WO2007033529A1

WO2007033529A1 - A gene encoding antibacterial protein of fenneropenaeus chinensis and its recombination expression method and uses

Info

Publication number: WO2007033529A1
Application number: PCT/CN2005/001560
Authority: WO
Inventors: Jianhai Xiang; Jiquan Zhang; Huhua Li; Zaizhao Wang
Original assignee: Institute Of Oceanology Chinese Academy Of Sciences
Priority date: 2005-09-19
Filing date: 2005-09-23
Publication date: 2007-03-29
Also published as: CN100485036C; CN1936000A

Abstract

A gene encoding antibacterial protein of Fenneropenaeus chinensis, its recombination expression method and uses are disclosed. The gene has the base sequence of SEQ ID No. 1 and encodes the protein having the amino acid sequence shown in SEQ ID No. 2. A vector pCR®T7/NT TOPO®TA recombination and host bacterium BL21 (DE3) pLysS which can express the antibacterial protein in vitro using prokaryotic expression system are obtained. The recombination protein can be expressed in high efficiency by the host bacterium. On the basis of the characteristic of inclusion of several disulfide bonds in the recombination protein, oxidation-reduction system consisting of oxidative glutathion and reductive glutathion is used in the isolation, purification and renaturation of the expressed protein. Thus a recombination protein having antibiotic activity is acquired. The high efficiency expression and renaturation of the recombination protein can be achieved. In vitro verification of biological function of the recombination protein confirmed that it has strong inhibiting effect on gram-positive bacteria and has some inhibiting effect on gram-negative bacteria as well.

Description

一种中国明对虾抗菌蛋白基因及重组表达和应用技术领域 Antibacterial protein gene of Chinese prawn and its recombinant expression and application

本发明涉及基因工程领域中抗菌蛋白（crustin)基因的表达，具体地说是一种中国明对虾抗菌蛋白基因及重组表达和应用。背景技术 The invention relates to the expression of a crustin gene in the field of genetic engineering, in particular to an antibacterial protein gene of Chinese prawn and its recombinant expression and application. Background technique

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。二十世纪九十年代以来，疾病的大规模爆发使海水养殖业蒙受了巨大的损失，抗生素的滥用与环境的恶化使得海水养殖动物的病害难以从根本上得到控制。 Shrimp culture is one of the pillar industries of marine aquaculture. As the market demand for high quality aquatic products continues to grow, shrimp farming plays an increasingly important role in aquaculture. Since the 1990s, the large-scale outbreak of disease has caused huge losses to the marine aquaculture industry. The abuse of antibiotics and the deterioration of the environment have made it difficult to control the diseases of marine aquaculture animals.

海水养殖品种生活于富含各种微生物的水环境中，长期的环境压力使其形成了有效的防御功能。抗菌肽（蛋白）被认为是鱼、虾、贝等防御***的主要成分之一。通过研究海水养殖品种的抗菌肽基因的结构和功能，进而通过基因工程技术在体外进行重组表达，将有希望成为能够替代抗生素的新型药物。 Marine aquaculture species live in a water environment rich in various microorganisms, and long-term environmental pressures make them effective defense functions. Antibacterial peptides (proteins) are considered to be one of the main components of defense systems such as fish, shrimp, and shellfish. By studying the structure and function of the antimicrobial peptide gene of marine aquaculture species, and then performing recombinant expression in vitro through genetic engineering technology, it will hopefully become a new drug that can replace antibiotics.

