Peptidoglycan recognition protein-d, method of preparation and use thereof

Abstract

The application discloses a kind of peptidoglycan recognition protein-D, preparation method and application, belong to biological medicine technical field.The application obtains natural peptidoglycan recognition protein-D from tussah by protein separation and purification technology, realizes the expression of peptidoglycan recognition protein-D and its derivative or analogue or partial fragment gene in host cell after resolving its primary structure (gene and protein) using genetic engineering technology, and the antibody of purified recombinant peptidoglycan recognition protein-D and its derivative or analogue or partial fragment is obtained by immunizing animal.The natural, recombinant peptidoglycan recognition protein-D and its derivative or analogue or partial fragment and its antibody of the application can be widely used for prevention, detection diagnosis, treatment and other biological medicine fields of microorganism, microorganism related molecular pattern.

Description

TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and relates to the structure of peptidoglycan recognition protein-D and a method for obtaining the same, a new physiological function and application in the field of biological medicine. Specifically, the present application relates to the structure of peptidoglycan recognition protein-D and an analog or an active fragment thereof, a production method and a function thereof, and application in the field of biological medicine such as detection and diagnosis of microorganisms and related molecular patterns, promotion of melanin production in insect blood lymph, influence on synthesis of different types of antibacterial peptides in insects, preparation of an antibody of peptidoglycan recognition protein-D and the analog or the active fragment thereof, and application in the field of biological medicine. BACKGROUND

[0004] As a member of the pattern recognition receptor (PPR) family, peptidoglycan recognition protein (PGRP) can recognize pathogen-related molecular patterns (PAMPs) existing only on the surface of pathogenic organisms to realize the perception of foreign pathogens, and then selectively activate the Toll pathway, the IMD pathway, the JAK-STAT pathway, reactive oxygen metabolism or melanization reaction to clear the pathogens.

[0005] Peptidoglycan recognition proteins (PGRPs) were first discovered in the silkworm, Bombyx mori, by Yoshida et al. (Yoshida, H.; Kinoshita, K.; Ashida, M. Purification of a Peptidoglycan Recognition Protein from Hemolymph of the Silkworm, Bombyx Mori. J. Biol. Chem. 1996, 271 (23), 13854-13860.) in 1996, and then PGRPs were found in mollusks, echinoderms and vertebrates. PGRPs contain one or more PGRP domains, which are about 165 amino acid residues in length. The PGRP domain is homologous to N-acetylmuramoyl-alanine amidase and has about 30% similarity to the bacteriophage T7 lysozyme. The PGRP domain is a reverse L-shaped groove structure composed of five β-strands in the central region and three α-helices in the periphery, as revealed by structural analysis. PGRPs are classified into different types according to the molecular weight of the protein and amidase activity. PGRPs are classified into long, medium and short types according to the molecular weight of the protein. PGRPs are classified into catalytically active PGRPs and non-catalytically active PGRPs according to whether they have amidase activity. Catalytically active PGRPs can cleave the amide bond between N-acetylmuramic acid and L-lysine of peptidoglycan, cleaving peptidoglycan into smaller fragments that have no immunostimulatory activity, and thus can reduce the stimulation of the IMD pathway by bacteria. Non-catalytically active PGRPs cannot cleave peptidoglycan, but can act as recognition receptors to participate in other immune responses.

[0006] TmPGRP-D of Tenebrio molitor can bind to PGN and interact with TmGNBP1 and serine protease MSP to activate Spätzle precursor processing enzyme, which cleaves Spätzle precursor into mature Spätzle, which binds to Toll receptor in the Toll pathway and activates the Toll pathway to synthesize antibacterial peptides. Similar to Tenebrio molitor, in Drosophila, PGRP-D, SD and Gram-negative binding protein-1 (GNBP-1) synergistically detect bacterial cell wall extracellular Lys-type PGN to initiate activation of the Toll pathway; and BmPGRP-S1 is involved in activation of the IMD pathway to induce expression of AMPs. The phenoloxidase cascade leads to melanin formation and is involved in the defense of insects against invading pathogenic microorganisms. Some PGRPs with receptor function can participate in the activation of the phenoloxidase cascade by recognizing microorganisms or PAMPs. Overexpression of DmPGRP-LE with receptor function and continuous activation of DmPGRP-LC with receptor function can trigger melanization. In Bombyx mori, BmPGRP-S1 can participate in the activation of the phenoloxidase cascade after binding to PGN; in Manduca sexta, MsPGRP-1 can not only recognize PGN, but also participate in the phenoloxidase cascade of Manduca sexta; PGRP-S of Ostrinia nubilalis can activate the phenoloxidase cascade after binding to PGN; and PGRP-A of Helicoverpa armigera can also participate in the activation of the phenoloxidase cascade. PGRP-SC1, -SC2 and -LB secreted by Drosophila have amidase activity and can cleave PGN molecules. Three PGRP genes have been identified in zebrafish, namely PGLYRP-2 (or zfPGRP2), PGLYRP-5 (zfPGRP-SC) and PGLYRP-6 (zfPGRP6), and all the three PGRP genes exhibit amidase activity and antibacterial activity. S. aureus and B. thuringiensis After activation of the phenoloxidase cascade, melanin is formed and is involved in the defense of insects against invading pathogenic microorganisms. Some PGRPs with receptor function can participate in the activation of the phenoloxidase cascade by recognizing microorganisms or PAMPs. Overexpression of DmPGRP-LE with receptor function and continuous activation of DmPGRP-LC with receptor function can trigger melanization. In Bombyx mori, BmPGRP-S1 can participate in the activation of the phenoloxidase cascade after binding to PGN; in Manduca sexta, MsPGRP-1 can not only recognize PGN, but also participate in the phenoloxidase cascade of Manduca sexta; PGRP-S of Ostrinia nubilalis can activate the phenoloxidase cascade after binding to PGN; and PGRP-A of Helicoverpa armigera can also participate in the activation of the phenoloxidase cascade. PGRP-SC1, -SC2 and -LB secreted by Drosophila have amidase activity and can cleave PGN molecules. Three PGRP genes have been identified in zebrafish, namely PGLYRP-2 (or zfPGRP2), PGLYRP-5 (zfPGRP-SC) and PGLYRP-6 (zfPGRP6), and all the three PGRP genes exhibit amidase activity and antibacterial activity.

