CN107602686A - A kind of polypeptide resistant to gram-positive bacteria - Google Patents

Abstract

The present invention provides a kind of polypeptide with anti-microbial property separated from silvery pomfret, and the sequence of the polypeptide is SEQ ID NO:1；The gene of encoding such polypeptides, its nucleotides sequence are classified as SEQ ID NO:2.The present invention infects silvery pomfret by the gram-positive microorganism commonly used in aquatic products field, and the polypeptide resistant to gram-positive bacteria is obtained by subtractive hybridization method, and this can be used as the feed addictive of silvery pomfret to use.

Description

Translated fromChinese

一种对革兰氏阳性菌具有抗性的多肽A polypeptide resistant to gram-positive bacteria

技术领域technical field

本发明属于抗菌肽筛选技术领域，具体涉及一种对革兰氏阳性菌具有抗性的多肽。The invention belongs to the technical field of antimicrobial peptide screening, and in particular relates to a polypeptide resistant to Gram-positive bacteria.

背景技术Background technique

银鲳(Pampus argenteus)属鲈形目(Perciformes)，鲳亚目(Stromateoidei)鲳科(Stromateidae)，鲳属(Pampus)，为暖水性集群性鱼类，在河口入海处咸淡水混合区域繁殖，在暖流经过的外海过冬，具有洄游的习性。其肉质鲜美、细嫩，肉厚刺软，深受消费者喜爱，是一种具有较高商业价值但尚未大规模成功养殖的鱼类。Silver Pomfret (Pampus argenteus) belongs to Perciformes (Perciformes), Stromateoidei (Stromateoidei) Pomfret family (Stromateidae), Pomfret (Pampus). It spends the winter in the open sea where the warm current passes, and has the habit of migrating. Its meat is delicious, tender, thick and soft, and is deeply loved by consumers. It is a fish with high commercial value but has not yet been successfully cultured on a large scale.

自上世纪90年代以来，由于过度捕捞及海洋环境恶化，银鲳资源迅速衰退。进入本世纪后，国内学者对银鲳的资源分布及其变动作了许多研究，结果表明，目前银鲳资源量呈明显衰退，伏休制度也未能从根本上改变银鲳的资源现状。最近10余年，徐善良、施兆鸿等人开始了对银鲳工厂化养殖和繁育技术的探索，并于2011年取得重大突破，采用温度、光照综合诱导方法调控银鲳性腺发育成熟，使人工养殖的银鲳亲鱼达到自行产卵，获得了批量受精卵。2015年5月又在国内首次实现了全人工子一代规模化繁育，育苗成活率得到提高。但目前有关银鲳亲鱼性腺发育及产卵调控技术尚不稳定，易受病害、营养、水质等条件影响。因此，病害防治作为银鲳养殖的一个重要环节以提上了研究日程。Since the 1990s, due to overfishing and deterioration of the marine environment, silver pomfret resources have declined rapidly. After entering this century, domestic scholars have done a lot of research on the resource distribution and changes of silver pomfret. The results show that the current silver pomfret resources have declined significantly, and the Fuxiu system has not fundamentally changed the status quo of silver pomfret resources. In the past 10 years, Xu Shanshan, Shi Zhaohong and others began to explore the industrial breeding and breeding technology of silver pomfret, and made a major breakthrough in 2011, using a comprehensive induction method of temperature and light to regulate the maturation of silver pomfret gonads, so that the artificial breeding Silver pomfret broodstock achieved self-spawning and obtained batches of fertilized eggs. In May 2015, the large-scale breeding of the first generation of all artificial sons was realized for the first time in China, and the survival rate of seedlings was improved. However, at present, the gonad development and spawning regulation technology of silver pomfret broodstock is not stable, and is easily affected by diseases, nutrition, water quality and other conditions. Therefore, disease control has been put on the research agenda as an important part of silver pomfret breeding.

目前在水产养殖领域中，相比于革兰氏阴性菌，革兰氏阳性菌的数目较少，但其发病所造成的经济损失相对更大。而我国水产领域中常用的抗菌药基本上都是针对革兰氏阴性菌筛选的，所谓的广谱抗菌药物也是主要针对革兰氏阴性菌。因此，开发对革兰氏阳性菌具有抵抗效果的制品就成为水产领域中的研究热点之一。At present, in the field of aquaculture, compared with Gram-negative bacteria, the number of Gram-positive bacteria is small, but the economic loss caused by the disease is relatively greater. The antibacterial drugs commonly used in the aquatic industry in my country are basically screened against Gram-negative bacteria, and the so-called broad-spectrum antibacterial drugs are also mainly targeted at Gram-negative bacteria. Therefore, the development of products with resistance to Gram-positive bacteria has become one of the research hotspots in the field of aquatic products.

