CN110066330B - Apostichopus japonicus glucan binding protein and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the fields of molecular biology and genetic engineering technology, and particularly discloses apostichopus japonicus selenka glucan binding protein and a preparation method and application thereof. The apostichopus japonicus glucan binding protein AJ-GBP is obtained for the first time by using an in-vitro recombinant expression technology, and the apostichopus japonicus glucan binding protein (AJ-GBP) is preferably shown as an amino acid sequence in a sequence table SEQ ID NO. 1. The recombinant protein has agglutination effect on bacillus subtilis, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, brevibacterium luteum and vibrio anguillarum, inhibition effect on bacillus subtilis, escherichia coli, brevibacterium luteum and vibrio anguillarum, and potential application value in the aspects of development of novel broad-spectrum antibacterial drugs, feed additives and the like.

Description

A kind of Apostichopus japonicus glucan binding protein and preparation method and application thereof

technical field

The invention belongs to the technical field of molecular biology and genetic engineering, and in particular relates to a glucan-binding protein of Apostichopus japonicus and a preparation method and application thereof.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Apostichopus japonicus belongs to Echinodermata, Holothuroidea, Aspidochirotida, Stichopodidae. It is known as "ginseng in the sea" and has high nutritional value. and medicinal value. It is widely distributed in the sea areas near Shandong, Liaoning and Hebei in my country. In recent years, with the improvement of people's social living conditions and the enhancement of health awareness, the market demand for Apostichopus japonicus has increased year by year, and the scale of aquaculture has been continuously stimulated. However, the problems of diseases encountered in the breeding process have always been an important restrictive factor that plagues the further development of the industry. The chemical drugs used, such as chlorine dioxide, potassium permanganate, formalin and antibiotics, have certain toxic effects on Apostichopus japonicus to varying degrees. Therefore, the use of biotechnology to prevent and control diseases has attracted more and more attention.

Natural antibacterial substances widely exist in animals and plants. At present, the antibacterial substances isolated from animals and plants mainly include alkaloids, terpenes, flavonoids, polyphenols, polysaccharides, organic acids, polypeptides, proteins, etc. Different active ingredients have different structural compositions and different antibacterial and bacteriostatic mechanisms. . Among them, antibacterial protein is a kind of macromolecular protein with broad-spectrum antibacterial, fungal, virus, protozoal, anti-tumor cell and other activities, which is encoded by specific genes of various biological cells and induced by external conditions. However, the inventors found that there are currently more studies on small-molecule antimicrobial peptides that are easier to synthesize, but less research on macromolecular antimicrobial proteins. In particular, there are few reports on the preparation of related antimicrobial proteins based on the imitation of sea cucumber, which is not conducive to imitation. The quality and safety of sea cucumber products and the healthy development of related food industries.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a pseudo-Apostichopus japonicus glucan binding protein and a preparation method and application thereof. Through the research on the β-1,3-glucan-binding protein of Apostichopus japonicus, it provides ideas for finding new strategies for disease control and breeding of Apostichopus japonicus, which is conducive to promoting the health and development of the aquaculture industry of Apostichopus japonicus in my country. sustainable development.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

The first aspect of the present invention provides a pseudo-Apostichopus japonicus glucan-binding protein, the amino acid sequence of the pseudo-Apostichopus japonicus glucan-binding protein AJ-GBP and SEQ ID NO. ) sequence homology; more preferably, the amino acid sequence of the AJ-GBP-like Apostichopus japonicus glucan binding protein is SEQ ID NO.1.

The second aspect of the present invention provides a DNA molecule encoding the above amino acid sequence.

Further, the present invention provides a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 having the nucleotide sequence shown in SEQ ID NO.2, or having at least 90% sequence identity with SEQ ID NO.2 source, and can express the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.1.

The third aspect of the present invention provides a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium containing a DNA molecule encoding the amino acid sequence of the Apostichopus japonicus glucan-binding protein.