[0007] Peptidoglycan recognition protein is one of the few pattern recognition proteins that are highly conserved from lower animals to higher animals, and is of great significance for understanding host immune regulation and immune-related diseases. However, there is currently no relevant research on the structure, preparation, biological function and application of peptidoglycan recognition protein-D of insects in the family Saturniidae of Lepidoptera. SUMMARY

[0009] The present application is directed to PGRP-D in the insect body of Saturniidae in Lepidoptera, and studies the preparation method, primary structure (gene and protein), biological function of natural PGRP-D, and its application. Recombinant PGRP-D and its analogues or active fragments and their biological functions and applications are obtained by using genetic engineering technology. In addition, natural, recombinant PGRP-D and its analogues or active fragments are used as antigens to stimulate the body to produce antibodies, and the application of the antibodies is also studied.

[0010] The present application first uses protein extraction, separation and purification technology to isolate and purify natural PGRP-D from insects of Saturniidae in Lepidoptera. Secondly, the primary structure (gene and protein) of PGRP-D is analyzed and its gene is obtained by using protein chemistry technology and molecular biology technology. Thirdly, the expression of PGRP-D gene in host cells is realized by using genetic engineering technology, and recombinant PGRP-D is obtained by combining protein extraction, separation and purification technology. At the same time, analogues or partial fragments of PGRP-D are obtained by using genetic recombination technology. Natural, recombinant PGRP-D and its analogues or partial fragments can specifically recognize various microbial related molecular patterns such as lipopolysaccharide, β-1, 3-glucan, peptidoglycan, lipoteichoic acid and mannan, as well as bacteria and fungi. The above combination can activate the prophenoloxidase activation system in insect humoral immunity on the one hand, and affect the synthesis of different types of insect antibacterial peptides on the other hand. The biological functions of natural, recombinant PGRP-D and its analogues or partial fragments and their antibodies of the present application can be widely applied in the biological and medical fields of prevention, detection and diagnosis, treatment, etc. against microorganisms; at the same time, the antibacterial peptides prepared by using natural, recombinant PGRP-D and its analogues or partial fragments of the present application to induce the expression of antibacterial peptides in insects can be applied in the biological and medical fields of prevention, detection and diagnosis, treatment, etc. against microorganisms.

[0011] The insect referred to in the present application is a Lepidoptera insect, preferably a Saturniidae insect, Saturniidae selected from the group consisting of tussah, castor silk moth, tussah moth, Indian tussah, amber moth, American tussah, oak silkworm, mountain silkworm, American tussah, camphor silkworm, maple silkworm. The insect is any natural or artificially released or artificially bred insect in any region. In order for the professional technical personnel to more comprehensively and clearly understand the present application, tussah is used as a representative to describe the following content, and the selection of tussah as a representative is not intended to limit the scope of the claims of the present application in any way.

[0012] The PGRP-D and the active fragment of PGRP-D are obtained by genetic engineering expression, including (1) an expression vector of a prokaryotic system, and an expression host cell is an E. coli cell or a B. subtilis cell or a lactic acid bacteria, (2) an expression vector of a yeast cell system, and an expression host cell is a yeast cell, (3) an expression vector of an insect cell system, and an expression host cell is an insect cell, (4) an expression vector of a mammalian cell system, and an expression host cell is a mammalian cell. The above expression forms are intracellular expression or secreted form expression, and the expression product in the above expression system is used as a source for preparing PGRP-D, a PGRP-D analogue or an active fragment.

[0013] The host cell includes a prokaryotic cell and a eukaryotic cell, and examples of a commonly used prokaryotic host cell include E. coli, B. subtilis and the like. Commonly used eukaryotic host cells include a yeast cell, an insect cell and a mammalian cell and the like.

[0014] The microorganism and the related molecular pattern referred to in the present application are fungi, gram-positive bacteria and gram-negative bacteria and the related molecular pattern. In order for the person skilled in the art to more comprehensively and clearly understand the present application, the following contents are described by taking Pichia pastoris, Candida albicans, Staphylococcus aureus, Escherichia coli, Micrococcus luteus, B. subtilis and the like as representatives of the microorganism (fungi, gram-positive bacteria and gram-negative bacteria), and Lys-PGN, DAP-PGN, lipoteichoic acid, mannan, β-1, 3-glucan, lipopolysaccharide and the like as representatives of the related molecular pattern of the microorganism, and the selection of the above specific microorganism or specific related molecular pattern of the microorganism as a representative is not in any way to limit the scope of the claims of the present application.

[0015] The present application is realized by the following technical scheme:

[0016] The present application provides a peptidoglycan recognition protein-D, and the amino acid sequence is shown as SEQ ID NO: 1.

[0017] Based on the above technical scheme, further, the peptidoglycan recognition protein-D is derived from a Lepidoptera Saturniidae insect, and is selected from one of Attacus atlas, Gonometa, Bombyx mori, Antheraea indica, Antheraea pernyi, Antheraea yamamai, Antheraea mylitta, Bombyx mandarina, Bombyx mandarina, Bombyx mandarina and Bombyx mandarina.

[0018] The present application further provides a gene encoding the peptidoglycan recognition protein-D.

[0019] Based on the above technical scheme, further, the nucleotide sequence of the gene of the peptidoglycan recognition protein-D is shown as SEQ ID NO: 2.