常见的水产领域中的革兰氏阳性菌病菌，例如诺卡氏菌，是一种典型的危害严重的慢性细菌病，多发于海水鱼和淡水养殖的黑鲈、大黄鱼等；近年来在我国南方如浙江、广东、福建等养殖区域都有发病的报道。发病的鱼会浮上水面，其反应迟钝、食欲下降，在鱼鳍的基部和腹部有溃烂出血，腹部肿大。腹腔内会有积水，肾脏和肝脏上由一颗颗乳白色的突起。诺卡氏菌潜伏期长，从鱼类感染至发病死亡需要15-20个月，发病前期无症状或症状不明显，发生死亡时已感染多时，病情持续时间长，发病率和死亡率都较高。Common Gram-positive bacterial pathogens in the field of aquatic products, such as Nocardia, are a typical chronic bacterial disease with serious hazards, which are mainly found in seawater fish and freshwater cultured black bass and large yellow croaker; There have been reports of disease in breeding areas such as Zhejiang, Guangdong, and Fujian in the south. Affected fish will float to the surface of the water, their reaction is slow, their appetite is decreased, there is festering and bleeding at the base of the fins and abdomen, and the abdomen is enlarged. There will be fluid in the abdominal cavity, and there are milky white protrusions on the kidneys and liver. Nocardia has a long incubation period. It takes 15-20 months from fish infection to onset and death. There are no symptoms or no obvious symptoms in the early stage of the disease. When death occurs, it has been infected for a long time. The disease lasts for a long time, and the morbidity and mortality are high. .

另外一种报道的革兰氏阳性菌的病菌为无乳链球菌，在鲻鱼和鲷鱼中都有发病的报道。Another reported gram-positive pathogen is Streptococcus agalactiae, which has been reported in both mullet and snapper.

在水产养殖病害防治技术领域中，抗生素作为一种对病原菌疗效显著的药物一直得到广泛应用。但随着抗生素的长期使用，大量耐药性菌株不断产生而导致抗生素疗效下降、剂量提高，同时抗生素的滥用情况不断加剧等问题也引起了人们的重视。鉴于新药开发的速度较慢，因此抗菌肽作为一种新型的抗生素替代物就成为了研究热点。In the technical field of aquaculture disease prevention and control, antibiotics have been widely used as a drug with significant curative effect on pathogenic bacteria. However, with the long-term use of antibiotics, a large number of drug-resistant strains continue to emerge, which leads to the decline in the efficacy of antibiotics and the increase in dosage. In view of the slow development of new drugs, antimicrobial peptides have become a research hotspot as a new type of antibiotic substitute.

在水产领域中，抗菌肽常被用作饲料添加剂来使用。但外源的抗菌肽常会因为生物不相容性使饲喂的动物产生应激反应；使养殖动物出现生长率降低等问题。因此，有必要筛选养殖动物自身来源的抗菌多肽，并对其结构、功能和作用机理进行分析就具有现实研究意义。In the field of aquaculture, antimicrobial peptides are often used as feed additives. However, exogenous antimicrobial peptides often cause stress reactions in fed animals due to bioincompatibility, and cause problems such as reduced growth rates in farmed animals. Therefore, it is necessary to screen the antibacterial peptides derived from the farmed animals themselves, and to analyze their structure, function and mechanism of action is of practical research significance.

发明内容Contents of the invention

本发明提供一种对革兰氏阳性菌具有抗性的多肽，该多肽对水产领域中常见的无乳链球菌、诺卡氏菌的抗菌效果，可与其它对革兰氏阴性菌有抗菌性的多肽进行联合使用。The invention provides a polypeptide with resistance to Gram-positive bacteria. The polypeptide has antibacterial effects on Streptococcus agalactiae and Nocardia common in the field of aquatic products, and can be combined with other antibacterial effects on Gram-negative bacteria. The peptides are used in combination.