The fourth aspect of the present invention provides the application of the DNA sequence, the recombinant expression vector, the transgenic cell system or the transgenic recombinant bacteria in the preparation of the A. japonicus glucan-binding protein AJ-GBP.

The fifth aspect of the present invention provides a preparation method of glucan-binding protein AJ-GBP, comprising the following steps:

Using Apostichopus japonicus cDNA as a template, PCR amplification was carried out with primers F1 and R1;

After purification, the PCR product was ligated with the E. coli expression vector pEASY-E2 vector, the ligated product was transformed into E. coli, and the recombinant was identified by sequencing to obtain the expression vector pEASY-AJ-GBP;

The above-mentioned expression vector pEASY-AJ-GBP was transferred into Trans BL21 (DE3) pLysS chemically competent cells, and the transformants were screened. The transformants were inoculated in LB medium containing ampicillin, and the expression was induced with IPTG, and then the ultrafiltration tube was used. The recombinant protein was purified to obtain the glucan-binding protein AJ-GBP.

The sixth aspect of the present invention provides the application of the above-mentioned Apostichopus japonicus glucan binding protein, wherein the AJ-GBP imitation sea cucumber glucan binding protein is used to prepare a broad-spectrum antibacterial drug preparation or a feed additive.

Further, the Apostichopus japonicus glucan-binding protein is used to prepare pharmaceutical preparations or feed additives against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Brevibacterium luteus and Vibrio eel.

The present invention has the following beneficial technical effects:

The present invention uses the in vitro recombinant expression technology to obtain the AJ-GBP imitation sea cucumber glucan binding protein for the first time. It is verified by experiments that the recombinant protein has obvious inhibitory effect on Gram-positive bacteria and Gram-negative bacteria. Specifically, The recombinant protein has agglutination effect on Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus luteus and Vibrio eel, and inhibits Bacillus subtilis, Escherichia coli, Bacillus luteus and Vibrio eel Therefore, it has potential application value in the development of new broad-spectrum antibacterial drugs and feed additives.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

Fig. 1 is the protein expression map detected by SDS-PAGE in Example 1 of the present invention, 1 is MARKER1, the molecular weight is 100kDa, 60kDa, 45kDa, 28kDa, 18kDa in descending order, 2 is MAKER2, the molecular weight is from large to small The sequence is 97.2kDa, 66.4kDa, 44.3kDa, 29kDa, 20.1kDa, 14.3kDa, the 3rd lane is the protein in the supernatant after the induced expression cells are broken, the 4th lane is the protein of the uninduced cell, and the 5th lane is the inclusion body after the induced expression The purified protein was dissolved, lane 6 was the inclusion body protein after induction and expression, lanes 7, 8, 9, and 10 were the first, second, third, and fourth filtrates in the ultrafiltration process, respectively.

Fig. 2 is a graph of the agglutination effect of the pseudo-Apostichopus japonicus glucan-binding protein on bacteria provided in Example 2 of the present invention, wherein Fig. 2(1) is Bacillus subtilis, Fig. 2(2) is Staphylococcus aureus, Fig. 2 ( 3) is B. luteus, Fig. 2(4) is Vibrio eel, Fig. 2(5) is Escherichia coli, and Fig. 2(6) is Pseudomonas aeruginosa.

Fig. 3 is a graph showing the antibacterial activity of the glucan-binding protein of Apostichopus japonicus provided in Example 3 of the present invention, wherein Fig. 3(1) is a growth curve diagram of Bacillus subtilis, and Fig. 3(2) is a growth curve diagram of Bacillus luteus , Figure 3 (3) is a growth curve of Staphylococcus aureus, Figure 3 (4) is a growth curve of Vibrio eel, Figure 3 (5) is a growth curve of Escherichia coli, Figure 3 (6) is a growth curve of Pseudomonas aeruginosa Graph.

