CN106749619B - Short peptidoglycan recognition protein, preparation method thereof, isolated nucleic acid, application thereof and antibacterial drug - Google Patents
Short peptidoglycan recognition protein, preparation method thereof, isolated nucleic acid, application thereof and antibacterial drug Download PDFInfo
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Abstract
The invention discloses a short peptidoglycan recognition protein and a preparation method thereof, isolated nucleic acid and application thereof, and an antibacterial drug, and belongs to the technical field of molecular biology. The amino acid sequence of the short peptidoglycan recognition protein disclosed by the invention is SEQ ID NO. 1, or is a derivative sequence which is obtained by replacing and/or deleting one or more amino acid residues of the amino acid sequence shown by SEQ ID NO. 1 and has the same activity as the amino acid sequence shown by SEQ ID NO. 1. The short peptidoglycan recognition protein disclosed by the invention has obvious bacteriostatic effect on intracellular bacteria and extracellular bacteria, and has potential application value in the aspects of developing novel antibacterial drugs, immunopotentiators, health-care products, feed additives and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a short peptidoglycan recognition protein and a preparation method thereof, isolated nucleic acid and application thereof and an antibacterial drug.
Background
Peptidoglycan recognition proteins (PGRPs) are important pattern recognition receptors in the innate immune system and play an important role in the body's immune response against bacterial pathogenic microorganisms. Currently, PGRPs have been reported in molluscs, arthropods, echinoderms and vertebrates. The reported molecules of PGRPs all contain one or more PGRP domains consisting of 160 amino acids.
Invertebrate PGRPs function primarily through several aspects: 1) recognizing a pattern recognition receptor, activating a Toll or immunodeficiency signal channel, and regulating the expression of a related antibacterial peptide gene; 2) inducing a prophenoloxidase cascade reaction to generate antimicrobial products such as melanin and the like to wrap microorganisms so as to limit infection; 3) amidase activity, which can hydrolyze amide bond between peptidoglycan N-acetylmuramic acid and L-alanine, and convert active PGN molecules into inactive PGN fragments to exert antibacterial activity. GRPs of lower vertebrate fish also have PGN recognition, amidase activity and direct bactericidal activity. Mammalian PGRPs also have PGN recognition function, while natural animal short and intermediate PGRPs have direct bactericidal activity.
Chinese giant salamanders (Andrias davidianus) are the second type of protective aquatic wild animals in China and are the largest tailed amphibians existing in the world. The giant salamander is an amphibious original class group and has important scientific value in researching the system evolution of terrestrial tetrapod animals. In recent years, through the continuous efforts of scholars in China, the artificial breeding technology and the industrialization scale of giant salamanders are continuously expanded, and the giant salamanders become important famous and special-quality breeding varieties in the middle and western regions in China. However, with the continuous enlargement of the giant salamander culture scale, the continuous improvement of the culture intensification degree and the increasingly frequent exchange of seedlings and seeds, the disease problem of the giant salamander becomes more and more prominent. The main pathogens of giant salamander diseases are bacteria, viruses, parasites and the like, and diseases caused by bacterial pathogens are the most common and cause the greatest loss.
Therefore, the research and application of the PGRPs antibacterial function in the giant salamander are important practical significance for deeply developing the research on the pathogenic bacteria defense mechanism of the giant salamander and protecting the germ plasm resources of the giant salamander and promoting the sustainable and healthy development of the giant salamander breeding industry.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a short-type peptidoglycan recognition protein which has obvious bacteriostatic action on both intracellular bacteria and extracellular bacteria.
Another object of the present invention is to provide an isolated nucleic acid encoding the short peptidoglycan recognition protein described above.
Another object of the present invention is to provide a vector containing the above-mentioned nucleic acid encoding a short peptidoglycan recognition protein.
Another object of the present invention is to provide a recombinant cell or a recombinant bacterium containing the above vector.
Another object of the present invention is to provide a method for producing the above-mentioned short peptidoglycan-recognizing protein.
The invention also aims to provide the application of the short-type peptidoglycan recognition protein in preparing antibacterial drugs, immunopotentiators, health products or feed additives.
Another object of the present invention is to provide an antibacterial agent, wherein the active ingredient of the antibacterial agent is the above-mentioned short peptidoglycan recognition protein.