本发明所提供的具有抗菌活性的多肽，其氨基酸序列为SEQ ID NO:1；The polypeptide with antibacterial activity provided by the present invention has an amino acid sequence of SEQ ID NO: 1;

编码上述多肽的基因，其核苷酸序列为SEQ ID NO:2；The gene encoding the above-mentioned polypeptide, its nucleotide sequence is SEQ ID NO: 2;

本发明的多肽用于制备抑制革兰氏阳性菌的制品；The polypeptide of the present invention is used to prepare products for inhibiting Gram-positive bacteria;

所述的制品为饲料添加剂；The product is a feed additive;

所述的饲料添加剂，为银鲳的人工饲料添加剂。The feed additive is an artificial feed additive for silver pomfret.

本发明通过水产领域中常用的革兰氏阳性微生物感染银鲳，通过消减杂交方法获得了对革兰氏阳性菌具有抗性的多肽，该可用作银鲳的饲料添加剂来使用。The present invention infects silver pomfret with gram-positive microorganisms commonly used in the field of aquatic products, and obtains a polypeptide with resistance to gram-positive bacteria through a subtractive hybridization method, which can be used as a feed additive for silver pomfret.

附图说明Description of drawings

图1：tester和driver的cDNA电泳图，其中M为1Kb DNA ladder，泳道1-3为tester3个样品，4-6为driver的三个样品；Figure 1: cDNA electrophoresis of tester and driver, where M is 1Kb DNA ladder, lanes 1-3 are three samples of tester, and lanes 4-6 are three samples of driver;

图2：抑制性消减杂交后，消减杂交产物的PCR扩增电泳图，其中M为100bp DNAmarker，1为tester扩增产物，2为消减杂交的扩增产物，3为driver的扩增产物；Figure 2: After suppression subtractive hybridization, PCR amplification electrophoresis of the subtractive hybridization product, where M is a 100bp DNA marker, 1 is the tester amplification product, 2 is the subtractive hybridization amplification product, and 3 is the driver amplification product;

图3：阳性克隆的电泳图；Figure 3: Electropherogram of positive clones;

图4：PA-G⁺-1多肽在抗菌肽数据库中的比较图。Figure 4: Comparison of PA-G⁺ -1 peptides in the antimicrobial peptide database.

具体实施方式detailed description

为了获得具有抗革兰氏阴性菌的抗菌肽，申请人通过对银鲳进行水产领域中致病革兰氏阳性菌注射感染；然后通过消减杂交方法获得目的抗菌肽，从而促成了本发明。In order to obtain antimicrobial peptides against Gram-negative bacteria, the applicant infected silver pomfret by injection with pathogenic Gram-positive bacteria in the field of aquatic products; and then obtained the target antimicrobial peptides by subtractive hybridization, thus contributing to the present invention.

实施例1：抗菌肽的筛选及检测Embodiment 1: Screening and detection of antimicrobial peptides

1、菌株活化1. Strain activation

1)蛳鱼诺卡氏菌(Nocardia seriolae)由宁波大学王国良教授提供，将蛳鱼诺卡氏菌接种到培养基中(葡萄糖0.02g/ml、酵母粉0.015g/ml、磷酸二氢钾0.75x10^-3g/ml、氯化钙0.2x10^-3g/ml、pH值为6.5)；然后在25℃下培养24h，然后3000rpm离心后获得菌沉淀，用无菌生理盐水稀释至蛳鱼诺卡氏菌浓度为1×10⁵cfu/mL的蛳鱼诺卡氏菌液备用；1) Nocardia seriolae was provided by Professor Wang Guoliang of Ningbo University. Nocardia seriolae was inoculated into the culture medium (glucose 0.02g/ml, yeast powder 0.015g/ml, potassium dihydrogen phosphate 0.75x10^-3 g/ml, calcium chloride 0.2x10^-3 g/ml, pH value 6.5); then cultivated at 25°C for 24h, then centrifuged at 3000rpm to obtain bacterial precipitate, diluted with sterile normal saline to The Nocardia spp. concentration is 1×10⁵ cfu/mL for later use;

2)将获得自中国水产科学研究院珠江水产研究所的无乳链球菌在血琼脂平板上进行活化，然后接种在脑心浸出液培养基(BHI)中扩大培养；30℃下培养24h，然后3000rpm离心后获得菌沉淀，用无菌生理盐水稀释至菌浓度为1×10⁵cfu/mL的无乳链球菌菌液备用；2) The Streptococcus agalactiae obtained from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences was activated on a blood agar plate, and then inoculated in the brain-heart infusion medium (BHI) for expansion; cultured at 30°C for 24h, then 3000rpm After centrifugation, the bacterial precipitate was obtained, and the Streptococcus agalactiae bacterial solution was diluted with sterile physiological saline to a bacterial concentration of 1×10⁵ cfu/mL for use;

分别取等体积的蛳鱼诺卡氏菌菌液和无乳链球菌菌液混合后作为实验用的混合菌液。Take equal volumes of Nocardia agalactiae bacteria solution and Streptococcus agalactiae bacteria solution respectively and mix them as the mixed bacteria solution for the experiment.

