亚砜(1∶1∶1，V∶V∶V，体积比)为底物，Cs+作为轰击粒子，电流为1mA，发射电压为25Kv。结果表明反相高压液相层析第III峰含有4个分子，它们的分子量分别为3377.17道尔顿、3850.67道尔顿、5117.64道尔顿和5174.18道尔顿，见附图5。
第三步：人精液凝固蛋白I衍生肽的鉴定：测定的反相高压液相层析第III峰氨基酸序列His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser进行BLAST分析的结果揭示它来源于人精液凝固蛋白I的降解，是人精液凝固蛋白I的衍生产物。FAB-MS质谱的精确分子量测定证明分子量为3377.17道尔顿的多肽来源于成熟人精液凝固蛋白I，其剪切位点为成熟人精液凝固蛋白I的第85位和第113位。所得到衍生片段在本发明中命名为SgI-29，其全序列为：His AsnLys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly GlyLys Ala His。SgI-29理论平均分子量[M+H]+为3377.74，测定分子量[M+H]+为3377.17。实验结果充分证明了SgI-29是由人精液凝固蛋白I被水解后产生的。
实施例2
1、人源性抗菌肽的制备及分离纯化：
本实施例采用固相化学合成法进行，利用全自动多肽合成仪分别合成它们的全序列，各合成抗菌肽的名称及其全序列氨基酸一级结构如下：
SgI-29(SEQ ID NO：1)：
His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile HisHis Lys Gly Gly Lys Ala His
SgI-29R7K(SEQ ID NO：2)：
His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile HisHis Lys Gly Gly Lys Ala His
SgI-25(SEQ ID NO：3)：
Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His LysGly Gly Lys。
通过HPLC反相C18柱层析脱盐、纯化。
2、人源性抗菌肽的鉴定：
纯化的人源性抗菌肽用高效液相色谱HPLC方法鉴定其纯度，分子量测定采用快原子轰击质谱法，用自动氨基酸测序仪测定氨基酸序列结构。
实施例3
1、人源性抗菌肽SgI-29基因在大肠杆菌中的表达
根据已知的编码成熟人精液凝固蛋白I的第86位到第112位的基因序列，将其克隆到表达载体pMAL-p2X质粒上，然后转化大肠杆菌DH5α，经IPTG诱导表达MBP-SgI-29融合蛋白于大肠杆菌周质，细菌周质部分蛋白经低渗处理后过MBP亲和柱，得到纯化MBP-SgI-29融合蛋白，再经FXa切割后得到抗菌肽SgI-29。
实施例中使用的高保真pfu酶，，T4DNA连接酶购自美国Promega公司，限制性内切酶Xmn I、Hind III、亲和树脂(Amylose resin)以及表达载体pMAL-p2X购自英国New EnglandBioLabs公司，胰蛋白胨、酵母提取物购自Oxoid公司，丙烯酰胺、甲叉双丙烯酰胺、过硫酸胺、异丙基-β-D-硫代半乳糖苷(IPTG)等购自Sigma公司。FXa购自Novagen公司。
人精囊腺取自一位56岁因膀胱尿路上皮癌行膀胱，前列腺，精囊腺全切除术男性患者，病理检查证实精囊腺未被肿瘤侵犯。将手术切下的精囊腺立即置于液氮中保存，直到提取总RNA。
2、总RNA的提取和cDNA合成
总RNA采用Qiangen公司总RNA提取试剂盒提取。cDNA合成采用TAKALA公司试剂盒，具体操作按说明书进行。
3、引物设计与合成
根据SgI基因的序列设计引物，SgI-1 5′GGT TTT CCA AGC AAG ATG AAG 3′；SgI-1R：5′AGG TGG TGT CAT CCA TGG ACC AAG 3′；SgI-EX1 5′CAT AAT AAA CAAGAA GGC AGA GAC C 3′；SgI52-EXR 5′CCT AAC AAG CTT TTA ATA TTG ACT GGATAT TCC 3′由北京赛百胜公司合成。其中AAG CTT是HindIII的酶切位点，其前面的碱基CCTAAC为保护序列。
4、SgI52基因扩增及克隆