The invention is realized by the following steps:
a short peptidoglycan recognition protein, the amino acid sequence of which is shown in (1) or (2) below:
(1) 1 is shown in SEQ ID NO;
(2) a derivative sequence which is obtained by replacing and/or deleting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 1 and has the same activity as the amino acid sequence shown in SEQ ID NO. 1.
An isolated nucleic acid encoding the short peptidoglycan recognition protein described above.
A vector comprising the nucleic acid.
Recombinant cells or recombinant bacteria containing the vector.
A method for preparing the short peptidoglycan recognition protein, which comprises the following steps: expressing the nucleic acid encoding the short peptidoglycan recognition protein in host cells, and purifying to obtain the short peptidoglycan recognition protein.
The short peptidoglycan recognition protein is applied to the preparation of antibacterial drugs, immunopotentiators, health products or feed additives.
An antibacterial drug contains the short peptidoglycan recognition protein as an active ingredient.
The short peptidoglycan recognition protein and the isolated nucleic acid provided by the invention, the application and the antibacterial drug have the beneficial effects that: the invention clones cDNA for coding the short peptidoglycan recognition protein from Chinese giant salamanders (Andrias davidianus) for the first time, the amino acid sequence of the short peptidoglycan recognition protein is shown as SEQ ID NO:1, and the function of the short peptidoglycan recognition protein is verified through cell experiments, and the short peptidoglycan recognition protein has stronger bacteriostatic function NO matter inside or outside cells, thereby having important practical significance for deeply developing the research on the pathogenic bacteria defense mechanism of the giant salamanders, protecting the germplasm resources of the giant salamanders and promoting the sustainable and healthy development of the giant salamander breeding industry. Meanwhile, a foundation is laid for the development of potential application fields such as antibacterial drugs, immunopotentiators, health care products, feed additives and the like based on PGRPs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the effect of short peptidoglycan recognition protein of Chinese giant salamander on proliferation of extracellular Edwardsiella tarda according to example 1 of the present invention;
FIG. 2 shows the effect of the short peptidoglycan recognition protein of Chinese giant salamander on proliferation of Edwardsiella tarda in the cells, which is provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The short peptidoglycan recognition protein, the preparation method thereof, the isolated nucleic acid, the application thereof and the antibacterial agent of the embodiments of the present invention are specifically described below.
The invention unexpectedly discovers for the first time that the amino acid sequence of the short-type peptidoglycan recognition protein (also called as the Chinese giant salamander short-type peptidoglycan recognition protein) from the Chinese giant salamander (Andrias davidianus) is shown as SEQ ID NO:1, the short-type peptidoglycan recognition protein has strong bacteriostatic effect both inside and outside cells, and simultaneously, the nucleic acid sequence coding the short-type peptidoglycan recognition protein is firstly cloned and separated.
On one hand, the invention provides a short-type peptidoglycan recognition protein, and the amino acid sequence of the short-type peptidoglycan recognition protein is shown as SEQ ID NO. 1.
The inventor verifies that the short peptidoglycan recognition protein has stronger inhibition effect on bacteria in cells or outside the cells through cell experiments.
It is readily understood that derivative sequences obtained by substitution and/or deletion of one or more amino acid residues based on SEQ ID NO. 1 and having the same activity (e.g., bacterial inhibition) as SEQ ID NO. 1 are also within the scope of the present invention.
In another aspect, the present invention provides an isolated nucleic acid encoding the short peptidoglycan recognition protein described above.
Based on the degeneracy of the codon, any short-form peptidoglycan recognition protein encoding the sequence based on the determination of the amino acid sequence of the short-form peptidoglycan recognition protein is within the scope of the present invention.
Further, the invention provides an optimized nucleic acid for coding the short-type peptidoglycan recognition protein, and the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 2.
In another aspect, the present invention provides a vector comprising the nucleic acid described above.
Further, the vector may be a cloning vector or an expression vector. Still further, the expression vector may be a prokaryotic expression vector or a eukaryotic expression vector. Still further, the expression vector is p2 xFLAG-CMV-14.
Further, the above-mentioned nucleic acid was ligated between EcoR I and Xba I cleavage sites of the expression vector p2 xFLAG-CMV-14.
It is easily understood that the short peptidoglycan-recognizing protein can be obtained in high purity by transforming cells or bacteria with the vector containing the nucleic acid by genetic engineering techniques to express the short peptidoglycan-recognizing protein, followed by isolation and purification.