2、腹腔注射实验2. Intraperitoneal injection experiment

将饲养的银鲳进行腹腔注射蛳鱼诺卡氏菌菌和无乳链球菌的混合菌液作为tester，而腹腔注射等体积的无菌生理盐水作为driver；进行抑制性消减杂交来筛选银鲳对革兰氏阳性菌侵染后应激反应的过量表达或特异性表达的基因的cDNA。The reared silver pomfret were injected intraperitoneally with a mixed bacterial solution of Nocardia ichthyosa and Streptococcus agalactiae as a tester, and an equal volume of sterile saline was injected intraperitoneally as a driver; inhibitory subtractive hybridization was carried out to screen silver pomfret pairs cDNAs of genes overexpressed or specifically expressed in the stress response following infection with Gram-positive bacteria.

具体步骤如下：Specific steps are as follows:

1)选择体重范围0.53～288.78g，体长范围3.0～19.4cm、身体健康的银鲳作为实验鱼，注射时先将鱼的腹腔部位用酒精擦拭消毒,然后取一次性注射器排尽空气,吸取0.5ml的混合菌液,接着将针头沿腹鳍基部插入进行腹腔注射免疫；把注射好的鱼恢复活力再进行第二次注射；1) Choose a healthy silver pomfret with a body weight ranging from 0.53 to 288.78g and a body length ranging from 3.0 to 19.4cm as the experimental fish. When injecting, first wipe and disinfect the abdominal cavity of the fish with alcohol, then take a disposable syringe to exhaust the air, and inhale 0.5ml of mixed bacterial solution, and then insert the needle along the base of the pelvic fin for intraperitoneal injection of immunization; revive the injected fish and then perform the second injection;

其中tester组取3只银鲳鱼注射混合菌液；而driver组的3只银鲳鱼注射等量的无菌生理盐水；Among them, 3 silver pomfrets in the tester group were injected with the mixed bacterial solution; and 3 silver pomfrets in the driver group were injected with the same amount of sterile saline;

2)总RNA提取和mRNA纯化2) Total RNA extraction and mRNA purification

在第二次注射后5d后，取出tester(注射菌液)和driver(无菌生理盐水)的肝胰脏,然后用生理盐水把肝胰脏表面的血液清洗干净并放入无水乙醇中脱水,接着将肝胰脏剪成小块放入液氮中氮研磨后,以总RNA提取试剂盒Trizol(Invitrogen)的方法提取总RNA,以Qiagen公司的Oligotex mRNA kit分离纯化mRNA。将提取得到的总RNA和mRNA溶于一定体积的无RNase的水中。用GeneQuantII(Pharmacia Biotech)检测RNA质量和浓度。After 5 days after the second injection, take out the hepatopancreas of the tester (injected bacterial solution) and driver (sterile normal saline), then clean the blood on the surface of the hepatopancreas with normal saline and put them in absolute ethanol for dehydration , and then the liver and pancreas were cut into small pieces and put into liquid nitrogen for nitrogen grinding. The total RNA was extracted with the method of the total RNA extraction kit Trizol (Invitrogen), and the mRNA was isolated and purified with the Oligotex mRNA kit of Qiagen Company. Dissolve the extracted total RNA and mRNA in a certain volume of RNase-free water. RNA quality and concentration were detected with GeneQuantII (Pharmacia Biotech).

