CN116987173A - Application of litopenaeus vannamei mannose receptor LvMR recombinant protein - Google Patents
Application of litopenaeus vannamei mannose receptor LvMR recombinant protein Download PDFInfo
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- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 49
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 49
- 241000238553 Litopenaeus vannamei Species 0.000 title claims abstract description 39
- 108010031099 Mannose Receptor Proteins 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 19
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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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/7056—Lectin superfamily, e.g. CD23, CD72
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to application of a litopenaeus vannamei mannose receptor LvMR recombinant protein. The application of the recombinant protein of the litopenaeus vannamei mannose receptor LvMR in identification and aggregation preparations of pathogenic bacteria, novel immune preparations and feed additives. The recombinant protein can be used for producing identification preparations, bacterial agglutination preparations and the like of pathogenic bacteria, is applied to the treatment of relevant shrimp and crab diseases in the aquaculture process, or is used for producing feed additives, preservatives or antistaling agents and the like, and can also provide a basis for further researching the immune defense mechanism of the prawns and provide references for disease control and gene auxiliary breeding of the litopenaeus vannamei.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to application of a litopenaeus vannamei mannose receptor LvMR recombinant protein.
Background
Mannose Receptor (MR) belongs to the C-lectin superfamily, and is an important pattern recognition receptor in the innate immune system, and plays an important role in the processes of recognizing pathogens, inducing cytokines, maintaining homeostasis, and the like. Vertebrate MR structures are relatively conserved, typically consisting of an extracellular cysteine-rich CR domain, a type II fibronectin (FN II) domain, and 8 tandem C-type lectin-like domains (CRDs) and transmembrane domains, which mediate phagocytosis of pathogens, participate in antigen presentation and transport, interact with ligands and trigger a series of signaling cascades within the cell, etc. In invertebrates, however, few reports on mannose receptors have been found only in procambarus clarkia. Procambrus clarkia PcMR contains 14 CTLDs and a transmembrane domain, which is significantly different from the vertebrate MR structure. The structural and immune function studies of MR in invertebrates remain in progress.
The litopenaeus vannamei has the advantages of short growth period, high-quality protein content, high-density transportation resistance and the like, and is an important mariculture variety in China. Along with the continuous expansion of the cultivation scale, diseases caused by bacteria and viruses frequently occur, and huge losses are caused for the shrimp cultivation industry. Therefore, the research on the disease-resistant mechanism of the litopenaeus vannamei and the development of novel antibacterial drugs for immune control are urgently needed from the immune defense factors of the litopenaeus vannamei. At present, the research on mannose receptors of litopenaeus vannamei is not clear, and the functions of genes are not clear.
Disclosure of Invention
The invention aims to provide an application of a litopenaeus vannamei mannose receptor LvMR recombinant protein.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the application of the litopenaeus vannamei mannose receptor LvMR recombinant protein in identification and aggregation preparations of pathogenic bacteria, novel immune preparations and feed additives.
The litopenaeus vannamei mannose receptor LvMR recombinant protein contains an amino acid sequence shown in SEQ ID NO. 1.
The litopenaeus vannamei mannose receptor LvMR recombinant protein is obtained by amplifying a gene fragment encoding litopenaeus vannamei mannose receptor LvMR mature peptide by a PCR technology, cloning the gene fragment into a pET32a (+) expression vector, realizing in-vitro recombinant expression in escherichia coli BL21 (DE 3), purifying by a TALON column and dialyzing.
SEQ ID NO. 1.
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFCNPPFTMVGSKCLYVESDLRLSWPEARDFCKGLAGDGGGADMASFPSCDNFVAFSRYLEFNAPNISGMWVGAHSAFTPNMWQWISGEDLQTGVPFWYYNEDYDGTRDCVSISLDYYNRLVDAHCELPLSFVCEKHLARKDEAEQAATKRARDMECENHGILVGDFCYVFNDRLETWEEAERNCRSQYDEVGGELYYPSSCNEFTHMAHHLEAAEKANSYWVGGVDISGNEEWTWVNGENIPGGPPYWATGEPSHSHDNQPREHCTVMTAEKRYYLQDVHCGDRHPYICKLRFLKLAAALEHHHHHH
The application of the litopenaeus vannamei mannose receptor LvMR recombinant protein in preparing a pathogen recognition preparation.