Fig. 4 is a graph showing the bacterial binding activity of the glucan-binding protein of Apostichopus japonicus provided in Example 4 of the present invention, wherein 4A is the protein bound by bacterial cells, and Fig. 4B is the protein of the fourth washing solution; 1 is MAKER, 2 is Escherichia coli, 3 is Bacillus subtilis, 4 is Staphylococcus aureus, 5 is Vibrio eel, 6 is Bacillus luteus, and 7 is Pseudomonas aeruginosa.

5 is a graph of the glucan-binding activity of the Apostichopus japonicus glucan-binding protein provided in Example 5 of the present invention.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

As mentioned above, there are few research results on the preparation of related antibacterial proteins based on Apostichopus japonicus, so it is not conducive to the quality and safety of Apostichopus japonicus products and the healthy development of related food industries.

The study found that β-1,3-glucan-binding proteins (β-1,3-glucan-binding proteins, referred to as GBP), also known as β-1,3-glucan recognition proteins (beta-1,3- glucan recognition protein, GRP for short), and lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) belong to different subtypes of the same gene. GBP has the functions of recognizing non-hexyl substances, activating the prophenoloxidase system, activating the coagulation cascade system, and activating the degranulation of blood cells leading to the release of a series of cytotoxic factors/cytolytic factors/antibacterial factors. However, there is no report on the antibacterial effect of Apostichopus japonicus glucan-binding protein. Therefore, it is of great theoretical and practical significance to explore the functional genes of Apostichopus japonicus and prepare biological antibacterial drugs, which will help to solve the problem of antibiotic resistance in current breeding.

In view of this, in a specific embodiment of the present invention, there is provided a pseudo-Apostichopus japonicus glucan-binding protein, and the amino acid sequence of the pseudo-Apostichopus japonicus glucan-binding protein AJ-GBP has 90% of the amino acid sequence of SEQ ID NO.1 The above sequences are homologous; more preferably, the amino acid sequence of the AJ-GBP-like Apostichopus japonicus glucan-binding protein is SEQ ID NO.1.

In yet another specific embodiment of the present invention, a DNA molecule having the above-mentioned amino acid sequence is provided. Due to the degeneracy of codons, there can be a wide variety of nucleotide sequences capable of encoding the glucan-binding proteins of the present invention. As for the DNA molecule encoding the amino acid sequence of the Apostichopus japonicus glucan-binding protein of the present invention, those skilled in the art can easily manufacture and synthesize it using the existing well-known methods. For example, by selecting codons corresponding to the amino acid residues constituting the designed amino acid sequence, a DNA molecule corresponding to the amino acid sequence of the Apostichopus japonicus glucan-binding protein can be easily determined and provided.

In yet another specific embodiment of the present invention, there is provided a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 having the nucleotide sequence shown in SEQ ID NO.2, or having at least 90% homology, and can express the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.1.

It should be noted that the term "homologous" mainly refers to homology in sequence, that is, it is used to indicate that two or more protein or DNA sequences have the same ancestor. Homologous sequences generally have similar functions. The homology of protein and DNA is often determined by the similarity of their sequences. Similarity refers to the ratio of the same DNA base or amino acid residue sequence between the test sequence and the target sequence in the sequence alignment process. .

In another specific embodiment of the present invention, a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacteria containing a DNA molecule encoding the amino acid sequence of the Apostichopus japonicus glucan-binding protein is provided.

The recombinant expression vector is a recombinant expression vector for expressing the AJ-GBP-like Apostichopus japonicus glucan binding protein obtained by inserting the DNA sequence into the E. coli expression vector.

The E. coli expression vector is pEASY-E2.

In yet another specific embodiment of the present invention, a transgenic recombinant bacterium is provided, and the recombinant bacterium is obtained by introducing the recombinant expression vector into Escherichia coli, and selecting the transgenic recombinant bacterium.

The application of the DNA sequence, the recombinant expression vector, the transgenic cell system or the transgenic recombinant bacteria in the preparation of the AJ-GBP imitation sea cucumber glucan binding protein.