3)cDNA消减文库的构建3) Construction of cDNA subtractive library

分别以注射菌液的实验组cDNA混合液(包含三个个体，图1)为tester,注射生理盐水的对照组的cDNA混合液(包含三个个体)为driver。使用Clontech PCR-Select cDNASubtraction Kit，按照试剂盒的步骤进行抑制性消减杂交。将第二次PCR扩增后的正向消减产物(图2)进行纯化后，与pGEM-T载体连接,4℃过夜；用化学转化法转化感受态细胞TOP10(Tiangen),根据蓝白斑检测文库克隆的重组率,筛选出有插入片段的阳性克隆。The cDNA mixture of the experimental group injected with bacterial solution (including three individuals, Figure 1) was used as the tester, and the cDNA mixture of the control group injected with normal saline (including three individuals) was used as the driver. Using the Clontech PCR-Select cDNASubtraction Kit, follow the steps of the kit for suppression subtractive hybridization. Purify the positive subtraction product (Figure 2) after the second PCR amplification, connect it to the pGEM-T vector, and overnight at 4°C; transform the competent cell TOP10 (Tiangen) by chemical transformation, and detect the library according to the blue-white spot The recombination rate of the clones was used to screen out positive clones with inserts.

4)文库的测序与分析4) Sequencing and analysis of the library

随机挑选阳性克隆,用碱裂解法提取质粒，送生物公司测序，测序结果在GenBank(http://www.ncbi.nlm.nih.gov/blast)进行同源性比对。Positive clones were randomly selected, plasmids were extracted by alkaline lysis, and sent to Biological Company for sequencing. The sequencing results were compared for homology in GenBank (http://www.ncbi.nlm.nih.gov/blast).

在构建的消减cDNA文库中共挑取了620个克隆，对所有克隆进行PCR扩增，电泳结果显示插入片段大小主要分布在150-500bp之间(图3)。将PCR产物纯化后，剔除含有污染质粒和空质粒的克隆，获得有效克隆547个。A total of 620 clones were picked from the constructed subtractive cDNA library, and all clones were amplified by PCR. The results of electrophoresis showed that the size of the inserts was mainly distributed between 150-500bp (Fig. 3). After the PCR products were purified, clones containing contaminating plasmids and empty plasmids were eliminated, and 547 effective clones were obtained.

对547个克隆全部进行测序，序列的cDNA用SeqMan拼接后得到436个非冗余cDNA。考虑到抗菌肽通常是由小于50个氨基酸残基组成的多肽,分子量约为2000-5000道尔顿。因此，从银鲳抑制性消减杂交后获得的cDNA文库中选择片段大小为200bp或以下的46个cDNA片段进行定量PCR。检测结果表明，其中13个cDNA的表达量在混合菌液注射的个体中显著性上调。All 547 clones were sequenced, and the sequenced cDNAs were spliced with SeqMan to obtain 436 non-redundant cDNAs. Considering that antimicrobial peptides are usually polypeptides consisting of less than 50 amino acid residues, the molecular weight is about 2000-5000 Daltons. Therefore, 46 cDNA fragments with a fragment size of 200bp or less were selected from the cDNA library obtained after suppression subtractive hybridization in silver pomfret for quantitative PCR. The detection results showed that the expression levels of 13 cDNAs were significantly up-regulated in the individuals injected with the mixed bacterial solution.

对这13个基因编码的多肽的氨基酸序列输入到抗菌肽数据库(,APD http://aps.unmc.edu/AP/main.php)进行相关分析，对相似度高于30％的核苷酸片段进行进一步的抗菌性分析；通过微量肉汤稀释法获得具有对革兰氏阳性菌具有抗性的多肽。The amino acid sequences of the polypeptides encoded by these 13 genes were entered into the antimicrobial peptide database (APD http://aps.unmc.edu/AP/main.php) for correlation analysis, and the nucleotides with a similarity higher than 30% Fragments were further analyzed for antibacterial activity; the polypeptide with resistance to Gram-positive bacteria was obtained by micro broth dilution method.

最终获得的一条多肽命名为PA-G⁺-1，其氨基酸序列为The finally obtained polypeptide is named PA-G⁺ -1, and its amino acid sequence is

DSPETGATVVFNTTTVSPALPTSTDCMDTYNEISVFK(SEQ ID NO:1)；DSPETGATVVFNTTTVSPALPTSTDCMDTYNEISVFK (SEQ ID NO: 1);

编码上述多肽的核苷酸片段，其序列如下：The sequence of the nucleotide fragment encoding the above-mentioned polypeptide is as follows:

GACTCTCCTGAAACCGGCGCTACTGTCGTATTCAACACCACCACCGTCAGCCCCGCCCTACCGACAAGTACCGACTGCATGGACACATACAACGAAATCTCCGTGTTCAAG(SEQ ID NO:2)，GACTTCTCCTGAAACCGGCGCTACTGTCGTATTCAACACCACCGTCAGCCCCGCCCTACCGACAAGTACCGACTGCATGGACACATACAACGAAATCTCCGTGTTCAAG (SEQ ID NO: 2),

PA-G⁺-1多肽在抗菌肽数据库(antimicrobial peptide database,APD，http://aps.unmc.edu/AP/main.php)中与编号为AP02052的抗菌肽的相似度最高，为35.13％(图4)，表明其为从银鲳中分离的新型多肽。In the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php), the PA-G⁺ -1 polypeptide has the highest similarity with the antimicrobial peptide numbered AP02052, which is 35.13% (FIG. 4), indicating that it is a novel polypeptide isolated from silver pomfret.