Therefore, in another aspect, the present invention also provides a recombinant cell or a recombinant bacterium containing the vector. The recombinant cell or recombinant bacterium can obtain the recombinant expression short peptidoglycan recognition protein (SEQ ID NO: 2).
Furthermore, the type of the recombinant bacteria can be escherichia coli, and can also be saccharomycetes or other bacteria and the like; further, the type of recombinant cell may be an insect cell line or a vertebrate cell line. Still further, the vertebrate cell line can be a human cell line, or a mouse cell line, or a grass carp Kidney cell line (CIK).
It is easily understood that the vector can be transformed into different bacteria or cells by those skilled in the art according to actual requirements, and thus any recombinant cell or recombinant bacterium containing the vector is within the scope of the present invention.
In another aspect, the present invention also provides a method for preparing the short peptidoglycan recognition protein, comprising: expressing the nucleic acid for coding the short-type peptidoglycan recognition protein in host cells, and purifying to obtain the short-type peptidoglycan recognition protein.
Further, the preparation method may include, for example:
amplifying an open reading frame fragment of a cDNA sequence of the giant salamander short-type peptidoglycan recognition protein in China by using primers adpgRP-F and adpgRP-R with restriction endonuclease sites to obtain a PCR amplification product;
carrying out double enzyme digestion on the PCR amplification product and the p2xFLAG-CMV-14 plasmid by EcoR I and Xba I, and transforming an escherichia coli Top10 competent cell after connection to obtain a recombinant bacterium;
and culturing the recombinant bacteria, and extracting plasmids to obtain the adpgRP-Flag eukaryotic expression plasmid.
Electrotransfection of the adpgRP-Flag eukaryotic expression plasmid into CIK cells to obtain recombinant cells;
the short peptidoglycan recognition protein can be obtained after the recombinant cells are cultured and purified.
It is easily understood that, on the premise of the amino acid sequence of the short-form peptidoglycan recognition protein provided by the present invention, a person skilled in the art can also obtain the short-form peptidoglycan recognition protein of the present invention by other genetic engineering techniques, which also fall into the scope of the present invention.
In still another aspect, the invention also provides the application of the short peptidoglycan recognition protein in the preparation of antibacterial drugs, immunopotentiators, health products or feed additives.
It is easy to understand that the amino acid sequence of the giant salamander short-type peptidoglycan recognition protein is shown in SEQ ID NO. 1 through cell experiments for the first time, and the giant salamander short-type peptidoglycan recognition protein has a strong antibacterial effect both inside cells and outside cells. Therefore, the Chinese giant salamander short-type peptidoglycan recognition protein can be applied to the fields of preparing antibacterial drugs, immunopotentiators, health-care products or feed additives and the like. The obtained main body of the antibacterial drug, the immunopotentiator, the health product or the feed additive containing the giant salamander short-type peptidoglycan recognition protein can have the effects of resisting bacterial infection, enhancing immunity and the like.
In still another aspect, the invention further provides an antibacterial drug, wherein the active ingredient of the antibacterial drug is the short peptidoglycan recognition protein (amino acid sequence is shown in SEQ ID NO: 1). The antibacterial medicine has antibacterial, antiinflammatory, and immunity enhancing effects.
The invention also provides an immunopotentiator, the active component of which is the short peptidoglycan recognition protein (the amino acid sequence is shown in SEQ ID NO: 1). The immunopotentiator has antibacterial, antiinflammatory, and immunity enhancing effects.
The invention also provides a health product, the active component of which is the short peptidoglycan recognition protein (the amino acid sequence is shown as SEQ ID NO: 1). The health product has antibacterial, antiinflammatory, and immunity enhancing effects.
The invention also provides a feed additive, the active component of which is the short peptidoglycan recognition protein (the amino acid sequence is shown as SEQ ID NO: 1). The feed additive has antibacterial, antiinflammatory, and immunity enhancing effects.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a Chinese giant salamander short-type short peptidoglycan recognition protein, the amino acid sequence (shown as SEQ ID NO:1) of which is:
MGECLLLHAAVTDQDDIQVPTLLSSPRFALKVPRMCPLALLLSALCTVTYGCPSIISRSQWGARNVSCLAPMKTPVPYVIIHHTEGRACTTVPACAALVRGIQDFHISERKRCDIGYSFLVGEDGNVYERRSWDYVGTHAPNYNNRSIGISIIGTFTEKT PTLLP, respectively; consists of 165 amino acid residues.