免疫防御体系是动物抗病力的基础，特别是低等无脊椎动物，它们的免疫反应要比高等动物简单得多，主要依赖于内源的非特异性免疫反应。近几年的研究表明，低等无脊椎动物在保护自身免受外源有害微生物（如细菌、真菌或病毒）伤害时，其机体中的非特异性抗菌因子如抗菌肽（蛋白）、溶菌酶、转铁蛋白等起到非常重要的作用。其中，抗菌肽（蛋白）是由动物和植物细胞特定基因编码产生的一类小分子多肽 (蛋白），具有抗菌活性。 20世纪 70年代中期，瑞典科学家 Boman等在用 £ coli 诱导惜古比天蚕 Hyatophora cecropia) 时分离得到了世界上第一种抗菌肽一天蚕素（_CeCropin)。此后，人们相继在其它昆虫、两栖动物、哺乳动物、人、植 1、甲壳动物、软体动物等生物体内都发现存在类似具有抗菌活性的多肽（蛋白）。 crustin 是最早由 Relf 等从滨蟹 Carcinus imems) 体内分离纯化得到的一种具有很强抑制革兰氏阳性菌活性的小分子蛋白，其自身不易失活，甚至在被煮沸后仍能保持很高活性 (J. M. Relf, J. R. S. Chisholm, G. D. Kemp and V. J. Smith, Purification and characterization of a cysteine- rich 11. 5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur. J. Biochem. 1999 ; 264 (2) : 350- 357)。此后，人们先后从凡纳滨对虾、白滨对虾、日本囊对虾和斑节对虾等虾类中发现了 crustin或抗菌蛋白类似物.（crustin- like )基因的存在。 The immune defense system is the basis of animal disease resistance, especially low invertebrates. Their immune response is much simpler than that of higher animals, mainly relying on endogenous non-specific immune responses. Studies in recent years have shown that low-level invertebrates protect themselves from harmful microorganisms (such as bacteria, fungi or viruses), and non-specific antibacterial factors such as antimicrobial peptides (proteins), lysozyme, Transferrin and the like play a very important role. Among them, the antimicrobial peptide (protein) is a small molecule polypeptide (protein) encoded by animal and plant cell specific genes, and has antibacterial activity. In the mid-1970s, Swedish scientist Boman et al. isolated the world's first antimicrobial peptide, CeC ropin, when using £ coli to induce _Hyatophora cecropia. Since then, people have found similar polypeptides (proteins) with antibacterial activity in other organisms such as insects, amphibians, mammals, humans, plants 1, crustaceans, and mollusks. Crustin is a small molecule protein that has been strongly isolated and purified from Relf et al. from Carcinus imems. It is not easy to inactivate itself and remains high even after being boiled. Activity (JM Relf, JRS Chisholm, GD Kemp and VJ Smith, Purification and characterization of a cysteine-rich 11. 5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur. J. Biochem. 1999; 264 (2) : 350-357). Since then, people have discovered the presence of crustin or antibacterial protein analogs (crustin-like) from shrimps such as vannamei, white prawn, prawn and prawns.

crustin的作用机理与传统抗生素不同，它的靶位点主要是病原体细胞膜，因此不易产生耐药性。目前，许多病原菌对现有抗生素逐步产生耐药性，而新型抗生素的发现又极其困难，因此， crustin的研究为开发新型抗菌药物开辟了广阔的前景。 The mechanism of action of crustin is different from that of traditional antibiotics. Its target site is mainly the pathogen cell membrane, so it is not easy to produce drug resistance. At present, many pathogens gradually develop resistance to existing antibiotics, and the discovery of new antibiotics is extremely difficult. Therefore, crustin research has opened up broad prospects for the development of new antibacterial drugs.

总之， crustin以及其他类型抗菌肽（蛋白）的研究不仅具有重要的理论意义， In short, the study of crustin and other types of antimicrobial peptides (proteins) is not only of great theoretical significance,

1 1

确认本在实践上也具有巨大的应用潜力，因此，它已成为当前生物医药研究的热点之一发明内容 Confirmation It also has great application potential in practice. Therefore, it has become one of the hotspots of current biomedical research.

目的在于提供一种中国明对虾抗菌蛋白基因及其高效重组表达和应用。 The aim is to provide an antibacterial protein gene of Chinese prawn and its high efficient recombinant expression and application.

本发明技术方案如下： The technical scheme of the present invention is as follows:

中国明对虾抗菌蛋白基因：具有序列表 SEQ ID No. 1中的碱基序列及 SEQ ID No. 2 所示的氨基酸序列。 Chinese prawn antibacterial protein gene: has the nucleotide sequence of SEQ ID No. 1 and the amino acid sequence of SEQ ID No. 2.