Further, the identification of pathogenic bacteria is the identification and combination of pathogenic bacteria cell wall conserved molecules, namely pathogenic related molecular modes.
The pathogen-associated molecular pattern is a gram-negative pathogen-associated molecular pattern lipopolysaccharide; gram-negative and gram-positive bacteria pathogen-associated molecular pattern peptidoglycans; pathogen-associated molecular pattern glucans of fungi; viral infection-associated molecular pattern double stranded RNA analogs poly (I: C).
The recombinant protein is applied to the preparation of recognition preparations of pathogen-associated molecular pattern lipopolysaccharide of gram-negative bacteria, pathogen-associated molecular pattern peptidoglycan of gram-negative bacteria and gram-positive bacteria, pathogen-associated molecular pattern glucan of fungi or virus infection-associated molecular pattern double-stranded RNA analogue poly (I: C).
The application of the litopenaeus vannamei mannose receptor LvMR recombinant protein in preparing an agglutination preparation of gram-negative bacteria, gram-positive bacteria or fungi.
Further, at 10mmol L -1 Ca 2+ In the presence, the recombinant protein LvMR has obvious agglutination effect on gram-negative bacteria, gram-positive bacteria or fungi.
The gram negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram positive bacteria are staphylococcus aureus or micrococcus luteus; the fungus is pichia pastoris.
The invention has the advantages that:
the invention uses PCR technology to amplify the gene segment of the mature peptide of the litopenaeus vannamei mannose receptor LvMR, clones the gene segment into a pET32a (+) expression vector, and realizes in vitro recombinant expression in escherichia coli BL21 (DE 3). After the recombinant protein LvMR is purified and dialyzed by a TALON column, the recombinant protein LvMR has binding activity on pathogen-related molecular pattern lipopolysaccharide of gram-negative bacteria, pathogen-related molecular pattern peptidoglycan of gram-negative bacteria and gram-positive bacteria, pathogen-related molecular pattern glucan of fungi and virus infection-related molecular pattern poly (I: C). The recombinant protein LvMR has obvious agglutination effect on vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus, micrococcus luteus and pichia pastoris.
The recombinant protein can be used for producing identification preparations, bacterial agglutination preparations and the like of pathogenic bacteria, is applied to the treatment of relevant shrimp and crab diseases in the aquaculture process, or is used for producing feed additives, preservatives or antistaling agents and the like, and can also provide a basis for further researching the immune defense mechanism of the prawns and provide references for disease control and gene auxiliary breeding of the litopenaeus vannamei.
Drawings
FIG. 1 shows the induced and purified LvMR recombinant protein of the mannose receptor of litopenaeus vannamei provided by the example of the invention (M: protein marker;1: protein expressed in the LvMR thallus is not induced; 2: protein expressed by LvMR after IPTG induction; 3: purified LvMR recombinant protein; 4: protein expressed in the pET-32a thallus is not induced; 5: protein expressed by pET-32a after IPTG induction; 6: purified pET-32a recombinant protein).
FIG. 2 is a diagram showing ELISA analysis of four pathogen-associated molecular patterns (lipopolysaccharide LPS, peptidoglycan PGN, dextran GLU and poly (I: C)) by Litopenaeus vannamei LvMR recombinant proteins of the present invention.
FIG. 3 is a graph showing the activity of the recombinant protein of LvMR of litopenaeus vannamei on bacterial and fungal agglutination (detection of fluorescently labeled Vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa, micrococcus luteus, staphylococcus aureus and Pichia pastoris, rLvMR, rTrx, caCl) according to an example of the invention 2 And EDTA are 0.2mg L, respectively -1 、0.2mg L -1 、10mmol L -1 And 10mmol L -1 Tris-HCl and rTrx served as blank and negative controls, respectively).
Detailed Description
The following description of the embodiments of the present invention is further provided in connection with the accompanying examples, and it should be noted that the embodiments described herein are for the purpose of illustration and explanation only, and are not limiting of the invention.
Example 1.