[0020] Another aspect of the present application provides the derivative or analog or active fragment of the peptidoglycan recognition protein-D, comprising the amino acid sequence as shown in SEQ ID NO: 1 or part of the sequence, and having the biological activity of the peptidoglycan recognition protein-D.

[0021] Based on the above technical solution, further, the derivative or analog or active fragment of the peptidoglycan recognition protein-D is selected from Met-PGRP-D sequence, Met-His6 tag-PGRP-D sequence, Met-PGRP-D-His6 tag sequence, Met-His6 tag-thrombin cleavage site-PGRP-D sequence, Met-GST tag-thrombin cleavage site-PGRP-D sequence, Met-PGRP-D-thrombin cleavage site-GST tag sequence, Met-PGRP-D-Flag tag sequence, Met-Flag tag-PGRP-D sequence, Met-His6 tag-SUMO tag-thrombin cleavage site-PGRP-D sequence, Met-SUMO tag-thrombin cleavage site-PGRP-D-His6 tag sequence.

[0022] Another aspect of the present application provides the preparation method of the peptidoglycan recognition protein-D, using one or more than two combinations of hemolymph, blood, hemocyte lysate, lymph, homogenate of lepidopteran Saturniidae insect as raw material solution, obtaining electrophoretically pure or even HPLC pure peptidoglycan recognition protein-D by one or more than two methods of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method;

[0023] Or the gene encoding the peptidoglycan recognition protein-D is cloned into a recombinant expression vector, introduced into a host cell, and the recombinant expressed peptidoglycan recognition protein-D is obtained.

[0024] Based on the above technical solution, further, the ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method is:

[0025] (1) the operating temperature is 0℃-45℃, preferably 0℃-10℃;

[0026] (2) the pH of the solution is pH2-pH12, preferably pH4-pH10;

[0027] (3) the reagent for adjusting the pH of the solution is a conventional and general acid, base, acid solution or base solution, the acid or acid solution is preferably HCl, HAc, phosphoric acid, citric acid, sulfuric acid, boric acid or a mixed solution thereof, the base or base solution is preferably NaOH, KOH, Tris, sodium or potassium citrate, sodium or potassium phosphate, borax or a mixed solution thereof;

[0028] (4) The buffer is a conventional and general buffer ion pair, preferably a citrate buffer ion pair, an HCl-Tris buffer ion pair, a citrate-phosphate buffer ion pair, a phosphate buffer ion pair, an acetate buffer ion pair, a borate buffer ion pair, a boric acid-Tris buffer ion pair, or a combination of the above buffer ions;

[0029] (5) The ionic strength of the solution or buffer is 0.001-0.5 mol / L, preferably 0.01-0.1 mol / L.

[0030] Another aspect of the present application provides a method for preparing the derivative or analog or active fragment of the peptidoglycan recognition protein-D, which comprises cloning a gene encoding the derivative or analog or active fragment of the peptidoglycan recognition protein-D into a recombinant expression vector, introducing the vector into a host cell, and isolating and purifying the recombinant expression of the derivative or analog or active fragment of the peptidoglycan recognition protein-D.

[0031] Based on the above technical solution, further, the expression system includes a prokaryotic system and an insect cell system, the host cell of the prokaryotic system is an E. coli cell or a B. subtilis cell, and the host cell of the insect cell system is an insect cell; the expression form is intracellular expression or secreted form expression.

[0032] Another aspect of the present application provides an antibody of the peptidoglycan recognition protein-D or the derivative or analog or active fragment thereof, which is prepared by stimulating the immune system of a mouse, rat, rabbit, dog, sheep, horse or cow with the natural peptidoglycan recognition protein-D or the derivative or analog or active fragment of the peptidoglycan recognition protein-D as an antigen.

[0033] Another aspect of the present application provides an application of the peptidoglycan recognition protein-D or the derivative or analog or active fragment thereof or the antibody in affecting the phenol oxidase proactivator activation system, antibacterial peptide synthesis, microorganisms and related molecular patterns.

[0034] Based on the above technical solution, further, the related molecular pattern includes lipopolysaccharide, β-1, 3-glucan, peptidoglycan, lipoteichoic acid and mannan.

[0035] The present application has the following beneficial effects over the prior art:

[0036] The natural, recombinant PGRP-D and partial fragments thereof can be obtained by the method which is conventional, simple and high in yield, and can be widely applied in the biological and medical fields of prevention, detection, diagnosis and treatment of microorganisms and microorganism related molecular patterns, the detection and tracking of natural peptide glycan recognition protein-D, recombinant peptide glycan recognition protein-D, recombinant peptide glycan recognition protein-D analogues or active fragments, and the influence of the insect antibacterial peptide synthesis and the application of the obtained antibacterial peptide in the biological and medical fields. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced as follows.

[0039] Figure 1 The figure is a separation and purification electrophoretogram of natural PGRP-D, wherein Lane M represents molecular weight markers; lane 1 represents natural PGRP-D purified by method 1; lane 2 represents natural PGRP-D purified by method 2; and lane 3 represents natural PGRP-D purified by method 3.

[0040] Figure 2 The figure is a separation and purification electrophoretogram of recombinant PGRP-D (prokaryotic expression system), wherein Lane M represents molecular weight markers; lane 1 represents PGRP-D with a C-terminal post-fusion histidine tag; lane 2 represents PGRP-D with a C-terminal post-fusion histidine tag and a thrombin cleavage site; lane 3 represents PGRP-D with an N-terminal pre-fusion GST tag; and lane 4 represents PGRP-D with an N-terminal pre-fusion histidine-SUMO tag and a thrombin cleavage site.