所述中国明对虾抗菌蛋白基因的重组表达：从中.国明对虾 cDNA文库中克隆到一种抗菌蛋白基因，利用其成熟肽基因构建原核表达载体，筛选出一种用于高效表达中国明对虾抗菌蛋白基因的重组表达体系，获得含二硫键的原核表达重组蛋白高效复性的缓冲液***，从而获得有活性的重组抗菌蛋白；具体为： Recombinant expression of the antibacterial protein gene of the Chinese prawn: An antibacterial protein gene was cloned from the Chinese prawn cDNA library, and the mature peptide gene was used to construct the prokaryotic expression vector, and a high-efficiency expression of the antibacterial protein of P. chinensis was screened. The recombinant expression system of the gene obtains a buffer system for efficiently reconstituting the recombinant protein containing the disulfide bond, thereby obtaining an active recombinant antibacterial protein; specifically:

1. 基因克隆 Gene cloning

根据中国明对虾 EST的提示，利用 RACE技术，从血细胞 cDNA文库中克隆目的基因； According to the EST of Chinese prawn EST, the target gene was cloned from the blood cell cDNA library using RACE technology;

2. 重组表达载体及宿主菌的选择 2. Selection of recombinant expression vector and host bacteria

确定以包含体形式进行重组表达的表达载体 pCIf T7/NT T0P0* TA及能够缓解目的基因表达产物毒性的宿主菌 BL21 (DE3 ) pLysS; The expression vector pCIf T7/NT T0P0* TA which is recombinantly expressed in the form of inclusion body and the host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the target gene expression product are determined;

3. 重组表达载体的构建 ' 3. Construction of recombinant expression vector '

利用表达载体为 T载体的特征，采用一对特异性引物克隆成熟肽基因， PCR产物回收后直接与表达进行连接，构建重组表达载体； Using the expression vector as the characteristic of the T vector, a pair of specific primers were used to clone the mature peptide gene, and the PCR product was directly recovered and ligated to form a recombinant expression vector;

4. 转化与筛选 4. Conversion and screening

将构建得到的重组表达载体转化大肠杆菌 BL21 (DE3 ) pLysS, 筛选工程菌并进行诱导表达； The constructed recombinant expression vector was transformed into Escherichia coli BL21 (DE3) pLysS, and the engineered bacteria were screened and induced to express;

5. 表达产物的分离纯化 5. Isolation and purification of the expressed product

釆用诱导剂异丙基 - β -D-硫代半乳糖苷诱导表达工程菌，超声波破碎细胞，利用金属螯合柱层析的方法纯化抗菌蛋白，蛋白复性，融合标签切除，获得有活性的重组抗菌蛋白；表达 Inducing agent isopropyl-β-D-thiogalactoside induced expression of engineering bacteria, ultrasonic disruption of cells, purification of antibacterial proteins by metal chelate column chromatography, protein renaturation, fusion tag excision, obtained active Recombinant antibacterial protein;

所述蛋白复性采用含二硫键的原核表达重组蛋白高效复性的缓冲液***，在复性缓冲溶液中添加氧化型谷胱甘肽和还原型谷胱甘肽；其浓度比：复性缓冲溶液（PBS) : 氧化型谷胱甘肽：还原型谷胱甘肽 =50: 1： 10；复性缓冲溶液 pH值为 7. 2~7. 4；所述一对特异性引物为： The protein renaturation uses a disulfide-containing prokaryotic expression recombinant protein to efficiently refold the buffer system, and oxidized glutathione and reduced glutathione are added to the renaturation buffer solution; concentration ratio: renaturation Buffer solution (PBS): oxidized glutathione: reduced glutathione = 50: 1: 10; renaturation buffer solution pH 7. 2~7. 4; The pair of specific primers are:

CD-F: 5， CAG AAT AAA GAC GAT ACT CG 3 ' ； CD-F: 5, CAG AAT AAA GAC GAT ACT CG 3 ';

CD-R: 5， CTA TCC CTC AGA ACC CAG 3，。 CD-R: 5, CTA TCC CTC AGA ACC CAG 3, .

所述中国明对虾抗菌蛋白基因对革兰氏阳性菌和革兰氏阴性菌的生长均具有抑制作用。本发明有如下优点-The antibacterial protein gene of the Chinese prawn has an inhibitory effect on the growth of Gram-positive bacteria and Gram-negative bacteria. The invention has the following advantages -

1. 本发明确定了一种中国明对虾抗菌蛋白基因的全部编码序列和非编码序列，从而也弄清了它所编码的氨基酸序列及其信号肽切割位点，利用所推导的成熟肽氨基酸序列对该基因进行重组表达。 1. The present invention determines the entire coding sequence and non-coding sequence of an antibacterial protein gene of P. chinensis, thereby clarifying the amino acid sequence and its signal peptide cleavage site, and using the deduced amino acid sequence of the mature peptide. The gene is expressed recombinantly.