The Chinese giant salamander short-type short peptidoglycan recognition protein provided by the embodiment has a strong antibacterial effect both inside and outside cells. The Chinese giant salamander short-type peptidoglycan recognition protein can be applied to the fields of preparing antibacterial drugs, immunopotentiators, health products or feed additives and the like.
Example 2
Construction and transformation of eukaryotic expression vectors
(1) Extracting total RNA of Chinese giant salamander (Andrias davidianus, purchased from GmbH of Caryu fish Xiangde aquatic product, Hubei province) by using kit
Reagents (Invitrogen corporation, USA).
The method comprises the following specific steps: fully homogenizing 100mg of giant salamander spleen tissues on ice; centrifuging at 4 deg.C and 12000g for 10min, transferring supernatant into a centrifuge tube without RNase, and standing at room temperature for 10 min; adding chloroform 0.2mL, vortexing for 30sec, and standing at room temperature for 3-5 min; centrifuging at 12000g for 10min at 4 deg.C; sucking the supernatant to an EP tube without RNase, adding isopropanol with the same volume, uniformly mixing, and standing at room temperature for 5 min; centrifuging at 12000g for 12min at 4 deg.C; discarding the supernatant, adding 800 μ L75% ethanol for resuspension and precipitation, centrifuging at 4 deg.C and 12000g for 5 min; and discarding the supernatant, and adding a proper amount of DEPC water to dilute the total RNA after the ethanol is completely volatilized.
Total RNA was reverse transcribed into cDNA using the RevertAIdTM First Strand cDNA Synthesis Kit.
The method comprises the following specific steps: mu.l oligo (dT) was added to 2. mu.g of total RNA18Adding DEPC water into the primer until the final volume is 11 mu L, centrifuging, treating at 70 ℃ of a PCR (polymerase chain reaction) amplification instrument for 5min, and then rapidly carrying out ice bath; mu.L of 5 × reaction buffer, 1. mu.L of RNase Inhibitor (ribonuclear Inhibitor), 2. mu.L of dNTPmix (10mmol/L each), 1. mu. L M-MuLV reverse transcriptase were added, and the mixture was centrifuged, reacted at 42 ℃ for 60min, and terminated at 70 ℃ for 10min to obtain cDNA as a template for the subsequent PCR.
(2) Specific primers adpgRP-F and adpgRP-R are adopted to amplify a cDNA sequence capable of coding the giant salamander short-type short-chain peptidoglycan recognition protein (adpgRP) in China. The amino acid sequence of the Chinese giant salamander short-type short-form peptidoglycan recognition protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the cDNA sequence for coding the Chinese giant salamander short-form peptidoglycan recognition protein is shown as SEQ ID NO. 2.
Wherein the base sequence of the upstream primer adPGRP-F is as follows:
5’-CCGGAATTCATGGGTGAGTGTCTGTTAT-3', the EcoR I cleavage site is underlined;
the base sequence of the downstream primer adPGRP-R is as follows:
5’-TGCTCTAGACGGTAACAGGGTCG-3', Xba I cleavage sites are underlined.
(3) And (3) PCR reaction system: the 25 μ L reaction volume included: 2.5. mu.L of dNTP mix (10mM each of dNTPs); 1. mu.L of each of the upstream and downstream primers (10. mu.M); 0.25. mu.L of Ex Taq enzyme (5U/. mu.L); 1 μ L andrias davidianus spleen cDNA; water was added to the total volume of 25. mu.L.
(4) The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 50s at 60 ℃, 50s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.
(5) And recovering and purifying the amplified target fragment. The expression vector p2xFLAG-CMV-14 was digested with EcoRI and XbaI. Using E.Z.N.ATMThe enzyme-digested product was purified and recovered by cycle-pure kit gel recovery kit (Omega Co.). Connecting the enzyme-cut target fragment with the enzyme-cut p2xFLAG-CMV-14 by T4 ligase overnight; the expression vector p2xFLAG-CMV-14-adpGRP was obtained and then transformed into competent cells of E.coli Top 10.