用已得到的cDNA为模版，采用SgI-1及SgI-1R为引物进行PCR扩增，条件：cDNA 1μl，10 X buffer 5μl，dNTP(10mM)1μl，SgI-1(20p/μl)1μl，SgI-1R(20p/μl)1μl，pfu 0.25μl，ddH2O 40.75μl。PCR扩增条件：94℃ 2min；92℃ 15s，50℃ 30s，72℃ 1min，35循环；72℃ 10min。PCR产物行琼脂糖凝胶电泳，将1.3Kb处明显条带用Tiangen DNA胶回收试剂盒进行DNA回收。用回收的DNA做底物，以SgI-EX1和SgI52-EXR做引物进行PCR扩增SgI-52基因片段。条件：回收的SgI DNA 0.5μl，10 X buffer 5μl，dNTP(2.5mM)4μl，SgI-EX1(20p/μl)1μl，SgI52-EXR(20p/μl)1μl，pfu 0.25μl，H2O 38.25μl。PCR扩曾条件：94℃ 2min；92℃ 15s，50℃ 30s，72℃ 20s，35循环；72℃ 2min。PCR产物经电泳发现150bp附近有明显条带。PCR产物经HindIII酶切，切胶纯化基因片段。采用试剂盒抽提大肠杆菌JM109中的pMAL-p2X质粒，用内切酶HindIII及XmnI切开质粒，电泳证实。用T4连接酶将质粒与已经用内切酶HindIII切好的SgI-52基因进行连接。4℃过夜。转化感受态大肠杆菌DH5α。挑取菌落进行鉴定，并提取质粒测序。
5、SgI-52多肽的诱导表达
过夜培养带有SgI-52基因的pMAL-p2X质粒大肠杆菌DH5α。加1/100过夜培养的大肠杆菌DH5 α进入RB培养基1000ml，37℃200rpm摇菌约2-3小时至O.D.600为0.5。加入IPTG(终浓度为0.3mM)继续培养到诱导3小时，以未加IPTG诱导的各重组质粒转化细菌培养物为对照。
6、重组蛋白检测和鉴定
将细菌培养物1升于4000g离心10min收集细菌，用30mM Tris-HCl+20％Sucrose pH8.0重悬细菌(每g菌加80ml)。加0.5M的EDTA使终浓度为1mM，室温摇5-10分钟。4℃8000g离心10分钟。用2.5ml冰冷的5mM的MgSO4重悬。冰浴下摇10分钟。4℃8000g离心10分钟。上清液即为周质表达的蛋白质。加适当的蛋白上样缓冲液，煮沸5min后进行SDS-PAGE电泳，并经考马斯亮蓝染色，确定重组蛋白有无表达。用第X因子将重组蛋白从融合蛋白上切除下来，进行质谱分析和序列测定。
实施例4
抗菌活性检测，参照文献方法(Lehrer et al.，1991.Ultrasensitive assays for endogenousantimicrobial polypeptides.J.Immunol.Methods 137：167-173)，采用琼脂糖双层打孔法测定最低有效浓度MEC(Minimal Effective Concentration)。本实施例中的样品采用固相法化学合成，各合成抗菌肽的名称及其全序列氨基酸一级结构见实施例2。
其中，SgI-29的核苷酸序列见附图6、氨基酸序列见附图7、合成的人源性抗菌肽SgI-29的质谱图见附图8。
底层培养基配方为：1％低熔点琼脂糖(SigmaA6013)，0.3mg/ml胰蛋白冻(Oxoid产品)溶于10mM pH7.4 Na2HPO4-NaH2PO4缓冲液中。底层培养基20ml于42℃时分别加入过夜培养对数生长期细菌(约105-106CFU)，摇匀后使其在直径76mm培养皿中均匀摊布作为底层。待凝固后，底层培养基上打3mm的圆孔，每孔加5μl不同浓度的抗菌肽水溶液。37℃孵育3小时后，在底层上覆盖一层营养琼脂(顶层培养基：1％Sigma A6013低熔点琼脂糖，0.6mg/ml Oxoid胰蛋白冻溶于10mM pH7.4 Na2HPO4-NaH2PO4缓冲液中)，37℃继续孵育12-16小时。测量无菌生长的透明环直径。抗菌活性的计算：抗菌活性单位U＝(抗菌环的直径mm-3)X10。以抗菌肽浓度的对数值为横坐标，抗菌活性为纵坐标作回归方程，并计算能抑制细菌生长的抗菌肽的最小浓度MEC。细菌菌株来源于昆明医学院第一附属医院，此试验重复四次，取平均值，结果如表1。
表1.人源性抗菌肽SgI-29的抑菌活性
| Microorganisms | 最低有效浓度MEC(μg/ml) | ||
| SgI-29 | SgI-29R7K | SgI-25 | |
