Serine protease homolog sulp-1, methods of making and uses thereof

Abstract

The application discloses serine protease homolog SLP-1 and a preparation method and application thereof, and belongs to the technical field of biological medicine. The natural serine protease homolog SLP-1 is obtained by using protein separation and purification technology to separate and purify the natural serine protease homolog SLP-1 from a tussock moth. After the primary structure (gene and protein) of the serine protease homolog SLP-1 is analyzed, the expression of the serine protease homolog SLP-1 and derivatives or analogs or partial fragments thereof in a host cell is realized by using genetic engineering technology. The recombinant serine protease homolog SLP-1 and derivatives or analogs or partial fragments thereof are obtained by purification. Animals are immunized to obtain corresponding antibodies. The natural, recombinant serine protease homolog SLP-1 and derivatives or analogs or partial fragments thereof and the antibodies thereof can be widely applied to the fields of prevention, detection, treatment drugs and the like for microorganisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to the structure of serine protease homolog SLP-1 and a method for obtaining and application thereof. Specifically, the present application relates to the structure of serine protease homolog SLP-1 and derivatives, analogs and active fragments thereof, a preparation method thereof, and application thereof in the detection of microorganisms and related molecular patterns, the induction of insects to produce antibacterial peptides, the preparation of antibodies against serine protease homolog SLP-1 and derivatives, analogs and active fragments thereof. BACKGROUND

[0002] The main function of serine proteases and their homologs in insect hemolymph is to participate in innate immune response. From the 15 protease genes identified in Bombyx mori to the 42 protease genes in Manduca sexta and the 63 protease genes in Aedes aegypti, serine proteases have formed a large gene family (serine protease superfamily) in the insect genomes studied so far. In the current reports on the role of serine proteases and their homologs in immunity, serine proteases, as a supergene protein family, are expressed in large quantities in various tissues of insects and rely on precise interactions between proteins to form a cascade reaction system to play the role of immune defense. Scientists have found through the study of model insects such as Drosophila, Manduca sexta and Tribolium castaneum that there are 5 serine proteases involved in the Toll signal transduction pathway of Drosophila; at least 7 serine proteases and their homologs have been found in Manduca sexta to participate in the activation of prophenoloxidase, and some serine proteases with definite functions can participate in the activation of the Toll pathway; MSP in Tribolium castaneum, as the first serine protease in the signal pathway, can induce a cascade amplification reaction of serine proteases and ultimately activate prophenoloxidase. In the study of serine proteases, it has also been found that some serine proteases can also play an antiviral function in the process of viral infection, which also indicates that this type of enzyme is a potential antiviral factor.

[0003] Innate immunity is a hot topic in current immunology research. The prophenoloxidase activation system and the Toll pathway are important innate immune mechanisms in insects, and serine proteases and their homologs are the most important link in the immune system, playing a crucial role in the response of insects to the invasion and infection of pathogenic microorganisms. Currently known serine protease homologs are relatively few, and the structural basis of the activation of this type of protein, the activation mechanism and necessary activation conditions, and the biological functions such as the mechanism of action with upstream molecules and downstream substrates have not been systematically explained. There is no research on the structure, preparation and biological functions of serine protease homologs of insects in the family Saturniidae of Lepidoptera. SUMMARY

[0004] The present application is directed to serine protease homolog SLP-1 in insects of the family Saturniidae in Lepidoptera, and studies the preparation method, primary structure (gene and protein), biological function of natural serine protease homolog SLP-1, and its application. Recombinant serine protease homolog SLP-1 and its derivatives, analogs, active fragments, and their biological functions and applications are obtained by using genetic engineering technology. In addition, natural, recombinant serine protease homolog SLP-1 and its derivatives or analogs or active fragments are used as antigens to stimulate the body to produce antibodies, and the application of the antibodies is also studied.

[0005] In the present application, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells, etc.