[0041] Figure 3 The figure is a separation and purification electrophoretogram of recombinant PGRP-D (insect expression system), wherein Lane M represents molecular weight markers; lane 1 represents recombinant PGRP-D obtained by pFastBac1-sf9 insect expression system; and lane 2 represents PGRP-D obtained by pMIB / V5-His-Sf21 insect expression system.

[0042] Figure 4 The figure is an analysis of the binding ability of PGRP-D to microorganisms, wherein (A) represents the binding ability of natural PGRP-D to different bacteria; and (B) represents the binding ability of recombinant PGRP-D to different bacteria.

[0043] Figure 5 The figure is the correlation between the expression amount of PGRP-D mRNA and immunity, wherein (A) represents the expression amount of PGRP-D mRNA in vivo after induction of bacteria; and (B) represents the expression amount of PGRP-D mRNA in vivo after induction of bacteria. ApChanges of PGRP-D expression over time; (B): Changes of PGRP-D expression in different tissues after bacteria induction Ap Changes of PGRP-D expression, Mg: midgut; Fb: fat body; Mt: Malpighian tubule; Hc: hemocyte; Em: epidermis.

[0044] Figure 6 Effects of exogenous recombinant PGRP-D on prophenoloxidase activating system, HL: hemolymph of the silkworm; PGRP: recombinant Ap- PGRP-D protein; Micro: microbe; Error bars represent mean ± SD, n = 3; * represents t test P < 0.05, ** represents t test P < 0.01, *** represents t test P < 0.001, **** represents t test P < 0.0001.

[0045] Figure 7 Inhibitory effects of PGRP-D antibody on prophenoloxidase activating system, HL: hemolymph of the silkworm; Ab: rabbit-derived Ap- PGRP-D polyclonal antibody; Micro: microbe; Error bars represent mean ± SD, n = 3; * represents t test P < 0.05, ** represents t test P < 0.01, *** represents t test P < 0.001, **** represents t test P < 0.0001.

[0046] Figure 8 Inhibitory effects of endogenous PGRP-D on prophenoloxidase activating system, DEPC: control group injected with DEPC water; dsEGFP : group injected with EGFP double-stranded RNA; ds PGRP: group injected with Ap- PGRP-D double-stranded RNA; Error bars represent mean ± SD, n = 3; ** represents t test P < 0.01, *** represents t test P < 0.001, **** represents t test P < 0.0001.

[0047] Figure 9 Effects of interfering PGRP-D on the production of antimicrobial peptides, (A): effects of interfering PGRP-D on E. coli induced production of antimicrobial peptides; (B): effects of interfering PGRP-D on S. aureus induced production of antimicrobial peptides; (C): effects of interfering PGRP-D on C. albicans induced production of antimicrobial peptides, Error bars represent mean ± SD, n = 3; * represents t test P < 0.05, ** represents t test P < 0.01. DETAILED DESCRIPTION:

[0049] The following examples can make the professional more fully understand the present application, but not in any way limit the scope of the present application claims.

[0050] Example 1: Isolation and purification of native PGRP-D

[0051] In this example, the silkworm is repeatedly washed with distilled water or deionized water, and blood lymph is collected at 10°C to -5°C by using conventional methods such as the wax disc method, centrifugation, back blood vessel blood collection, perfusion, squeezing, homogenization, reflex flow blood collection, tearing, cutting, shearing, and puncturing.

[0052] 1. Method 1

[0053] The blood lymph is collected, centrifuged at 12,000 x g, and the supernatant is taken. Ammonium sulfate precipitation is performed at 50%, and the precipitate is resuspended in 50 mM citric acid buffer solution pH 5.2, 100 mM NaCl, and 5% glycerol. After centrifugation, the supernatant is passed through a Sephacryl S-200 column, and the eluate containing the target protein is collected. After dialysis with 50 mM PB pH 8.0, the eluate containing the target protein is collected by elution with a gradient of 0-3 M NaCl using a PGRP antibody affinity column.

[0054] The test results are as follows Figure 1 lane 1, the purity of native PGRP-D reaches electrophoretic purity.

[0055] 2. Method 2

[0056] A mixture of fungi (Candida albicans), gram-positive bacteria (Micrococcus luteus), and gram-negative bacteria (Escherichia coli) (10 μl) dissolved in insect physiological saline (120 mM NaCl, 0.9 mM CaCl2, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM NaHCO3, 1 mM NaH2PO4, 38.8 mM glucose) is injected into the silkworm, and the bacteria-induced blood lymph is collected after 24-48 hours of induction. The blood lymph is diluted 10 times with 50 mM Tris-HCl buffer solution pH 5.5, and passed through a Mono-Q ion exchange chromatography column. Linear gradient elution is performed using 0-3 M NaCl in 50 mM Tris-HCl buffer solution pH 5.5. The target component is collected, concentrated to 1 mL by ultrafiltration, diluted 10 times with 10 mM sodium phosphate buffer solution pH 4.5, and passed through an Octyl-sepharose 4-Fast Flow column. Linear elution is performed using 10-500 mM sodium sulfate buffer solution pH 4.5 to obtain the target protein component.

[0057] The test results are as follows Figure 1lane 2, the purity of native PGRP-D reached electrophoretic purity.

[0058] 3. Method 3

[0059] The blood lymph of the Antheraea pernyi was precipitated by 70% ammonium sulfate, and the supernatant was discarded after centrifugation at 8000xg for 15 min. The precipitate was dissolved in a phosphate buffer solution, and was eluted by a hydroxyapatite column using a phosphate ion gradient. The fraction containing the target protein was collected. The above fraction was dialyzed in a phosphate buffer solution, and was eluted by an anion exchange column HiTrapTM Q using a NaCl concentration gradient (0-1M). The fraction containing the target protein was collected. The above fraction was added with (NH4)2SO4 to a concentration of 2M, and was eluted by a phenyl hydrophobic column using a (NH4)2SO4 concentration reduction gradient (0-50%). The fraction containing the target protein was collected. The above fraction was subjected to PGN-sepharose affinity chromatography, and the fraction containing the target protein was collected.