Example 3
Plasmid purification was performed from the above-mentioned Escherichia coli containing p2xFLAG-CMV-14-adpGRP using QIAGEN plasmid Mini Kit, and CIK cells (Ctenophagogon idella cell line) were transfected with the purified plasmid p2xFLAG-CMV-14-adpGRP using a cell electrotransfection Kit from Lonza. The method comprises the following specific steps:
(1) taking CIK cells with good growth state, adding appropriate amount of culture medium, repeatedly blowing to disperse into single cells, counting, and taking about 1 × 106The individual cells were sub-packaged in sterile EP tubes;
(2) centrifuging at room temperature of 400g for 10min, and removing the supernatant;
(3) suspending cells by using an electrotransfer buffer solution, adding 3 mu g of plasmid to be transfected, uniformly mixing, transferring all the cells into an electrotransfer cup, placing the cells into a clamping groove of an electrotransfer instrument, and running a T20 program to perform electrotransfer; sucking out the cell mixed solution from the electric rotating cup, adding the cell mixed solution into a 24-pore plate to which 2ml of fresh culture medium is added in advance, and culturing at 28 ℃; CIK cells containing p2xFLAG-CMV-14-adpGRP are obtained.
Example 4
This example provides a method for preparing the short peptidoglycan recognition protein (SEQ ID NO:1) of the Chinese giant salamander of example 1.
CIK cells containing p2xFLAG-CMV-14-adpGRP were cultured in the same manner as in example 3. And continuously culturing the CIK cells, and separating and purifying to obtain the short type peptidoglycan recognition protein containing Chinese giant salamanders.
Example 5
And (3) a bacteriostatic activity experiment to verify the antibacterial action of the Chinese giant salamander short-type short peptidoglycan recognition protein provided in example 1. The experimental procedure is as follows.
(1) Inoculating Edwardsiella tarda (Edward siellatarda) into TSB culture medium, and standing at 28 deg.C until OD540Taking bacterial liquid, centrifuging and collecting the bacterial body, and suspending and diluting the bacterial body in a proper amount of serum-free M199 culture medium, wherein the bacterial body is about 0.5;
(2) removing the cell culture medium (CIK cells containing p2 xFLAG-CMV-14-adpPGRP as experimental group obtained after culturing in the step (3) of the example 3) in a 24-well plate, respectively adding 500. mu.l of diluted M199 culture medium containing Edwardsiella tarda, and centrifuging at 170g for 6min to ensure that the Edwardsiella tarda fully contacts the cells;
(3) after incubation in an incubator at 25 ℃ for 90min, the extracellular bacteria are counted by sucking the culture medium for 3h and 6h respectively;
(4) for intracellular bacteria counting, removing the culture medium, gently rinsing the cells twice by using a serum-free culture medium, adding 500 mu l of M199 culture medium containing 100 mu g/ml gentamicin to kill extracellular bacteria, continuing to culture, removing the culture medium after 3h and 6h respectively, rinsing the cells four times by using the serum-free culture medium, adding 500 mu l of PBS buffer solution containing 1% Triton-X100 into each hole, and placing the cells on a shaking table to lyse the cells at room temperature for 20 min; performing 10-fold gradient dilution on the lysate by using TSB, and then performing bacterial counting;
(5) CIK cells of a transformation empty vector (p 2xFLAG-CMV-14 expression vector without a cDNA sequence encoding a Chinese giant salamander short-type short-chain peptidoglycan recognition protein (adpGRP)) were used as a control group, and the data were analyzed, and the results are shown in FIG. 1 and FIG. 2.
The results shown in FIG. 1 (in the figure,: "flag" represents a control group, and "PGRP 1" represents an experimental group, the abscissa represents the detection time, and the ordinate represents each 3X 105The number of extracellular Edwardsiella tarda at each CIK cell concentration, "+" indicates significant difference, p<0.05, ". x" indicates that the difference is extremely significant, p<0.01), rotationAfter the p2xFLAG-CMV-14-adpGRP expression plasmid is stained for 3h, the cell number of the extracellular Edwardsiella tarda of the CIK cells in the experimental group is 3.3 multiplied by 107A plurality of; the number of extracellular Edwardsiella tarda cells after 6h was 6.0X 107And (4) respectively. The number of extracellular bacteria is far lower than that of the control group (the transformation empty vector p2xFLAG-CMV-14) in corresponding time (3h is 3.9 multiplied by 10)7A plurality of; 6h is 1.0 multiplied by 108One);
the results shown in FIG. 2 (in the figure,: "flag" represents the control group, "PGRP 1" represents the experimental group, the abscissa represents the detection time, and the ordinate represents each 3X 105The number of Edwardsiella tarda in the cells at each CIK cell concentration, "+" indicates that the difference is significant, and p is<0.05, ". x" indicates that the difference is extremely significant, p<0.01) and the number of Edwardsiella tarda cells in CIK cells of the experimental group after transfecting the p2xFLAG-CMV-14-adpGRP expression plasmid for 3h is 1.2 multiplied by 106A plurality of; the number of cells of Edwardsiella tarda in cells after 6h is 8 multiplied by 106And (4) respectively. The number of intracellular bacteria was much lower than that of the control group at the corresponding time (2.1X 10 in 3 h)6A plurality of; 6h is 1.4 multiplied by 107One).