2. 本发明筛选出能有效地进行抗菌蛋 '白重组表达的载体及宿主菌，对研究其他具有抗菌功能蛋白的重组表达提供了依据。 2. The present invention screens vectors and host bacteria which can effectively carry out recombinant expression of antibacterial egg white, and provides a basis for studying recombinant expression of other antibacterial functional proteins.

3. 本发明对含有二硫键的重组蛋白的分离纯化及蛋白质复性提供了一个合适的氧化还原体系，可以有效的保证重组蛋白中二硫键的正确形成。 3. The present invention provides a suitable redox system for the separation and purification of recombinant proteins containing disulfide bonds and protein renaturation, which can effectively ensure the correct formation of disulfide bonds in recombinant proteins.

4. 意义深远。通过本发明可开发有效的对虾细菌性疾病的防治药物同时，对其他具有抗菌活性蛋白的重组表达及易于氧化的重组蛋白的分离纯化的研究提供了新的思路。具体实施方式 4. Meaningful. Through the present invention, an effective drug for preventing and treating bacterial diseases of shrimp can be developed. At the same time, new ideas for the separation and purification of recombinant proteins with antibacterial activity and easily oxidized proteins are provided. detailed description

下面结合实施例对本发明作进一步详细说明。 The present invention will be further described in detail below with reference to the embodiments.

实施例 Example

、一种克隆的中国明对虾抗菌蛋白基因，具有如下序列： , a cloned antibacterial protein gene of Chinese prawn, having the following sequence:

(1) SEQ ID No 1 的信息（参见序列表） (1) Information of SEQ ID No 1 (see sequence listing)

' (a) 序列特征： ' (a) Sequence characteristics:

*长度： 523碱基对 *Length: 523 base pairs

*类型：核酸 *Type: Nucleic acid

*链型：双链 *chain type: double chain

*拓扑结构：线性 *Topology: Linear

(b ) 分子类型： cDNA (b) Molecular type: cDNA

( c ) 假设：否 (c) Assumption: No

( d) 反义：否 (d) Antisense: No

( e ) 最初来源：中国明对虫下 { Fenneropenaeus chinensis (e) Original source: Chinese Phyllostachys { Fenneropenaeus chinensis

序列描述： SEQ ID No. 1 Sequence Description: SEQ ID No. 1

AAAAAAAAAAAA (2) SEQ ID No. 2的信息 (参见序列表) AAAAAAAAAAAA (2) Information of SEQ ID No. 2 (see sequence listing)

(a) 序列特征 (a) Sequence characteristics

*长度： 134氨基酸 *Length: 134 amino acids

*类型：氨基酸 *Type: Amino acid

*链型：单链 *Chain type: single chain

*拓扑结构：线性 *Topology: Linear

(b) 分子类型：蛋白序列描述： SEQ ID No. 2

(b) Molecular type: Protein sequence description: SEQ ID No. 2

EG 中国明对虾基因的体外重组表达、分离纯化及生物活性分析： Recombinant expression, isolation, purification and biological activity analysis of EG Chinese prawn gene:

从中国明对虾 cDNA文库中筛选到一种抗菌蛋白（crustin)基因，利用其成熟肽基因构建原核表达载体，筛选出一种用于高效表达中国明对虾抗菌蛋白基因的重组表达体系，找到了一种用于含二硫键的原核表达重组蛋白高效复性的缓冲液***，获得了有活性的重组抗菌蛋白。具体如下： A crustin gene was screened from the cDNA library of P. chinensis, and the prokaryotic expression vector was constructed by using the mature peptide gene to screen a recombinant expression system for efficient expression of the antibacterial protein gene of P. chinensis. A buffer system for the efficient refolding of prokaryotic expression recombinant proteins containing disulfide bonds, and an active recombinant antibacterial protein is obtained. details as follows:

1 )基因克隆 1) Gene cloning

2)重组表达载体及宿主菌的选择 2) Selection of recombinant expression vector and host bacteria

由于目的基因的重组表达产物具有较强的抑菌活性，本发明对目前几种商业化的表达载体及宿主菌进行筛选，并确定了以包含体形式进行重组表达的表达载体 pCR⁸ T7/NT T0P0* TA及能够缓解目的基因表达产物毒性的宿主菌 BL21 (DE3) pLysS。 Since the recombinant expression product of the target gene has strong antibacterial activity, the present invention screens several commercial expression vectors and host bacteria, and determines the expression vector pCR ⁸ T7/NT which is recombinantly expressed in the form of inclusion bodies. T0P0* TA and host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the gene expression product of interest.