| Escherichia coli ATCC 25922Escherichia coli ML-3 5PEscherichia coli clinic strain 1Escherichia coli clinic strain 2Bacillus pyocyaneus ATCC 27853Bacillus pyocyaneus CMCCB 10104Bacillus pyocyaneus PA 01Bacillus pyocyaneus clinic strainStaphylococcus aureus ATCC 25923Staphylococcus aureus ATCC 43300 | 2.8317.4333.5563.9375.2927.33412.86410.405＞100＞100 | 3.0626.8824.0793.6384.9966.37810.63811.909未测定未测定 | 5.36110.2316.8237.23111.73415.63418.88525.362未测定未测定 |
| Candida albicans ATCC 10231Pichia pastoris GS 115 | 20.52318.244 | 25.76821.224 | 30.66524.315 |
实验所用细菌以及真菌菌株说明：
金黄色葡萄球菌(Staphylococcus aureus)ATCC 43300，是耐甲氧西林的金黄色葡萄球菌ATCC标准株，对青霉素类，β-内酰胺类耐药，对万古霉素敏感。
大肠杆菌(Escherichia coli)ML-35P，是氨苄耐药标准株。
大肠杆菌(Escherichia coli)临床耐药株1和2，均为产超广谱β-内酰胺酶的临床分离株，对青霉素，头孢1代、2代、3代以及4代耐药，对泰能、氨基糖甙类敏感。
绿脓杆菌(Bacillus pyocyaneus)临床耐药株，为多药耐药临床分离株，对目前临床使用各类抗生素均耐药，包括泰能，MEM。
真菌类的白色念珠菌(Candida albicans)ATCC 10231以及毕赤酵母(Pichia pastoris)GS115均为标准株。
由表1可见，合成的人源性抗菌肽SgI-29及其氨基酸替代以及氨基酸缺失衍生物具有显著的抑制细菌以及真菌生长的作用，可作为抗微生物物质用于制备抗微生物感染制剂。
序列表.SEQ
<110>中国科学院昆明动物研究所
<120>人源性抗菌肽及其抗微生物的应用
<160>6
<210>1
<211>29
<212>PRT
<213>智人(Homo sapiens)
<400>1
His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe
1 5 10 15
His Arg Val Val Ile His His Lys Gly Gly Lys Ala His
20 25
<210>2
<211>29
<212>PRT
<213>人工序列
<400>2
His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe
1 5 10 15
His Arg Val Val Ile His His Lys Gly Gly Lys Ala His
20 25
<210>3
<211>25
<212>PRT
<213>人工序列
<400>3
Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg
1 5 10 15
Val Val Ile His His Lys Gly Gly Lys
20 25
<210>4
<211>87
<212>DNA
<213>智人(Homo sapiens)
<400>4
cataataaac aagaaggcag agaccatgat aaatcaaaag gtcattttca cagggtagtt 60
atacaccata aaggaggcaa agctcat 87
<210>5
<211>87
<212>DNA
<213>人工序列
<400>5
cataataaac aagaaggcaa agaccatgat aaatcaaaag gtcattttca cagggtagtt 60
atacaccata aaggaggcaa agctcat 87
<210>6
<211>75
<212>DNA
<213>人工序列
<400>6
aaacaagaag gcaaagacca tgataaatca aaaggtcatt ttcacagggt agttatacac 60
cataaaggag gcaaa 75
Claims (10)