The preparation method of the litopenaeus vannamei mannose receptor LvMR recombinant protein comprises the following specific operations:
designing a homology arm primer LvMR_ReF containing a pET32a carrier sequence according to a cDNA sequence of Litopenaeus vannamei LvMR
(5'GCTGATATCGGATCCGAATTCTGCAACCCGCCGTTCACCATG 3') and LvMR_ReR (5'CTCGAGTGCGGCCGCAAGCTTGAGGAACCGCAGCTTGCAGAT 3'), amplifying a gene fragment encoding the LvMR mature peptide by PCR technique, the reaction being carried out in TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.), the reaction conditions being: 94 ℃ for 30s;98 ℃,10s,58 ℃,30s,72 ℃ and 1min for 32 cycles; 72 ℃ for 2min. The cells were cloned into a linear vector pET32a (+) expression vector digested with EcoR I and Hind III, transformed into E.coli BL21 (DE 3), sequenced to confirm that the expression was correct, inoculated with positive clones into LB medium, shake-cultured at 37℃until OD600 nm=0.4-0.6, added with IPTG to a final concentration of 0.5mM, and then centrifuged to collect the cells. The thalli are treated by ultrasonic waves of 200W, ultrasonic waves for 2s and ultrasonic waves for 2s under ice bath conditions for 45min. The supernatant was removed by centrifugation and the pellet was collected. After the precipitation was dissolved in 8M urea, the recombinant product was purified by using a TALON column of Clontech. The purified recombinant protein was transferred to a dialysis bag, dialyzed against a dialysis renaturation solution (ph=8.0) containing 2mM reduced glutathione, 0.2mM oxidized glutathione, 1mM EDTA, 50mM Tris-HCl, 50mM nacl, 10% glycerol and 1% glycine and gradient reduced urea (6, 5, 4, 3, 2, 0M) at 4 ℃ to renature the recombinant protein, and finally dialyzed 2 times against 50mM Tris-HCl (ph=8.0) buffer to remove other components of the solution. The recombinant protein after dialysis renaturation is concentrated by Microsep advanced ultrafiltration centrifugal concentration tube of PALL company, and the concentration of the Litopenaeus vannamei LvMR recombinant protein is 1.5mg/mL (see figure 1) measured by BCA protein concentration measuring kit of Biyun Tian company.
Example 2.
PAMPs binding experiment of Litopenaeus vannamei mannose receptor LvMR recombinant protein
The binding activity of the recombinant proteins to pathogen-associated molecular patterns PAMPs (pathen-associated molecular patterns, certain common highly conserved molecular structures on the surface of pathogenic microorganisms) was detected by enzyme-linked immunosorbent assay. And selecting pathogen-associated molecular pattern Lipopolysaccharide (LPS) of gram-negative bacteria, pathogen-associated molecular pattern Peptidoglycan (PGN) of gram-negative bacteria and gram-positive bacteria, pathogen-associated molecular dextran (GLU) of fungi and virus infection-associated molecular pattern poly (I: C) as study objects.
20mg of LPS, PGN and GLU, poly (I: C) were dissolved in 100mL of 50mM NaCO3-NaHCO3 buffer (pH 9.6), respectively. 100. Mu.L of LPS, GN and GLU solution (20. Mu.g) was added to the 96-well ELISA plate and coated at 4℃for 16-18h. The uncoated PAMPs in the 96-well ELISA plate were poured out, washed 3 times with PBS-T for 5min each, and blocked for 1h at 37℃with 200. Mu.L of 3% BSA in PBS. Washing 3 times with PBS-T for 5min each, adding 100 μl of recombinant protein diluted in gradient and 5mM CaCl at the same time 2 And 0.1mg/mL BSA, incubated at 18℃for 3h. Thereafter, the wells were washed 3 times with PBS-T for 5min each, 100. Mu.L of diluted anti-His-tag antibody (1:1000) was added to each well and incubated at 37℃for 1h. Then, PBS-T was usedWashing was performed 3 times, 5min each, 100. Mu.L of diluted HRP-labeled goat anti-mouse secondary antibody (1:1000) was added to each well, and incubated at 37℃for 45min. Washing with PBS-T for 3 times and 5min each time, preparing a chromogenic working solution by using a TMB chromogenic kit, incubating for 30min at room temperature, and reading at 450nm wavelength. Each experiment was repeated 3 times. As shown in FIG. 2, the recombinant protein LvMR has high binding activity to both LPS, PGN, GLU and poly (I: C), wherein the dissociation constant of the LvMR recombinant protein and LPS is 2.14X10 -7 M and PGN have dissociation constants of 1.15X10 -7 The dissociation constant for M and poly (I: C) was 5.72X10 -7 M and GLU have dissociation constants of 1.12X10 -4 M。
Example 3.