[0006] The present application provides a preparation method, structure, biological function, and application of a serine protease homolog SLP-1 obtained from insects of the family Saturniidae in Lepidoptera. First, natural serine protease homolog SLP-1 is obtained by using protein extraction, separation, and purification technology from insects of the family Saturniidae in Lepidoptera. Second, the primary structure (gene and protein) of the target protein is analyzed and its gene is obtained by using protein chemistry technology and molecular biology technology. Third, the expression of the serine protease homolog SLP-1 gene in host cells is realized by using genetic engineering technology, and recombinant serine protease homolog SLP-1 is obtained by combining protein separation and purification technology. At the same time, derivatives or analogs or partial fragments of serine protease homolog SLP-1 are obtained by using genetic recombination technology. Natural, recombinant serine protease homolog SLP-1 and its derivatives or analogs or partial fragments can specifically recognize various microbial associated molecular patterns such as lipopolysaccharide, β-1,3-glucan, peptidoglycan, lipoteichoic acid, and mannan, and can specifically activate the prophenoloxidase activation system induced by various microbial associated molecular patterns such as lipopolysaccharide, β-1,3-glucan, peptidoglycan, lipoteichoic acid, and mannan, and microorganisms such as bacteria and fungi. Natural, recombinant serine protease homolog SLP-1 and its derivatives or analogs or partial fragments and their antibodies of the present application can be widely used in the fields of prevention, detection, and therapeutic drugs against microorganisms; at the same time, the antibodies prepared by using natural, recombinant serine protease homolog SLP-1 and its derivatives or analogs or partial fragments as antigens can be applied in the fields of prevention, detection, and therapeutic drugs against microorganisms.

[0007] The present application provides the following technical solutions:

[0008] The present application provides a serine protease homolog SLP-1, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Based on the above technical solution, further, the serine protease homolog SLP-1 is derived from Lepidoptera Saturniidae insects, and is selected from one of Attacus atlas, Gonomphis sp., Bombyx mori, Antheraea pernyi, A. assamensis, A. yamamai, A. mylitta, A. automedon, A. zuleika, A. formosana and A. falcata.

[0010] The present application further provides a gene encoding the serine protease homolog SLP-1.

[0011] Based on the above technical solution, further, the nucleotide sequence of the gene of the serine protease homolog SLP-1 is shown in SEQ ID NO: 2.

[0012] The present application further provides a derivative or analog or active fragment of the serine protease homolog SLP-1, which comprises all or part of the sequence shown in SEQ ID NO: 1, and has the biological activity of the serine protease homolog SLP-1.

[0013] Based on the above technical solution, further, the derivative or analog or active fragment of the serine protease homolog SLP-1 is selected from Met-SLP-1, Met-His6 tag-SLP-1, Met-SLP-1-His6 tag, Met-His6 tag-thrombin cleavage site-SLP-1, Met-GST tag-thrombin cleavage site-SLP-1, Met-SLP-1-thrombin cleavage site-GST tag, Met-SLP-1-Flag tag, Met-Flag tag-SLP-1, Met-His6 tag-SUMO tag-thrombin cleavage site-SLP-1, Met-SUMO tag-thrombin cleavage site-SLP-1-His6 tag sequence.

[0014] The present application further provides a preparation method of the serine protease homolog SLP-1, which comprises:

[0015] The insect hemolymph is used as raw material, and one or two or more combinations of affinity chromatography, hydrophobic chromatography, ion exchange chromatography, gel filtration chromatography, salting-out and ultrafiltration are used for separation and purification to obtain SLP-1 with different purities, even electrophoretic purity or HPLC purity.

[0016] The basic conditions of the extraction, separation and purification system of SLP-1 are characterized by: (1) the operation temperature is 0-45°C, preferably 0-10°C; (2) the pH of the solution is pH 2-pH 12, preferably pH 4-pH 10; (3) the chemical reagent for adjusting the pH of the solution is a common and general acid or base and its solution, the acid and its solution are preferably HCl, HAc, phosphoric acid, citric acid, sulfuric acid and boric acid, and the base and its solution are preferably NaOH, KOH, Tris, sodium or potassium citrate, sodium or potassium phosphate and borax; (4) the buffer is a common and general buffer ion pair buffer, preferably citrate buffer ion pair, HCl-Tris buffer ion pair, citrate-phosphate buffer ion pair, phosphate buffer ion pair, acetate buffer ion pair, boric acid buffer ion pair, boric acid-Tris buffer ion pair and a combination of the above buffer ions; (5) the ionic strength of the solution or buffer is 0.001-0.6 mol / L, preferably 0.01-0.2 mol / L. The above conditions do not destroy the physicochemical properties of the medium used for extraction, separation and purification, and do not affect the biological activity of SLP-1.