[0060] The test results are shown in Table 1. Figure 1 lane 3, the purity of native PGRP-D reached electrophoretic purity.

[0061] Example 2: PGRP-D structure analysis and gene sequence analysis

[0062] According to the conventional protein chemistry and molecular biology techniques, methods and means, the structure of PGRP-D was analyzed, and the complete nucleotide sequence and the amino acid sequence of PGRP-D were obtained.

[0063] The amino acid sequence of native PGRP-D (mature peptide chain) is shown in SEQ ID NO: 1, and the gene sequence encoding the native PGRP-D is shown in SEQ ID NO: 2.

[0064] The full-length cDNA sequence of PGRP-D was obtained by using molecular biology techniques and methods, and is shown in SEQ ID NO: 3. The encoded amino acid sequence is shown in SEQ ID NO: 4.

[0065] Example 3: Obtaining recombinant PGRP-D and its analogs and active fragments by using a prokaryotic expression system

[0066] This example describes the construction strategy and basic method of expressing the gene of PGRP-D and its analogs and active fragments of the present application by using a prokaryotic expression system.

[0067] Structure of PGRP-D derivatives, analogs and active fragments

[0068] (1) Met-PGRP-D amino acid sequence

[0069] MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0070] (2) Met-His6 tag-PGRP-D sequence

[0071] MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0072] (3) Met-PGRP-D-His6 tag sequence

[0073] MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHH

[0074] (4) Met-His6 tag-thrombin cleavage site-PGRP-D sequence

[0075] MHHHHHHHLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0076] (5) Met-GST tag - Thrombin cleavage site - PGRP-D sequence

[0077] MHHHHHHHLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0078] (6) Met-PGRP-D - Thrombin cleavage site - GST tag sequence

[0079] MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESLVPRGSILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

[0080] (7) Met-PGRP-D-Flag tag sequence

[0081] MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESDYKDDDDK

[0082] (8) Met-Flag tag-MSPH sequence

[0083] MDYKDDDDKYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0084] (9) Met-His6 tag-SUMO tag-thrombin cleavage site-PGRP-D sequence

[0085] MDYKDDDDKYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

[0086] (10) Met-SUMO tag-thrombin cleavage site-PGRP-D-His6 tag sequence

[0087] MHHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHH.

[0088] The expression vector, expression host cell and expression strategy of the prokaryotic expression system are the conventional, general expression vector, expression host cell and expression strategy for genetic engineering expression.

[0089] The method for separating and purifying the expression product is the method, principle and strategy of Example 1.

[0090] 1. Construction of expression vector of PGRP-D

[0091] According to the N-terminal and C-terminal amino acid sequences of natural PGRP-D, corresponding oligonucleotide primers are designed, and restriction endonuclease hydrolysis site sequences are added to the 5' ends of the two oligonucleotide primers. Insect fat body cDNA pool is used as a template for PCR amplification, and the product is detected by agarose gel electrophoresis and the nucleic acid fragments are recovered by gel recovery. After restriction endonuclease digestion, the same double enzyme-digested expression plasmid is recombined and linked under the action of DNA ligase, and the competent cells of Escherichia coli are heat-transformed. After positive transformants are obtained by colony PCR and restriction endonuclease digestion verification and screening, a biotechnology service company is submitted for DNA sequence determination. Through the above genetic engineering method, the expression vector of PGRP-D gene is constructed.

[0092] The features of the expression vector construction of this embodiment are: 1. The expression vectors can be selected from pTYB11, pMAL-C2X, pET-28a, pGEX-2T, pBV220, pQE30, pET20b, etc. in E. coli as the host; 2. A peptide segment can be fused at the N-terminal of PGRP-D as an affinity chromatography tag (Tag); 3. A peptide segment can be fused at the C-terminal of PGRP-D as an affinity chromatography tag (Tag); 4. The tag can be selected from His-Tag (six or more consecutive histidines), GST-Tag, Flag-Tag, etc.; 5. The amino acid sequence of the proteolytic enzyme hydrolysis site, such as thrombin, enterokinase, blood coagulation factor X, etc. can be added between the affinity chromatography tag and PGRP-D to obtain a recombinant PGRP-D protein consistent with the structure of the natural PGRP-D protein.

[0093] 2. Obtaining of the recombinant PGRP-D protein and its derivatives, analogs, active fragments

[0094] The PGRP-D gene expression vector is transformed into E. coli by genetic engineering technology, and after picking a single colony, it is inoculated into LB containing antibiotics to induce the expression of the PGRP-D gene, thereby obtaining a culture solution or bacterial body containing PGRP-D. The bacterial body containing PGRP-D is first lysed by a lysis solution and ultrasonically broken to release the target protein, and then the supernatant is collected by centrifugation as the raw material solution of the recombinant PGRP-D for standby use.

[0095] The features of the expression of the target gene are: 1. The method of transforming the expression vector into the host can be selected from heat transformation and electroporation; 2. The induction method includes chemical induction-isopropyl β-D-thiogalactoside (IPTG) induction and warming induction; 3. The PGRP-D gene can be expressed in the cell or outside the cell; 4. The PGRP-D existing in the cell needs to be released into the solution by lysis solution lysis, ultrasonic breaking, etc.

[0096] According to the methods, principles, strategies, etc. of Example 1, the recombinant PGRP-D and its derivatives, analogs, active fragments are separated and purified from the above-mentioned PGRP-D-containing raw material solution to the desired purity, until reaching electrophoretic purity or HPLC purity.