所述重组表达体系可以高效地表达抗菌蛋白，表达效率达 845 mg/L。 The recombinant expression system can efficiently express antibacterial proteins with an expression efficiency of 845 mg/L.

3) 重组表达载体的构建 3) Construction of recombinant expression vector

利用表达载体为 T载体的特征，设计一对特异性引物（CD- F: 5' CAG AAT AAA GAC GAT ACT CG 3' ； CD-R: 5， CTA TCC CTC AGA ACC CAG 3， ) 克隆成熟肽基因， PCR 产物回收后直接与表达进行连接，构建重组表达载体。 Using the expression vector as a feature of the T vector, a pair of specific primers (CD-F: 5' CAG AAT AAA GAC GAT ACT CG 3'; CD-R: 5, CTA TCC CTC AGA ACC CAG 3, ) was designed to clone the mature peptide. The gene, PCR product is recovered and directly ligated to the expression to construct a recombinant expression vector.

4)诱导表达及诱导产物的筛选 4) Induction of expression and screening of induced products

将重组表达载体转化表达宿主菌 BL21 (DE3) pLysS, 在终浓度为 lOO y g/tnL的氨苄青霉素与 34 μ g/mL氯霉素的 LB培养基中培养菌体浓度至 0D₆。„为 0. 6左右（37°C， 250 r/min), 添加终浓度为 lmmol/L IPTG进行诱导表达 4h; 然后采用诱导剂异丙基 - e - D-硫代半乳糖苷（简称 IPTG)筛选诱导表达工程菌。 The recombinant expression vector was transformed into the expression host strain BL21 (DE3) pLysS, and the cell concentration was cultured to 0D ₆ in LB medium at a final concentration of 100 μg/tnL of ampicillin and 34 μg/mL of chloramphenicol. „ is about 0.6 (37 ° C, 250 r / min), adding a final concentration of lmmol / L IPTG for induction of expression for 4h; then using the inducer isopropyl - e-D-thiogalactoside (IPTG) was screened to induce expression of engineered bacteria.

5) 分离纯化 5) Separation and purification

诱导结束后，超声波破碎细胞，利用金属螯合柱层析的方法纯化抗菌蛋白，蛋白复性具体是：离心收集菌泥，称量湿重，按 1： 10加入 buffer A (50ramol/L磷酸钠， 6mol/L盐酸胍， 300醒 ol/L氯化钠， lmmol/L氧化型谷胱甘肽， 10mmol/L还原型谷胱甘肽， PH7. 2), 超声波破碎， 4。C， 12, OOOr/min下离心 30min，收集上清。利用 Co - NTA- Agarose在变性条件下进行分离纯化。分离纯化后的重组蛋白，依次在含有 8mol/L、 4raol/L、 2mol/L、 lmol/L及 0. 5mol/L尿素的 50mmol/L PBS缓冲液（lmmol/L 氧化型谷胱甘肽， lOmraol/L还原型谷胱甘肽， pH7. 4)中透析各 8h，将裂解液中的盐酸胍替换出来，在 50mmol/L pH7. 4 PBS缓冲液平衡的 Sephadex G- 75层析柱去除尿素，收集洗脱液。 After the end of induction, the cells were disrupted by ultrasonication, and the antibacterial protein was purified by metal chelate column chromatography. The protein refolding was as follows: Centrifugal collection of the bacterial sludge, weighing the wet weight, adding buffer A (10ramol/L sodium phosphate) at 1:10 , 6mol / L guanidine hydrochloride, 300 awake ol / L sodium chloride, lmmol / L oxidized glutathione, 10mmol / L reduced glutathione, PH7. 2), ultrasonic disruption, 4. Centrifuge for 30 min at C, 12, OOOr/min and collect the supernatant. Separation and purification were carried out under denaturing conditions using Co-NTA-Agarose. The purified recombinant protein was isolated in 50 mmol/L PBS buffer (1 mmol/L oxidized glutathione) containing 8 mol/L, 4 raol/L, 2 mol/L, 1 mol/L and 0.5 mol/L urea. lOmraol/L reduced glutathione, pH 7. 4) dialysis for 8 h, replace guanidine hydrochloride in the lysate, remove urea in Sephadex G-75 column equilibrated in 50 mmol/L pH7.4 buffer , collect the eluent.