1.一种分离的人源性抗菌肽，其特征在于所述人源性抗菌肽具有选自序列表SEQ IDNO：1所示的氨基酸序列：His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly HisPhe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His。
2.衍生自权利要求1的分离的人源性抗菌肽氨基酸残基替代且具有抗菌活性的抗菌肽，其特征在于所述抗菌肽具有选自序列表SEQ ID NO：2所示的氨基酸序列：His AsnLys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His LysGly Gly Lys Ala His。
3.衍生自权利要求1的多个氨基酸缺失且具有抗菌活性的抗菌肽，其特征在于所述抗菌肽具有选自序列表SEQ ID NO：3所示的氨基酸序列：Lys Gln Glu Gly Arg AspHis Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys。
4.权利要求1所述的人源性抗菌肽的分离方法，其特征在于分离抗菌肽具有SEQ IDNO：1所示的氨基酸序列。
5.权利要求1、2及3所述的人源性抗菌肽及其衍生物的制备方法，其特征在于采用固相化学法合成。
6.一种分离的核苷酸序列或其互补序列，其特征在于所述核苷酸序列编码权利要求1至3所述的抗菌肽。
7.一种构建物，其特征在于所述的构建物含有权利要求1至3所述的核苷酸序列。
8.一种细胞，其特征在于所述的细胞含有权利要求1至3所述的肽、权利要求6所述核苷酸序列或权利要求7所述的构建物。
9.一种制备权利要求1至3所述抗菌肽的方法，其特征在于该方法包括：
(1)在适合权利要求1至3所述的抗菌肽表达的条件下，培养权利要求8所述的细胞；
(2)分离出权利要求1至3所述的抗菌肽。
10.权利要求1至3所述的人源性抗菌肽及其衍生物的用途，其特征在于：所述的抗菌肽及其衍生物在制备抗微生物感染的药物中的应用。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906105A (zh) * | 2010-03-31 | 2013-01-30 | 诺瓦生命科学有限公司 | 肽及其用途 |
| CN105994251A (zh) * | 2016-06-20 | 2016-10-12 | 成都美强兽医技术服务有限公司 | 一种杀灭动物精液病毒细菌的稀释保存液 |
| CN110438139A (zh) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | 一种利用毕赤酵母菌制备抗菌肽t9w的方法 |
| CN112263712A (zh) * | 2020-10-14 | 2021-01-26 | 中山大学附属第六医院 | 一种具有抗菌性能的腹膜修复材料及其制备方法 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906105A (zh) * | 2010-03-31 | 2013-01-30 | 诺瓦生命科学有限公司 | 肽及其用途 |
| CN102906105B (zh) * | 2010-03-31 | 2017-05-10 | 诺瓦生命科学有限公司 | 肽及其用途 |
| CN105994251A (zh) * | 2016-06-20 | 2016-10-12 | 成都美强兽医技术服务有限公司 | 一种杀灭动物精液病毒细菌的稀释保存液 |
| CN105994251B (zh) * | 2016-06-20 | 2018-07-17 | 成都美强兽医技术服务有限公司 | 一种杀灭动物精液病毒细菌的稀释保存液 |
| CN110438139A (zh) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | 一种利用毕赤酵母菌制备抗菌肽t9w的方法 |
| CN112263712A (zh) * | 2020-10-14 | 2021-01-26 | 中山大学附属第六医院 | 一种具有抗菌性能的腹膜修复材料及其制备方法 |
| CN112263712B (zh) * | 2020-10-14 | 2022-04-29 | 中山大学附属第六医院 | 一种具有抗菌性能的腹膜修复材料及其制备方法 |
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