[0097] For example: (1) The pET-19b is used to construct the PGRP-D gene fused with the histidine tag at the C-terminal, the E. coli is heat-transformed, and the PGRP-D-His is expressed in the cell after IPTG induction. The bacterial body is resuspended with lysis buffer, ultrasonically broken, and centrifuged to obtain the supernatant as the raw material solution for further separation and purification of PGRP-D. According to the methods, principles, strategies, etc. of Example 1, the PGRP-D is separated and purified to electrophoretic purity (lane 1). Figure 2 lane 1).

[0098] (2) Using pET-28a(+) to construct PGRP-D gene with C-terminal post-fusion histidine tag and thrombin, using electrotransformation to transfer the expression vector into host cells, inducing expression by warming, resuspending the bacterial cells with lysis buffer (50 mmol / L PBS, 0.15 mol / L NaCl, 50 mmol / L imidazole), performing ultrasonic disruption, and centrifuging to obtain the supernatant as the raw material for further separation and purification of PGRP-D. According to the methods, principles, strategies, etc. of Example 1, PGRP-D is separated and purified to electrophoretic purity (lane 2). Figure 2

[0099] (3) Using pGEX4T-1 to construct PGRP-D expression vector with N-terminal pre-fusion GST tag, heat-transforming E. coli, inducing expression by warming, and expressing GST-PGRP-D in the cells. Resuspending the bacterial cells with lysis buffer, performing ultrasonic disruption, and centrifuging to obtain the supernatant as the raw material for further separation and purification of PGRP-D. According to the methods, principles, strategies, etc. of Example 1, PGRP-D is separated and purified to electrophoretic purity (lane 3). Figure 2

[0100] (4) Using pET-28a-SUMO to construct PGRP-D expression vector with N-terminal pre-fusion histidine-SUMO tag and thrombin cleavage site, heat-transforming E. coli, inducing expression by warming, and expressing His-SUMO-thrombin cleavage site-PGRP-D in the cells. According to the methods, principles, strategies, etc. of Example 1, PGRP-D is separated and purified to electrophoretic purity (lane 4). Figure 2

[0101] The above-mentioned purified expression products containing tags are subjected to hydrolysis by the above-mentioned conventional and general proteolytic enzymes (such as thrombin, enterokinase, blood coagulation factor X, etc.) to remove the fusion peptide segment in the expression product, and then separated and purified to obtain PGRP-D. The structure of the recombinant PGRP-D is the same as that of the natural PGRP-D.

[0102] Example 4: Obtaining recombinant PGRP-D and its analogs and active fragments using insect cell expression system

[0103] This example describes the construction strategy and basic method of expressing PGRP-D and its analogs and active fragments of the application using insect cell expression system.

[0104] The expression vector, expression host cells, and expression strategy of the insect cell expression system are all conventional and general expression vectors, expression host cells, and expression strategies for genetic engineering expression.

[0105] ​​​The present embodiment is to make the skilled person more fully understand the present application, and is not to limit the scope of the present application in any way.

[0106] For the separation and purification method of the expression product, the method, principle, strategy, etc. of Example 1 are adopted.

[0107] 1. Obtain recombinant PGRP-D and its analogs, active fragments by using pFastBacl-sf9 insect expression system

[0108] The PGRP-D and its analogs, active fragments gene is linked to the pFastBacl plasmid to construct the pFastBacl-PGRP-D recombinant expression plasmid. After transposition of E. coli DH10, Blue-gal and IPTG induction, the transposition recombinant bacmid is obtained by blue-white screening. The insect cells sf9 are transfected, and the Western blot verifies the expression of recombinant PGRP-D in the cells.

[0109] The cells are collected, resuspended with lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), and centrifuged after ultrasonic disruption to obtain the raw material liquid containing the target protein. Directly sample to the affinity chromatography column with anti-PGRP-D antibody-sepharose CL-6B as ligand, and gradient elution is carried out with 0 mol / L-3 mol / L NaCl lysis buffer. The recombinant protein is highly expressed and reaches electrophoretic purity. The electrophoretic identification result after purification is as follows Figure 3 Lane 1.

[0110] 2. Obtain recombinant PGRP-D and its analogs, active fragments by using pMIB / V5-His-Sf21 insect expression system

[0111] The PGRP-D and its analogs, active fragments gene is linked to the pMIB / V5-His plasmid to construct the pMIB / V5-His-PGRP-D recombinant expression plasmid. After transposition of E. coli DH5, Blue-gal and IPTG induction, the transposition recombinant bacmid is obtained by blue-white screening. The insect cells Sf21 are transfected, and the Western blot verifies the expression of recombinant PGRP-D in the cells.

[0112] The cells were collected, resuspended with lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), and centrifuged after ultrasonic disruption to obtain a raw material solution containing the target protein. The solution was directly loaded onto a pre-equilibrated metal ion chelation chromatography column, washed with 0.02 mol / L imidazole (pH 8.0) to remove a large amount of impurities, and eluted with 0.2 mol / L imidazole (pH 8.0). The recombinant protein was highly expressed and reached electrophoretic purity. The electrophoretic identification results after purification are shown in Figure 2. Figure 3 Lane 2.

[0113] Example 5: Obtaining of PGRP-D antibody

[0114] According to conventional and general antibody production techniques, the various PGRP-D obtained in Examples 1, 3, and 4 were used as antigens to stimulate the immune systems of mice or rats or rabbits or dogs or sheep or horses or cattle to produce corresponding antibodies.

[0115] Using conventional and general antibody detection methods, the production of PGRP-D antibodies in the sera of immunized mice or rats or rabbits or dogs or sheep or horses or cattle was detected.

[0116] When the immunized mice or rats or rabbits or dogs or sheep or horses or cattle produced PGRP-D antibodies, the sera of the immunized mice or rats or rabbits or dogs or sheep or horses or cattle were collected and stored using conventional and general animal serum collection and storage methods. The sera can be directly applied.