In another specific embodiment of the present invention, there is provided a preparation method of dextran-binding protein AJ-GBP, comprising the following steps:

(1) Using the Apostichopus japonicus cDNA as a template, PCR amplification was performed with primers F1 and R1, and it was set aside;

(2) After the PCR product is purified, it is connected with the E. coli expression vector pEASY-E2 vector, the ligated product is transformed into E. coli, and the recombinant is identified by sequencing;

(3) The above-mentioned expression vector pEASY-AJ-GBP was transferred into Trans BL21 (DE3) pLysS chemically competent cells, and the transformants were screened. The recombinant protein was purified by ultrafiltration to obtain the dextran-binding protein AJ-GBP.

The primers are:

F1: 5'-ATGACCGCCGGTGGTGGAGG-3' (SEQ ID NO. 3);

R1: 5'-GCACCAGGATCGACGCTCAAG-3' (SEQ ID NO. 4).

In another specific embodiment of the present invention, the application of the Apostichopus japonicus glucan binding protein is provided, and the AJ-GBP imitation sea cucumber glucan binding protein is used to prepare a broad-spectrum antibacterial drug preparation or a feed additive.

In another specific embodiment of the present invention, the Apostichopus japonicus glucan-binding protein is provided for the preparation of anti-Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Brevibacterium luteus and Vibrio eel. Pharmaceutical preparations or feed additives.

The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

Example 1: Prokaryotic recombinant expression of AJ-GBP coding region of Apostichopus japonicus

Specific steps are as follows:

1. Construction of prokaryotic recombinant expression vector

The present invention adopts the pEASY-E2 prokaryotic expression vector of Beijing TransGen Biotech Co., Ltd. The entire coding region of AJ-GBP was amplified by PCR with specific primers. The PCR reaction program was set as: ①94①pre-denaturation for 4min; ②94①denaturation for 30s; ③52①renaturation for 30s; ④72①extension for 1min; Perform steps ②③④ for 40 cycles. The PCR product was purified and recovered, and ligated with pEASY-E2 vector. The ligation product was transformed into Escherichia coli Trans BL21(DE3)pLysS, and 5 to 10 single colonies were selected and inoculated in LB liquid medium, and sent to a sequencing company for sequencing to verify the correctness of the reading frame. The specific primers were:

F1: 5'-ATGACCGCCGGTGGTGGAGG-3';

R1: 5'-GCACCAGGATCGACGCTCAAG-3'.

Expression of recombinant protein: Inoculate the correct strain in 50 mL of LB liquid medium, place it in a shaking incubator, cultivate at 37①200 rpm for 12 to 16 hours, and then inoculate this culture medium in a ratio of 1:100 to the new LB medium. In 500mL liquid medium, 37① was cultured to OD ₆₀₀ =0.6. IPTG was added to make the final concentration 0.8mM, and cultured at 37① for 5h. The cells were collected and washed with PBS buffer for 3 times and then resuspended. After the cells were disrupted, the cells were centrifuged at 10,000g for 20 min, and the precipitated inclusion body protein was washed twice with Buffer A (5mM EDTA, 50mM Tris-HCL), Buffer B (5mM EDTA, 50mM Tris-HCL) , 2M urea) was washed twice, dissolved in Buffer C (10mM Tris-HCL, 0.1M NaH ₂ PO ₄ , 8M urea), filtered with a 0.45 μm filter membrane, and purified using a Millipore 10kd ultrafiltration centrifuge tube to obtain the following The protein with the amino acid sequence shown in SEQ ID NO.1.