The results show that the number of bacteria in the experimental group is lower than that in the control group no matter in cells or outside the cells, and the Chinese giant salamander short-type short-chain peptidoglycan recognition protein (adpgRP) expressed on the p2xFLAG-CMV-14-adpgRP vector has strong bacteria inhibition effect and can inhibit the growth of bacteria at least Edwardsiella tarda.
In conclusion, the Chinese giant salamander peptidoglycan recognition protein provided by the invention is a short peptidoglycan recognition protein, and after eukaryotic cells are transfected, the extracted recombinant protein has strong antibacterial effect on both intracellular and extracellular Edwardsiella tarda. The invention firstly researches and applies the antibacterial function of PGRPs in the giant salamander, so that the antibacterial effect of the Chinese giant salamander peptidoglycan recognition protein (SEQ ID NO:1) is disclosed for deeply researching the pathogenic bacteria defense mechanism of the giant salamander, and the antibacterial function has important practical significance for protecting the germplasm resources of the giant salamander and promoting the sustainable and healthy development of the giant salamander breeding industry. Meanwhile, a foundation is laid for the development of potential application fields such as antibacterial drugs, immunopotentiators, health care products, feed additives and the like based on PGRPs.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> salt city industry institute
<120> short peptidoglycan recognition protein, preparation method thereof, isolated nucleic acid, application thereof and antibacterial drug
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<170>PatentIn version 3.5
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cccaactaca ataacaggtc cattggcatc agcatcattg gcaccttcac agaaaaaacg 300
ccgactctgc tgccctga 318
Claims (8)
1. A short peptidoglycan recognition protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. An isolated nucleic acid encoding the short-form peptidoglycan recognition protein of claim 1.
3. The nucleic acid of claim 2, wherein the nucleotide sequence of the nucleic acid is set forth in SEQ ID NO 2.
4. A vector comprising the nucleic acid of claim 2 or 3.
5. A recombinant cell or recombinant bacterium comprising the vector of claim 4.
6. A method for preparing the short peptidoglycan recognition protein of claim 1, comprising: expressing the nucleic acid for coding the short-type peptidoglycan recognition protein in host cells, and purifying to obtain the short-type peptidoglycan recognition protein.
7. The use of the short peptidoglycan recognition protein of claim 1 in the preparation of antibacterial drugs, immunopotentiators, health products or feed additives for inhibiting edwardsiella tarda.
8. An antibacterial agent, wherein the active ingredient of the antibacterial agent is the short-form peptidoglycan recognition protein of claim 1, and the bacterium is edwardsiella tarda.
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| CN116178521B (en) * | 2023-03-21 | 2024-04-09 | 中国海洋大学 | Fish-source peptidoglycan recognition protein mutant, preparation method and application thereof |
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2017
- 2017-02-13 CN CN201710077148.9A patent/CN106749619B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| involvement of a short-type peptidoglycan recognition protein (pgrp) from chinese giant salamanders andrias davidianus in the immune response against bacterial infection;huiyang et al.;《developmental and comparative immunology》;20181231;37-44 * |
| transcriptome analysis of the endangered chinese giant salamander (andrias davidianus): immune modulation in response to aeromonas hydrophila infection;zhitao qi et al.;《veterinary immunology and immunopathology》;20161231;85-95 * |
| 非洲爪蟾短型肽聚糖识别蛋白基因的克隆与鉴定;齐志涛等;《水生生物学报》;20100930;第34卷(第5期);922-926 * |
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