[0117] Using conventional and general antibody separation and purification techniques, such as salting-out, various types of chromatography media, and antibody affinity chromatography media, PGRP-D antibodies of different purities were separated and purified from the stored sera containing PGRP-D antibodies until electrophoretically pure or HPLC pure PGRP-D antibodies were obtained to meet different application requirements.

[0118] Example 6: Biological activity of natural PGRP-D and recombinant PGRP-D partial fragments

[0119] In this example, the natural PGRP-D and the active fragments of recombinant PGRP-D have the same biological activity. The biological activity experiments on insects of the order Lepidoptera are described using the moth as a representative. Professional technicians can further expand the application range of natural PGRP-D and active fragments of recombinant PGRP-D based on their biological activity.

[0120] 1. Specificity of natural PGRP-D and recombinant PGRP-D partial fragments in binding to microorganisms

[0121] (1) The binding specificity of natural PGRP-D to microorganisms

[0122] Western blotting was used to investigate the binding characteristics of natural PGRP-D with Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and fungi (Saccharomyces cerevisiae). Natural PGRP-D was incubated with equal amounts of microorganisms, eluted with 1M NaCl, and then eluted again with 2% SDS at high temperature (55℃). The binding of natural PGRP-D to different microorganisms was indirectly detected using a PGRP-D polyclonal antibody. Results are as follows: Figure 4 As shown in -A, natural PGRP-D binds to Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae.

[0123] (2) The binding specificity of recombinant PGRP-D and its partial fragments to microorganisms

[0124] The binding characteristics of the recombinant PGRP-D fragment with Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and fungi (Saccharomyces cerevisiae) were investigated using the Western-blotting method described in 1-(1) of Example 6. The results are as follows: Figure 4 As shown in Figure -B, recombinant PGRP-D binds to all three microorganisms mentioned above, and this experimental result is consistent with the experimental results of natural PGRP-D.

[0125] The above experiments demonstrate that natural and recombinant PGRP-D can specifically bind to Gram-positive bacteria, some Gram-negative bacteria, and some fungi. The same experimental results can be obtained using partial fragments of the recombinant PGRP-D described in Examples 3 and 4.

[0126] 2. Correlation between PGRP-D expression level and innate immunity

[0127] Will E. coli , S. aureus , C. albicans Three representative microorganisms were injected into silkworms in equal proportions. Using 18S rRNA from natural oak silkworm tissue as an internal reference gene, the expression of PGRP-D in oak silkworms at 3 h, 6 h, 9 h, 12 h, 18 h, and 24 h was detected using Real-time PCR. The results are as follows: Figure 5 As shown in -A, and the expression status in various tissues of the tussah silkworm, the results are as follows: Figure 5As shown in Fig. 1, the expression of PGRP-D mRNA in the body of M. separata increased gradually with the time of microbial induction, and reached the peak at 12 h. The expression of PGRP-D mRNA was the highest in the epidermis, followed by the fat body, midgut and hemocytes, and almost no expression in the midgut. The above results showed that PGRP-D was involved in the innate immune defense system of M. separata, and mainly played its role in the epidermis and fat body.

[0128] 3. Effects of native, recombinant PGRP-D and its antibodies on PPO-AS

[0129] (1) Effects of recombinant PGRP-D and its fragments on PPO-AS

[0130] The effects of recombinant PGRP-D on PPO-AS were investigated using six soluble PAMPs and three microorganisms, and the results are shown in Fig. 2. Compared with the control group of hemolymph + Tris-HCl buffer solution, PAMPs and microorganisms could significantly activate PPO-AS. After the addition of exogenous recombinant PGRP-D, the PO activity of each experimental group showed a significant increase. Figure 6

[0131] (2) Effects of in vivo interference of PGRP-D expression on PPO-AS

[0132] The effects of endogenous native PGRP-D on PPO-AS were further investigated by reducing the expression of endogenous PGRP-D using RNAi technology. The results are shown in Fig. 3. Compared with the DEPC water injection control group and the control group, the PO activity of each experimental group after the interference of native PGRP-D decreased significantly. Figure 8 dsEGFP

[0133] (3) Effects of anti-PGRP-D antibodies on PPO-AS

[0134] The effects of endogenous PGRP-D on PPO-AS were investigated by blocking the endogenous native PGRP-D protein using rabbit-derived anti-PGRP-D polyclonal antibodies. The results are shown in Fig. 4. Compared with the control group of hemolymph of M. separata + Tris-HCl buffer solution, PAMPs and microorganisms could significantly activate PPO-AS. After the addition of anti-PGRP-D polyclonal antibodies, the PO activity of each experimental group showed a significant decrease. Figure 7

[0135] 4. Effects of native, recombinant PGRP-D on the synthesis of antimicrobial peptides

[0136] To investigate the effects of PGRP-D on the synthesis of antimicrobial peptides, the expression of PGRP-D was injected into the hemolymph of M. separata, and the results are shown in Fig. 5. Compared with the control group, the expression of PGRP-D significantly reduced the synthesis of antimicrobial peptides.​​​​ds PGRP-D 72h down-regulate endogenous PGRP-D expression, and then injected into the larvae of E. coli , S. aureus , to investigate the changes in the level of antimicrobial peptide mRNA in the body of the tussah silkworm. The results are shown in Figure 9 , compared with the control group, the interference PGRP-D experimental group injected E. coli , the mRNA levels of antimicrobial peptides Defensin, Gloverin , Attacin , Lebocin were significantly down-regulated. And the interference PGRP-D experimental group injected S. aureus , the mRNA levels of antimicrobial peptides Defensin, , Gloverin , Attacin Lebocin were significantly up-regulated. The above results show that the influence of PGRP-D on the synthesis pathway of antimicrobial peptides induced by different pathogens is significantly different.