6) 酶切去除融合标签 6) Enzymatic removal of fusion labels

将洗脱液的蛋白浓度稀释至 2mg/mL，利用 Irwitrogen公司的 EKMax™试剂盒进行融合标签的切除（25 ， 24 h) 。 The protein concentration of the eluate was diluted to 2 mg/mL, and the fusion label was excised (25, 24 h) using Irwitrogen's EKMaxTM kit.

7) 抑菌实验 7) Antibacterial experiment

将去除融合标签后的蛋白浓缩至 64 μ mol/L，同时以 50mmol/L pH7. 4的 PBS为阴性对照。利用牛津杯琼脂扩散法检测其对革兰氏阳性菌金黄色葡萄球菌 ( Staphylococcus aureii、枯草芽孢杆菌 ( Bacillus subtil is^) 、藤黄微球菌 {Micrococcus luteus) 与革兰氏阴性菌大肠杆菌 ί Escherichia coif) , 鳗弧菌' ( Vibrio anguillarum) 的抑菌活性。 SP : 将待检测菌株分别于 37 °C， 200r/niin下培养过夜；次日离心菌体并用灭菌的 50mmol/L pH7. 4的 PBS洗涤菌体 2次，并利用 PBS稀释菌体至10 6113/1！^，取 100 μ ΐ均勾涂布于 LB平板表面，于 37°C培养箱中培养 2h后，取出平板在琼脂表面等距离放置牛津杯，向牛津杯中添加不同浓度梯度的重组蛮白，同时以 PBS作为阴性对照， 37°C培养箱中继续培养 22h，取出观察结果。 The protein after removal of the fusion tag was concentrated to 64 μmol/L while using 50 mmol/L pH 7.4 PBS as a negative control. Using the Oxford Cup agar diffusion method to detect Gram-positive bacteria Staphylococcus aureii, Bacillus subtil is^, Micrococcus luteus and Gram-negative Escherichia coli ί Escherichia Coif), Vibrio anguillarum's antibacterial activity. SP: The strain to be tested was cultured overnight at 37 ° C, 200 r/niin; the cells were centrifuged the next day and the cells were washed twice with sterilized 50 mmol/L pH 7.4 PBS, and the cells were diluted with PBS to 10 6113/1! ^, 100 μ ΐ was applied to the surface of the LB plate, and after culturing for 2 hours in a 37 ° C incubator, the plate was taken out and placed on the agar surface at an equal distance to the Oxford Cup, and the recombinant white of different concentration gradients was added to the Oxford Cup. At the same time, PBS was used as a negative control, and culture was continued for 22 hours in a 37 ° C incubator, and the observation results were taken out.

结果表明 4 mol/L的重组蛋白能有效地抑制所检测的革兰氏阳性菌（金黄色葡萄球菌、枯草芽孢杆菌与藤黄微球菌）的生长， 16 mol/L的重组蛋白能有效地抑制所检测的革兰氏阴性菌（大肠杆菌与鳗弧菌）的生长。 The results showed that 4 mol/L recombinant protein could effectively inhibit the growth of Gram-positive bacteria (S. aureus, Bacillus subtilis and Micrococcus luteus), and 16 mol/L recombinant protein could effectively inhibit Growth of Gram-negative bacteria (E. coli and Vibrio anguillarum) detected.

该基因的成功克隆与重组蛋白生物活性的鉴定，在新型抗菌药物的开发上具有重要应用前景。 The successful cloning of this gene and the identification of the biological activity of recombinant proteins have important application prospects in the development of new antibacterial drugs.

Claims

权利要求书 Claim

1. 一种中国明对虾抗菌蛋白基因，其特征在于：具有序列表 SEQ ID No. 1中的碱基序列及 SEQ ID No. 2所示的氨基酸序列。 An antibacterial protein gene of P. chinensis having the nucleotide sequence of SEQ ID No. 1 of the Sequence Listing and the amino acid sequence of SEQ ID No. 2.