PA-3多肽的氨基酸残基共有37个，具体如下：There are 37 amino acid residues in the PA-3 polypeptide, as follows:

D-S-P-E-T-G-A-T-V-V-F-N-T-T-T-V-S-P-A-L-P-T-S-T-D-C-M-D-T-Y-N-E-I-S-V-F-K；DSPETG-A -T-VVF -NTTT-V -SP-AL -PTSTD-CM -DTYNE-I -S-VF -K;

其中疏水性氨基残基(下划线标注)如下：I:1,V:4,L:1,F:2,C:1,M:1,A:2,W:0；Wherein the hydrophobic amino residues (underlined) are as follows: I:1, V:4, L:1, F:2, C:1, M:1, A:2, W:0;

阴离子残基如下：E:2,D:3；阴离子残基如下：K:1。The anionic residues are as follows: E:2, D:3; the anionic residues are as follows: K:1.

实施例2：PA-G⁺-1多肽的重组表达Embodiment 2: Recombinant expression of PA-G⁺ -1 polypeptide

1、在PA-G⁺-1多肽的核苷酸序列的(SEQ ID NO:1)连接入毕赤酵母表达载体中。将含抗菌肽基因的载体和酵母表达载体均用XhoI和XbaI双酶切，酶切产物回收并连接，进行PCR鉴定、测序。1. The nucleotide sequence of the PA-G⁺ -1 polypeptide (SEQ ID NO: 1) is linked into the expression vector of Pichia pastoris. Both the vector containing the antimicrobial peptide gene and the yeast expression vector were digested with XhoI and XbaI, and the digested products were recovered and ligated for PCR identification and sequencing.

2、阳性质粒经SacI单酶切线性化后加入毕赤酵母感受态细胞悬液中。电转化后均匀涂布于含100μg/mL Zeocin的YPDS选择平板上，30℃孵育3-5天。待YPDS平板上的阳性转化子生长较大，将各转化子依次点种至含Zeocin 200μg/mL、500μg/mL、1000μg/mL的YPDS选择平板，以在高浓度Zeocin平板上正常生长的菌落为可能高拷贝重组菌株。2. After the positive plasmid was linearized by SacI single enzyme digestion, it was added to the competent cell suspension of Pichia pastoris. After electroporation, spread evenly on YPDS selection plate containing 100 μg/mL Zeocin, and incubate at 30°C for 3-5 days. When the positive transformants on the YPDS plate grow larger, each transformant is inoculated onto the YPDS selection plate containing Zeocin 200 μg/mL, 500 μg/mL, and 1000 μg/mL in turn, and the colonies that grow normally on the high-concentration Zeocin plate are Possibly high copy recombinant strains.

3、将筛选到的阳性重组菌单菌落活化后按1％-10％接种量接种于三角瓶，28-30℃，200r/min摇床培养16-24h后以5％-20％接种量接入发酵罐中，温度28-30℃，转速500-1500r/min，培养基pH值5.0-6.0，通气量0.1-1.0VVM，溶氧>20％情况下进行发酵，在培养18-24h后流加50％甘油4h，待溶氧突然升至100％时流加甲醇至发酵结束，整个发酵持续48-72h。3. Activate the single colony of the positive recombinant bacteria screened and inoculate it into the Erlenmeyer flask with 1%-10% inoculum amount, and inoculate with 5%-20% inoculum amount after 16-24 hours at 28-30°C and 200r/min shaker. Put into the fermenter, the temperature is 28-30°C, the rotation speed is 500-1500r/min, the pH value of the medium is 5.0-6.0, the ventilation rate is 0.1-1.0VVM, and the dissolved oxygen is >20%. Add 50% glycerol for 4 hours, and when the dissolved oxygen suddenly rises to 100%, add methanol until the end of fermentation, and the whole fermentation lasts for 48-72 hours.