[0137] Example 7: Application of natural PGRP-D, recombinant PGRP-D active fragment and its antibody

[0138] This embodiment is described with PGRP-D as a representative, and PGRP-D active fragment also has the same biological activity. At the same time, the tussah silkworm is also described as a representative of the biological activity of insects in the order Lepidoptera. The skilled person can further expand the application range of PGRP-D and recombinant PGRP-D active fragment and its antibody based on the biological activity of PGRP-D and recombinant PGRP-D active fragment and its antibody.

[0139] 1. PGRP-D and its active fragment affect phenoloxidase activation system and antimicrobial peptide synthesis pathway

[0140] As described in Example 6, PGRP-D and its active fragment can activate the activation of phenoloxidase, and have different effects on the synthesis of antimicrobial peptides induced by different microorganisms. Based on this, it can be applied to the related fields of phenoloxidase and phenoloxidase activation system, and antimicrobial peptide synthesis.

[0141] 2. PGRP-D and its active fragment for detecting microorganisms

[0142] As described in Example 5, PGRP-D and its active fragment can bind to some microorganisms and related molecular patterns, based on which, by detecting whether the microorganisms and PGRP-D and its active fragment bind, whether the sample contains microorganisms or their related molecular patterns is detected.

[0143] 3. Application of PGRP-D and its active fragment antibody

[0144] Antibodies against PGRP-D and its active fragments obtained from Example 5 are used for immunological and molecular biological detection of PGRP-D in Lepidoptera samples. The antibodies are also used for immunological tracking of PGRP-D during isolation and purification of PGRP-D from Lepidoptera samples, and for qualitative and quantitative detection of PGRP-D. The antibodies are used in the above-mentioned Examples for isolation and purification of native and recombinant PGRP-D and its active fragments.

[0145] In any sample to be tested for microorganisms, a sufficient amount of antibodies against PGRP-D and its active fragments is added. The sample to be tested is then tested for microorganisms according to the method described in this Example for detection of microorganisms using PGRP-D and its active fragments. The results are as described above. If the sample contains microorganisms in an amount that can be detected, no microorganisms are detected (negative result). The design of this experiment is used as a negative control for detection of microorganisms in samples.

[0146] As described in Example 6, PGRP-D and its active fragments activate prophenoloxidase. Antibodies against PGRP-D and its active fragments can significantly inhibit the activity of prophenoloxidase. Based on this, the activity of prophenoloxidase can be adjusted using antibodies against PGRP-D and its active fragments, and this can be used in related fields using prophenoloxidase and the prophenoloxidase activation system.

[0147] The results described above show that antibodies against PGRP-D and its active fragments bind to PGRP-D and its active fragments and mask the biological activity of PGRP-D and its active fragments in combination with microorganisms and their associated molecular patterns, thereby eliminating the original biological activity of PGRP-D and its active fragments. This principle of binding and masking can be widely used.

Claims

1. A peptidoglycan recognition protein-D, having an amino acid sequence as shown in SEQ ID NO:

1. 2.A gene encoding the peptidoglycan recognition protein-D of claim 1.

3. The gene for peptidoglycan recognition protein-D according to claim 2, characterized by, The nucleotide sequence of the gene of the peptidoglycan recognition protein-D is shown in SEQ ID NO:

2.

4. The derivative or analogue or active fragment of peptidoglycan recognition protein-D according to claim 1, characterized in that, The derivative or analog or active fragment of the peptidoglycan recognition protein-D is selected from the group consisting of Met-PGRP-D sequence, Met-His6 tag-PGRP-D sequence, Met-PGRP-D-His6 tag sequence, Met-His6 tag-thrombin cleavage site-PGRP-D sequence, Met-GST tag-thrombin cleavage site-PGRP-D sequence, Met-PGRP-D-thrombin cleavage site-GST tag sequence, Met-PGRP-D-Flag tag sequence, Met-Flag tag-PGRP-D sequence, Met-His6 tag-SUMO tag-thrombin cleavage site-PGRP-D sequence, Met-SUMO tag-thrombin cleavage site-PGRP-D-His6 tag sequence; The Met-PGRP-D amino acid sequence is as follows: MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES The Met-His6 tag-PGRP-D sequence is as follows: MHHHHHHYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES The Met-PGRP-D-His6 tag sequence is as follows: MHHHHHHYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHHH The sequence of Met-His6 tag-thrombin cleavage site-PGRP-D is as follows: MHHHHHHLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES The sequence of Met-GST tag-thrombin cleavage site-PGRP-D is as follows: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES The sequence of Met-PGRP-D-thrombin cleavage site-GST tag is as follows: MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESLVPRGSILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD Met-PGRP-D-Flag tag sequence is as follows: MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESDYKDDDDK Met-Flag tag-MSPH sequence is as follows: MDYKDDDDKYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES Met-His6 tag-SUMO tag-thrombin cleavage site-PGRP-D sequence is as follows: MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESLVPRGSILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES HHHHHH. MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHH. Using one or more than two kinds of combination of blood lymph, blood, blood cell lysate, lymph, homogenate of the blood of the Antheraea pernyi as raw material liquid, the ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method or more than two kinds of combination of the methods are used to obtain the electrophoretically pure or HPLC pure peptidoglycan recognition protein-D; 5. The method of producing the peptidoglycan recognition protein-D according to claim 1, characterized by, Or the gene encoding the peptidoglycan recognition protein-D is cloned into a recombinant expression vector, introduced into a host cell, and the recombinant expressed peptidoglycan recognition protein-D is obtained. The gene encoding the peptidoglycan recognition protein-D derivative or analog or active fragment is cloned into a recombinant expression vector, introduced into a host cell, and the recombinant expressed peptidoglycan recognition protein-D derivative or analog or active fragment is obtained after isolation and purification.

6. A method for producing a derivative or an analogue or an active fragment of the peptidoglycan recognition protein-D according to claim 4, characterized in that, ​

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