>AJ-GBP-aa

MTAGGGGNWEFQYYTNNRSNSYVRDNTLYIKPTLTSEKEGEAFLTSGTLNLWGASPADLCTGNNWWGCERTGSFNNILNPIQSARLRTVNSFAFKHGRIEVFAQLPKGDWLWPAIWLLPKRNAYGGWPASGEIDLVESRGNRNLRAADGTHVGAEQVGMTLHWGPYWPLNGYPMTHNAKNLPDGQTFGDGFHNYTLVWTADSLDFYLDGEPILERRSWC

Sequence features:

Length: 219 amino acids

Type: Amino Acid

Chain type: single chain

Topology: Line Type

Features: Features: The molecular weight is 24.64kDa, and the isoelectric point is 5.94. Has two protein kinase C phosphorylation sites (S36EK, S83AR), three casein kinase II phosphorylation sites (S36EKE, S55PAD, T186FGD), one β-1,3-glucanase site (W127- E132-I133-D134), a β-1,3-glycan linkage recognition motif (FHNYTLVWTADSLDFYLDG), a polysaccharide binding motif (VFAQLPKGDWLWPAIWLLP) and a glucanase motif (WPASGEIDLVES). In addition, AJ-GBP does not have the cell adhesion site RGD, which is unique to shrimp β-GRP, while KGD is found at position 107 of the amino acid sequence.

Source: Imitation sea cucumber

Example 2: Analysis of bacterial agglutination activity of prokaryotic recombinant protein imitating the AJ-GBP coding region of Apostichopus japonicus

Against Bacillus subtilis, Staphylococcusaureus, Curtobacterium luteum (G ⁺ bacteria) and Vibrio anguillarum, Escherichia coli, Pseudomonas aeruginosa) (G ^- bacteria) Agglutination Experiment

Inoculate Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus luteus into LB liquid medium, 37① to cultivate to OD ₆₀₀ value to 0.6, Vibrio eel is cultivated to OD with TCBS medium at 28① ₆₀₀ value to 0.6, then diluted with sterile saline to an OD ₆₀₀ value of 0.001, then centrifuged at 5000 rpm for 10 minutes to collect the bacterial pellet, washed three times with TBS buffer and resuspended to adjust the bacterial concentration to 2 × 10 ⁹ cells /mL. 75 μL of recombinant protein (about 600 μg/ml) were added to 96-well plates, then 30 μL of bacterial solution was added, and incubated at room temperature for 1 h. In the negative control group, the recombinant protein was replaced with bovine serum albumin (BSA). The results showed that the recombinant protein could agglutinate the above-mentioned six test bacteria.

Experiments showed that the recombinant AJ-GBP protein could effectively agglutinate gram-positive and gram-negative bacteria.

Example 3: Antibacterial activity analysis of AJ-GBP prokaryotic recombinant protein from Apostichopus japonicus

Growth inhibition experiments on Staphylococcus aureus, Bacillus subtilis, Bacillus luteus (G ⁺ bacteria) and Vibrio eel, Escherichia coli, Pseudomonas aeruginosa (G- bacteria ⁾

Bacillus subtilis, Staphylococcus aureus, Bacillus luteus, Pseudomonas aeruginosa and Escherichia coli were inoculated into LB liquid medium, cultivated at 37① to an OD ₆₀₀ value of 0.6, and Vibrio eel was inoculated into TCBS medium at 28① and cultured to OD ₆₀₀ value to 0.6, diluted 20 times with fresh medium before use. Add 100 μl of bacterial solution and 100 μl of recombinant protein to each well of the 96-well cell culture plate and mix well, set the culture temperature to 37 ① After culturing for 3 hours, record the OD ₆₀₀ value with a microplate reader every 30 minutes, and reach the growth platform in about 7-8 hours. During the period, BSA was set as the control group, and the OD ₆₀₀ values were calculated to draw the growth curves of different groups, and three parallels were set for each group.

Experiments show that the recombinant AJ-GBP protein can inhibit gram-negative bacteria, and also has inhibitory effect on gram-positive bacteria, but the inhibitory effect on Staphylococcus aureus and Pseudomonas aeruginosa is not obvious.