4、发酵结束后100℃灭菌10-20min，放料，5000r/min离心10min，收集发酵上清即为抗菌肽半成品。抗菌肽半成品经微滤、超滤、喷雾干燥、冻干等方式获得粉剂。4. After fermentation, sterilize at 100°C for 10-20 minutes, discharge, centrifuge at 5000r/min for 10 minutes, and collect the fermentation supernatant, which is the semi-finished antimicrobial peptide. Antimicrobial peptide semi-finished products are obtained by microfiltration, ultrafiltration, spray drying, freeze-drying and other methods to obtain powder.

5、取诱导表达72h的发酵上清，经0.22μm滤膜过滤，通过SP-Sepharose阳离子交换层析进行纯化，用15mmol/L的pH6.5的磷酸钠缓冲液平衡层析柱，流速为1.5m L/min；待柱体平衡好后进行上样，之后用20mmol/L磷酸钠缓冲液冲洗柱子，然后用1.0mol/LNaCl进行梯度洗脱，并收集洗脱峰。电泳结果表明纯化后的重组蛋白为一条带，通过Folin酚法测定蛋白质浓度。纯化的PA-G⁺-1多肽用于抗菌性分析。5. Take the fermentation supernatant of induced expression for 72 hours, filter it through a 0.22 μm filter membrane, purify it by SP-Sepharose cation exchange chromatography, and equilibrate the chromatography column with 15 mmol/L sodium phosphate buffer solution of pH 6.5 at a flow rate of 1.5 m L/min; load the sample after the column is well-balanced, then wash the column with 20mmol/L sodium phosphate buffer, then perform gradient elution with 1.0mol/L NaCl, and collect the elution peaks. The results of electrophoresis showed that the purified recombinant protein was a band, and the protein concentration was determined by the Folin phenol method. The purified PA-G⁺ -1 peptide was used for antibacterial analysis.

实施例3：PA-G⁺-1多肽的抗菌性检测Embodiment 3: Antibacterial detection of PA-G⁺ -1 polypeptide

检测PA-G⁺-1多肽对无乳链球菌、诺卡氏菌、副溶血弧菌和迟缓爱德华氏菌的抗菌性能；同时使用氨基酸序列为SQGVKPSINVGSYSLATIIQDLL(SEQ ID NO:3)的pa1-1多肽作为对照。Detect the antibacterial properties of the PA-G⁺ -1 polypeptide against Streptococcus agalactiae, Nocardia, Vibrio parahaemolyticus and Edwardsiella lentus; simultaneously use the pa1-1 polypeptide whose amino acid sequence is SQGVKPSINVGSYSLATIIQDLL (SEQ ID NO: 3) as comparison.

具体步骤如下：Specific steps are as follows:

1、菌株的活化处理：通过在固体培养基上划线培养，获得无乳链球菌、诺卡氏菌、副溶血弧菌和迟缓爱德华氏菌的纯化单菌落。分别挑选于25ml液体LB培养基中扩大培养，将培养后的菌液稀释成浓度为5×10⁵CFU/mL，依次取60μl加入到96孔板的各孔内准备进行实验。1. Activation treatment of bacterial strains: Obtain purified single colonies of Streptococcus agalactiae, Nocardia, Vibrio parahaemolyticus and Edwardsiella tarda by streak culture on solid medium. Select and expand culture in 25ml liquid LB medium respectively, dilute the cultured bacterial solution to a concentration of 5×10⁵ CFU/mL, and add 60 μl to each well of a 96-well plate in order to prepare for the experiment.

2、将重组表达的PA-G⁺-1多肽和pa1-1多肽定量后，用液体培养基依次做倍比稀释。将稀释好的抗菌肽各取40μl依次加入到96孔板的各个孔中，此时每个孔的反应体系为100μl。96孔板盖盖后，28℃振荡培养24h后，观察并用酶标仪在600nm处测量OD值并记录实验结果。以抑制细菌生长的最小浓度来定义最小抑菌浓度(MIC)。利用发酵的菌液和没有发酵的液体培养基分别用来做阴性和阳性对照，各自代表抑菌率为0和100％(表1)。2. After the recombinantly expressed PA-G⁺ -1 polypeptide and pa1-1 polypeptide are quantified, they are sequentially diluted in liquid medium. 40 μl of the diluted antimicrobial peptides were sequentially added to each well of the 96-well plate, and the reaction system in each well was 100 μl at this time. After the 96-well plate was covered and cultured with shaking at 28°C for 24 hours, observe and measure the OD value at 600nm with a microplate reader and record the experimental results. The minimum inhibitory concentration (MIC) is defined as the minimum concentration that inhibits bacterial growth. The fermented bacterial liquid and the non-fermented liquid culture medium were used as negative and positive controls respectively, each representing a bacteriostatic rate of 0 and 100% (Table 1).