In vitro microbial agglutination experiment of litopenaeus vannamei mannose receptor LvMR recombinant protein
FITC staining: bacteria and fungi cultured to logarithmic phase in conventional manner (1.0 mL) were collected by centrifugation at 4000rpm at 4deg.C for 5min, and after discarding the culture medium, the bacteria were washed 3 times with PBS. Adding the solution with the final concentration of 0.1mg mL -1 Is stained slowly overnight in the dark. The cells were collected by centrifugation at 4000rpm at 4℃for 5min, and after discarding the medium, the cells were washed 3 times with a large volume of PBS to remove residual FITC.
Wherein, gram negative bacteria of vibrio parahaemolyticus, vibrio alginolyticus and pseudomonas aeruginosa; gram positive bacteria staphylococcus aureus and micrococcus luteus; the fungus pichia pastoris.
And (3) bacteria condensation experiment: FITC-labeled bacteria were resuspended using sterile PBS and the bacteria concentration was adjusted to 1X 10 8 One mL -1 . In the experimental group, 25 mu LLvMR recombinant protein and 20 mu L of FITC-labeled bacterial suspension are taken and mixed uniformly in a 1.5mL centrifuge tube; in the control group, 25. Mu.L of rTrx was mixed with 20. Mu.L of FITC-labeled bacterial suspension. To observe Ca 2+ Whether or not to affect the agglutination activity, 10mM CaCl was added to each of the experimental group and the control group 2 And chelating Ca with 10mM EDTA 2+ . The sample was incubated in the dark at 28℃for 2h with slow shaking, and 5. Mu.L was observed under a fluorescence microscope (see FIG. 3). The above example LvMR recombinant protein was found to be found in Ca 2+ In the presence of gram negative bacteria such as vibrio parahaemolyticus, vibrio alginolyticus, pseudomonas aeruginosa and gram positive bacteriaThe staphylococcus aureus and the micrococcus luteus and the pichia pastoris have obvious agglutination effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. A litopenaeus vannamei mannose receptor LvMR recombinant protein, characterized in that: the application of the recombinant protein of the litopenaeus vannamei mannose receptor LvMR in identification and aggregation preparations of pathogenic bacteria, novel immune preparations and feed additives.
2. The use of the litopenaeus vannamei mannose receptor LvMR recombinant protein of claim 1, wherein: the litopenaeus vannamei mannose receptor LvMR recombinant protein contains an amino acid sequence shown in SEQ ID NO. 1.
3. The use of the litopenaeus vannamei mannose receptor LvMR recombinant protein of claim 2, wherein: the litopenaeus vannamei mannose receptor LvMR recombinant protein is obtained by amplifying a gene fragment encoding litopenaeus vannamei mannose receptor LvMR mature peptide by a PCR technology, cloning the gene fragment into a pET32a (+) expression vector, realizing in-vitro recombinant expression in escherichia coli BL21 (DE 3), purifying by a TALON column and dialyzing.
4. The use of the litopenaeus vannamei mannose receptor LvMR recombinant protein of claim 1, wherein: the application of the litopenaeus vannamei mannose receptor LvMR recombinant protein in preparing a pathogen recognition preparation.
5. The use of the mannosylate receptor LvMR recombinant protein of claim 4, wherein: the recombinant protein is applied to the preparation of recognition preparations of pathogen-associated molecular pattern lipopolysaccharide of gram-negative bacteria, pathogen-associated molecular pattern peptidoglycan of gram-negative bacteria and gram-positive bacteria, pathogen-associated molecular pattern glucan of fungi or virus infection-associated molecular pattern double-stranded RNA analogue poly (I: C).
6. The use of the litopenaeus vannamei mannose receptor LvMR recombinant protein of claim 1, wherein: the application of the litopenaeus vannamei mannose receptor LvMR recombinant protein in preparing an agglutination preparation of gram-negative bacteria, gram-positive bacteria or fungi.
7. The use of the litopenaeus vannamei mannose receptor LvMR recombinant protein of claim 6, wherein: the gram negative bacteria are vibrio parahaemolyticus, vibrio alginolyticus or pseudomonas aeruginosa; the gram positive bacteria are staphylococcus aureus or micrococcus luteus; the fungus is pichia pastoris.
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