Example 4: Analysis of Bacterial Binding Activity of Prokaryotic Recombinant Protein of AJ-GBP Coding Region of Apostichopus japonicus

Binding experiments to Staphylococcus aureus, Bacillus subtilis, Bacillus luteus (G ⁺ bacteria) and Vibrio eel, Escherichia coli, Pseudomonas aeruginosa (G- bacteria ⁾

Bacillus subtilis, Staphylococcus aureus, Bacillus luteus, Pseudomonas aeruginosa and Escherichia coli were inoculated into LB liquid medium, cultivated at 37① to an OD ₆₀₀ value of 0.6, and Vibrio eel was inoculated into TCBS medium at 28① and cultured to The OD ₆₀₀ value reached 0.6, and then the bacterial pellet was collected by centrifugation at 5000 rpm for 10 minutes, washed three times with TBS buffer, and then resuspended to adjust the bacterial concentration to 3.5×10 ⁸ cells/mL. Take 200uL of bacterial suspension and mix with 200uL of recombinant protein (about 600μg/ml), and incubate with shaking at room temperature for 30min. Centrifuge at 5000 rpm for 10 minutes and discard the supernatant to collect the bacteria. After washing the bacteria for 4 times with TBS, the fourth washing solution and bacteria were added to the protein loading buffer in a boiling water bath for 10 min. After the supernatant was collected by centrifugation, SDS-PAGE and Western blotting detects the target band.

Experiments show that the recombinant AJ-GBP protein has obvious binding activity to Gram-negative bacteria and Gram-positive bacteria; and the recombinant AJ-GBP protein can effectively inhibit Gram-positive bacteria and Gram-negative bacteria. effect.

Example 5: Dextran, lipopolysaccharide and peptidoglycan binding activity analysis of prokaryotic recombinant protein imitating the AJ-GBP coding region of Apostichopus japonicus

Add 50 μl of 80 μl/ml dextran, lipopolysaccharide, and peptidoglycan to a high-affinity 96-well plate, 37① to dry overnight; 60① to incubate for 30 min the next day; 200 μl of 5% skimmed milk powder was added to each well. PBS, block at 37① for 2h; pour off the blocking solution, add 200 μl of PBST (20% Tween-20:PBS=1:100) to each well for four washes, 5 min each time; add 50 μl of protein (protein containing 1% Skim milk powder was diluted twice in PBS, a total of six groups of proteins; the control group used BSA); the protein was discarded, and 200 μl of PBST (20% Tween-20:PBS=1:100) was added to each well to wash four times, 5 min each time ; Dilute the primary antibody (rat anti-His-tag antibody) in PBS containing 1% nonfat milk powder at a ratio of 1:300, add 100 μl to each well, and react at room temperature for 2 hours; discard the primary antibody and add 200 μl to each well PBST (20% Tween-20:PBS=1:100) for four times, 5min each time; the horseradish peroxidase-labeled rabbit anti-rat secondary antibody was diluted 1:3000 in 1% degreasing solution In the PBS of milk powder, add 100 μl to each well, and react at room temperature for 2 h; discard the secondary antibody, add 200 μl of PBST (20% Tween-20:PBS=1:100) to each well and wash four times, 5 min each time; use Shanghai The color development was carried out with a Sanko EL-TMB color development kit, and the absorbance value was read at 450 nm on a microplate reader.

Experiments show that recombinant AJ-GBP protein has obvious binding activity to glucan, and the binding strength is proportional to the protein concentration, but has no obvious binding activity to lipopolysaccharide and peptidoglycan.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments. Those skilled in the art should understand that on the basis of the technical solutions of the present invention, those skilled in the art do not need to Various modifications or deformations that can be made with creative work are still within the protection scope of the present invention.

