WO2007033529A1 - Gène codant pour une protéine antibactérienne de fenneropenaeus chinensis, procédé d'expression et utilisations des recombinants - Google Patents

Gène codant pour une protéine antibactérienne de fenneropenaeus chinensis, procédé d'expression et utilisations des recombinants Download PDF

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Publication number
WO2007033529A1
WO2007033529A1 PCT/CN2005/001560 CN2005001560W WO2007033529A1 WO 2007033529 A1 WO2007033529 A1 WO 2007033529A1 CN 2005001560 W CN2005001560 W CN 2005001560W WO 2007033529 A1 WO2007033529 A1 WO 2007033529A1
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Prior art keywords
protein
gene
antibacterial protein
recombinant
antibacterial
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PCT/CN2005/001560
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English (en)
Chinese (zh)
Inventor
Jianhai Xiang
Jiquan Zhang
Huhua Li
Zaizhao Wang
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Institute Of Oceanology Chinese Academy Of Sciences
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Publication of WO2007033529A1 publication Critical patent/WO2007033529A1/fr

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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
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the antimicrobial peptide (protein) is a small molecule polypeptide (protein) encoded by animal and plant cell specific genes, and has antibacterial activity.
  • Swedish scientist Boman et al. Swedish scientist Boman et al.
  • 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.
  • crustin 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.
  • the aim is to provide an antibacterial protein gene of Chinese prawn and its high efficient recombinant expression and application.
  • Chinese prawn antibacterial protein gene has the nucleotide sequence of SEQ ID No. 1 and the amino acid sequence of SEQ ID No. 2.
  • 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:
  • the target gene was cloned from the blood cell cDNA library using RACE technology
  • 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;
  • the constructed recombinant expression vector was transformed into Escherichia coli BL21 (DE3) pLysS, and the engineered bacteria were screened and induced to express;
  • ⁇ Inducing agent isopropyl- ⁇ -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;
  • PBS renaturation Buffer solution
  • oxidized glutathione reduced glutathione
  • the pair of specific primers are:
  • CD-F 5, CAG AAT AAA GAC GAT ACT CG 3 ';
  • CD-R 5
  • CTA TCC CTC AGA ACC CAG 3
  • CTA TCC CTC AGA ACC CAG 3
  • 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 -
  • 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.
  • 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.
  • 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.
  • AAAAAAAAAAAAAA Information of SEQ ID No. 2 (see sequence listing)
  • 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:
  • the target gene was cloned from the blood cell cDNA library using RACE technology
  • 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.
  • the recombinant expression system can efficiently express antibacterial proteins with an expression efficiency of 845 mg/L.
  • 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.
  • 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 ⁇ g/tnL of ampicillin and 34 ⁇ g/mL of chloramphenicol. disturb 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.
  • IPTG inducer isopropyl - e-D-thiogalactoside
  • 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.
  • 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.
  • 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.
  • the protein after removal of the fusion tag was concentrated to 64 ⁇ mol/L while using 50 mmol/L pH 7.4 PBS as a negative control.
  • 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!

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Insects & Arthropods (AREA)
  • Oncology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un gène codant pour une protéine antibactérienne de Fenneropenaeus chinensis, le procédé d'expression des recombinants et leurs utilisations. Le gène possède la séquence de bases de SEQ ID NO: 1 et il code pour la protéine ayant la séquence d'acides aminés de SEQ ID NO : 2. On utilise un vecteur pCR®T7/NT TOPO®TA et une bactérie hôte, BL21 (DE3) pLysS, système d'expression procaryote capable d'exprimer la protéine antibactérienne in vitro. La protéine recombinante peut être exprimée par la bactérie hôte avec un rendement élevé. L'inclusion de plusieurs liaisons disulfure dans la protéine recombinante permet d'utiliser un système d'oxydoréduction consistant en glutathion oxydant et glutathion réducteur pour l'isolement, la purification et la renaturation de la protéine exprimée. Il est ainsi possible d'obtenir une protéine recombinante à activité antibiotique avec un rendement élevé d'expression et de renaturation de la protéine recombinante. Une vérification in vitro de la fonction biologique de la protéine recombinante a confirmé qu'elle possédait un puissant effet inhibiteur sur les bactéries Gram positif de même qu'un certain effet inhibiteur sur les bactéries Gram négatif.
PCT/CN2005/001560 2005-09-19 2005-09-23 Gène codant pour une protéine antibactérienne de fenneropenaeus chinensis, procédé d'expression et utilisations des recombinants WO2007033529A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNB2005100472287A CN100485036C (zh) 2005-09-19 2005-09-19 一种中国明对虾抗菌蛋白基因及重组表达和应用
CN200510047228.7 2005-09-19

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CN117327706A (zh) * 2023-10-13 2024-01-02 江苏三仪生物工程有限公司 一种用于提高水产抗病毒和免疫能力的组合物及其应用

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CN103539845B (zh) * 2013-11-01 2016-05-11 中国科学院海洋研究所 一种环状的合成多肽e及其抗病毒应用
CN103539846B (zh) * 2013-11-01 2016-05-11 中国科学院海洋研究所 一种环状的合成多肽f及其抗菌抗病毒应用
CN103539849B (zh) * 2013-11-01 2016-08-24 中国科学院海洋研究所 一种环状的合成多肽d及其抗菌抗病毒应用
CN105255900A (zh) * 2015-09-30 2016-01-20 中国科学院海洋研究所 一种改造的中国明对虾抗菌蛋白基因ALFm的重组表达及其应用
CN107201372B (zh) * 2017-04-28 2020-12-15 中国水产科学研究院南海水产研究所 斑节对虾过氧化物还原酶1编码基因序列及其用途
CN109627310B (zh) * 2019-01-10 2021-12-10 汕头大学 一种字纹弓蟹抗菌蛋白提取方法及其食品保鲜应用

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