表1：抗菌肽对多种水产致病菌的最低抑菌浓度Table 1: Minimum inhibitory concentrations of antimicrobial peptides against various aquatic pathogens

结果表明筛选的PA-G⁺-1多肽对无乳链球菌、诺卡氏菌具有明显的抑菌能力，而对于革兰氏阴性菌的副溶血弧菌和迟缓爱德华氏菌抑菌效果很差。因此，本发明所筛选的PA-G⁺-1多肽与其它抗革兰氏阴性菌的多肽联合使用作为银鲳饲料的添加剂来使用。The results show that the screened PA-G⁺ -1 polypeptide has obvious antibacterial ability to Streptococcus agalactiae and Nocardia, but has poor antibacterial effect on Gram-negative bacteria Vibrio parahaemolyticus and Edwardsiella tarda . Therefore, the PA-G⁺ -1 polypeptide screened in the present invention is used in combination with other polypeptides against Gram-negative bacteria as an additive for silver pomfret feed.

序列表sequence listing

<110> 宁波大学<110> Ningbo University

<120> 一种对革兰氏阳性菌具有抗性的多肽<120> A polypeptide resistant to Gram-positive bacteria

<160> 3<160> 3

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 37<211> 37

<212> PRT<212> PRT

<213> 银鲳(Pampus argenteus)<213> Silver Pomfret (Pampus argenteus)

<400> 1<400> 1

Asp Ser Pro Glu Thr Gly Ala Thr Val Val Phe Asn Thr Thr Thr ValAsp Ser Pro Glu Thr Gly Ala Thr Val Val Phe Asn Thr Thr Thr Val

1 5 10 151 5 10 15

Ser Pro Ala Leu Pro Thr Ser Thr Asp Cys Met Asp Thr Tyr Asn GluSer Pro Ala Leu Pro Thr Ser Thr Asp Cys Met Asp Thr Tyr Asn Glu

20 25 30 20 25 30

Ile Ser Val Phe LysIle Ser Val Phe Lys

35 35

<210> 2<210> 2

<211> 111<211> 111

<212> DNA<212>DNA

<213> 银鲳(Pampus argenteus)<213> Silver Pomfret (Pampus argenteus)

<400> 2<400> 2

gactctcctg aaaccggcgc tactgtcgta ttcaacacca ccaccgtcag ccccgcccta 60gactctcctg aaaccggcgc tactgtcgta ttcaacacca ccaccgtcag ccccgcccta 60

ccgacaagta ccgactgcat ggacacatac aacgaaatct ccgtgttcaa g 111ccgacaagta ccgactgcat ggacacatac aacgaaatct ccgtgttcaa g 111

<210> 3<210> 3

<211> 23<211> 23

<212> PRT<212> PRT

<213> 银鲳(Pampus argenteus)<213> Silver Pomfret (Pampus argenteus)

<400> 3<400> 3

Ser Gln Gly Val Lys Pro Ser Ile Asn Val Gly Ser Tyr Ser Leu AlaSer Gln Gly Val Lys Pro Ser Ile Asn Val Gly Ser Tyr Ser Leu Ala

1 5 10 151 5 10 15

Thr Ile Ile Gln Asp Leu LeuThr Ile Ile Gln Asp Leu Leu

20 20

Claims (6)

1. a kind of polypeptide with antibacterial activity, the amino acid sequence of described polypeptide is SEQ ID NO:1.

2. encode the nucleic acid fragment of the polypeptide described in claim 1.

3. nucleic acid fragment as claimed in claim 2, it is characterised in that the sequence of described nucleic acid fragment is SEQ ID NO:1.

4. application of the polypeptide in the product for suppressing gram-positive bacteria is prepared described in claim 1.

5. application as claimed in claim 4, it is characterised in that the antibacterial product is feed addictive.

6. application as claimed in claim 5, it is characterised in that described feed addictive, be that the man-made feeds of silvery pomfret addAgent.