2.一种按照权利要求 1所述中国明对虾抗菌蛋白基因的重组表达，其特征在于: 从中国明对虾 cDNA文库中克隆到一种抗菌蛋白基因，利用其成熟肽基因构建原核表达载体，筛选出一种用于高效表达中国明对虾抗菌蛋白基因的重组表达体系，获得含二硫键的原核表达重组蛋白高效复性的缓冲液***，从而获得有活性的重组抗菌蛋白；具体为： 2. A recombinant expression of an antibacterial protein gene of the Chinese prawn according to claim 1, wherein: an antibacterial protein gene is cloned from a cDNA library of P. chinensis, and a prokaryotic expression vector is constructed using the mature peptide gene to screen A recombinant expression system for efficiently expressing the antibacterial protein gene of P. chinensis is obtained, and a buffer system for efficiently reconstituting the prokaryotic expression recombinant protein containing disulfide bond is obtained, thereby obtaining an active recombinant antibacterial protein; specifically:

1 ) 基因克隆 1) Gene cloning

根据中国明对虾 EST的提示，利用 RACE技术，从血细胞 cDNA文库中克隆目的基 According to the EST of Chinese prawn EST, the RACE technology was used to clone the target base from the blood cell cDNA library.

'因； 'cause;

2) 重组表达载体及宿主菌的选择 2) Selection of recombinant expression vector and host bacteria

确定以包含体形式进行重组表达的表达载体 pCR^e T7/NT Τ0Ρ0^β ΤΑ及能够缓解目的基因表达产物毒性的宿主菌 BL21 (DE3) pLysS; The expression vector pCR ^e T7/NT Τ0Ρ0 ^β ΤΑ which is recombinantly expressed in the form of inclusion body and the host strain BL21 (DE3) pLysS capable of alleviating the toxicity of the expression product of the target gene are determined;

3)重组表达载体的构建 3) Construction of recombinant expression vector

4) 转化与筛选 4) Conversion and screening

5 )表达产物的分离纯化 5) Separation and purification of expression products

采用诱导剂异丙基- 硫代半乳糖苷诱导表达工程菌，超声波破碎细胞，利用金属螯合柱层析的方法纯化抗菌蛋白，蛋白复性，融合标签切除，获得有活性的重组抗菌蛋白。 The engineered bacteria were induced by the inducer isopropyl-thiogalactoside, the cells were disrupted by ultrasonic wave, and the antibacterial protein was purified by metal chelate column chromatography. The protein was renatured and the fusion tag was excised to obtain the active recombinant antibacterial protein.

3，按照权利要求 2所述中国明对虾抗菌蛋白基因的重组表达，其特征在于：所述蛋白复性采用含二硫键的原核表达重组蛋白髙效复性的缓冲液***，在复性缓冲溶液中添加氧化型谷胱甘肽和还原型谷胱甘肽 ·，其浓度比：复性缓冲溶液：氧化型谷胱甘肽：还原型谷胱甘肽 =50: 1： 10; 复性缓冲溶液 pH值为 7. 2~7. 4。 3. The recombinant expression of the antibacterial protein gene of the Chinese prawn according to claim 2, wherein the protein is renatured using a disulfide-containing prokaryotic expression recombinant protein, a refolding buffer system, in a renaturation buffer Oxidized glutathione and reduced glutathione are added to the solution at a concentration ratio: renaturation buffer solution: oxidized glutathione: reduced glutathione = 50: 1: 10; renaturation buffer 5〜7. 4。 The pH of the solution is 7. 2~7. 4.

4. 按照权利要求 2所述中国明对虾抗菌蛋白基因的重组表达，其特征在于：所述一对特异性引物为： 4. The recombinant expression of the antibacterial protein gene of the Chinese prawn according to claim 2, wherein: the pair of specific primers are:

CD-F: 5， CAG AAT AAA GAC GAT ACT CG 3，； CD-F: 5, CAG AAT AAA GAC GAT ACT CG 3, ;

CD-R: 5' CTA TCC CTC AGA ACC CAG 3 ' 。 CD-R: 5' CTA TCC CTC AGA ACC CAG 3 '.

5. 一种按照权利要求 1所述中国明对虾抗菌蛋白基因的应用，其特征在于：对革兰氏阳性菌和革兰氏阴性菌的生长均具有抑制作用。 An application of the antibacterial protein gene of the Chinese prawn according to claim 1, characterized in that it has an inhibitory effect on the growth of Gram-positive bacteria and Gram-negative bacteria.