SEQUENCE LISTING

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Met Thr Ala Gly Gly Gly Gly Asn Trp Glu Phe Gln Tyr Tyr Thr Asn

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Arg Ala Ala Asp Gly Thr His Val Gly Ala Glu Gln Val Gly Met Thr

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Asn Ala Lys Asn Leu Pro Asp Gly Gln Thr Phe Gly Asp Gly Phe His

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gaggcatttt taaccagtgg aacattgaac ctgtggggag cgtcacctgc tgacctctgt 180

accggaaaca attggtgggg ttgcgagaga accggaagtt tcaacaacat tctaaacccc 240

atacagtccg ccagactcag gacagttaac tctttcgctt tcaagcatgg acgaattgaa 300

gtctttgctc agttgcccaa aggagattgg ctttggccag caatttggct tctacccaaa 360

cgcaatgctt atggtggatg gcccgcatcc ggagagattg atctggttga atccagagga 420

aaccgaaact tgagagctgc agatggtacc cacgtcggag ctgagcaagt cggaatgacc 480

ttacattggg gaccctactg gcctctgaac ggctatccca tgacccataa tgccaagaac 540

ctaccagacg gacagacatt tggcgatggt ttccataact acacacttgt ctggactgct 600

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Claims (14)

1. An imitation Apostichopus japonicus glucan binding protein, characterized in that the amino acid sequence of the AJ-GBP imitation sea cucumber glucan binding protein is SEQ ID NO.1. 2. A DNA molecule encoding the amino acid sequence of the Apostichopus japonicus glucan-binding protein according to claim 1. 3. The DNA molecule of claim 2, wherein the DNA molecule is the nucleotide sequence shown in SEQ ID NO.2. 4. A recombinant expression vector or transgenic cell system comprising the DNA molecule of claim 2 or 3. 5. The transgenic recombinant bacteria containing the DNA molecule of claim 2 or 3. 6. The recombinant expression vector of claim 4, wherein the DNA molecule is inserted into the Escherichia coli expression vector and expresses the recombinant expression vector of AJ-GBP imitating Apostichopus japonicus glucan binding protein, wherein , the E. coli expression vector is pEASY-E2. 7. The transgenic recombinant bacterium according to claim 5, wherein the transgenic recombinant bacterium is obtained by introducing the recombinant expression vector into Escherichia coli for screening. 8. The application of the DNA molecule according to claim 2 in the preparation of AJ-GBP-like Apostichopus japonicus glucan-binding protein. 9. The application of the recombinant expression vector of claim 4 in the preparation of AJ-GBP imitation sea cucumber glucan binding protein. 10. The application of the transgenic cell system according to claim 4 in the preparation of AJ-GBP-like Apostichopus japonicus glucan-binding protein. 11. The application of the transgenic recombinant bacteria according to claim 5 in the preparation of AJ-GBP-like Apostichopus japonicus glucan-binding protein. 12. A preparation method of glucan-binding protein according to claim 1, characterized in that, the preparation method is as follows: Using Apostichopus japonicus cDNA as a template, PCR amplification was carried out with primers F1 and R1; After purification, the PCR product was ligated with the E. coli vector pEASY-E2, the ligated product was transformed into E. coli, and the recombinant was identified by sequencing; The expression vector pEASY-AJ-GBP was transferred into Trans BL21(DE3)pLysS chemically competent cells, and the transformants were screened. The transformants were inoculated in LB medium containing ampicillin, and the expression was induced by IPTG, and then purified by ultrafiltration tube. Recombinant protein, namely obtain glucan binding protein AJ-GBP; Wherein, the primers are: F1: 5'-ATGACCGCCGGTGGTGGAGG-3'; R1: 5'-GCACCAGGATCGACGCTCAAG-3'. 13 . The application of the Apostichopus japonicus-like glucan-binding protein according to claim 1 , wherein the AJ-GBP-like Apostichopus japonicus glucan-binding protein is used for preparing broad-spectrum antibacterial preparations or feed additives. 14 . 14. The application of the Apostichopus japonicus glucan-binding protein according to claim 1, wherein the AJ-GBP imitation sea cucumber glucan-binding protein is used for the preparation of resistance to Bacillus subtilis, Staphylococcus aureus, large intestine Bacillus, Pseudomonas aeruginosa, Brevibacterium luteus and Vibrio eel as pharmaceutical preparations or feed additives.

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