[0017] The separation and analysis method of the present application comprises one or a combination of two or more of the following:

[0018] 1. Ion exchange chromatography separation and purification of SLP-1

[0019] The insect hemolymph obtained by the above method is adjusted to the required condition range by an acidic or basic solution according to the basic conditions of the extraction, separation and purification system of SLP-1. The treated sample is loaded onto an ion exchange chromatography column which has been equilibrated with a buffer, and then washed with the buffer to remove the non-absorbed impurities. The elution mode can be a salt concentration stage mode, using 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, 1 mol / L, 2 mol / L and 3 mol / L salt solutions for stage elution, or a salt concentration gradient mode, with a gradient of 0.00-3 mol / L. The presence of the target protein is detected by anti-SLP-1 antibody, and the eluate containing SLP-1 is combined and stored for future use; or a common and general dialysis or ultrafiltration method is used to remove the salt from the combined eluate, or further dialysis or ultrafiltration treatment is performed with a low concentration buffer as needed, and the above sample solution is stored for future use.

[0020] The characteristics of ion exchange chromatography separation and purification are as follows: (1) the selection of cation exchange chromatography medium, such as CM-cation exchange chromatography medium or SP-cation exchange chromatography medium or S-cation exchange chromatography medium, and the pH of the buffer is selected in the range of pH 2 to pH 7; (2) the selection of anion exchange chromatography medium, such as Q-anion exchange chromatography medium or DEAE-anion exchange chromatography medium or QAE-anion exchange chromatography medium, and the pH of the buffer is selected in the range of pH 7 to pH 12; (3) the selection of buffer and its concentration, which is described in the above SLP-1 extraction, separation and purification system basic conditions; (4) the elution salt solution can be selected from the buffer with the required concentration or the neutral salt added in the buffer to the required concentration; (5) the selection of neutral salt, such as (NH4)2SO4 or Na2SO4 or NaCl or KCl, and NaCl is preferred; (6) the operation temperature of separation and purification, which is described in the above SLP-1 extraction, separation and purification system basic conditions. The above conditions do not affect the biological activity of SLP-1 and the separation and purification of the active ingredient.

[0021] 2. Affinity chromatography separation and purification of SLP-1

[0022] The insect hemolymph obtained by the above method is adjusted to the pH range required by the SLP-1 extraction, separation and purification system basic conditions by using an acidic solution or an alkaline solution. The treated sample solution is loaded onto an affinity chromatography column which is previously equilibrated with a buffer. The unabsorbed impurities are removed by washing with the buffer. The elution can be performed by using salt (or chemical reagent) concentration gradient (0.0 mol / L to 3.0 mol / L or 0.0 mol / L to 6.0 mol / L or 0.0 mol / L to 8.0 mol / L); or by using salt (or chemical reagent) stage concentration elution, such as 0.1 mol / L, 0.2 mol / L, 0.5 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L salt solution. The presence of the target protein is detected by using anti-SLP-1 antibody, and the eluate containing SLP-1 is combined and stored for future use. The salt (or chemical reagent) in the combined eluate can be removed by using conventional and general dialysis or ultrafiltration method, or further dialysis or ultrafiltration treatment with the required low concentration buffer, and the above sample solution is stored for future use.

[0023] The characteristics of affinity chromatography separation and purification are as follows: (1) the ligand of affinity filler is selected from SLP-1 antibody, lipopolysaccharide, heparin, concanavalin A, formaldehyde-fixed bacteria or fungi, serine protease inhibitor (such as benzamidine), sepharose CL-4B, etc.; (2) the temperature, buffer, and pH of separation and purification operation are selected according to the characteristics of SLP-1 extraction, separation, and purification system; (3) the elution salt (or chemical reagent) solution can be selected from buffer with required concentration or salt (or chemical reagent) added in buffer to the required concentration; (4) the elution salt (or chemical reagent) can be selected from (NH4)2SO4, Na2SO4, NaCl, KCl, urea, or guanidine hydrochloride; (5) the highest concentration of (NH4)2SO4, Na2SO4, NaCl, or KCl is 3.0 mol / L, the highest concentration of urea is 8.0 mol / L, and the highest concentration of guanidine hydrochloride is 6.0 mol / L; (6) if urea or guanidine hydrochloride is selected as the elution salt (or chemical reagent) to denature SLP-1, the SLP-1 can be refolded by conventional and general protein refolding method to obtain SLP-1.

[0024] 3. Hydrophobic chromatography separation and purification of SLP-1

[0025] The insect hemolymph obtained by the above method is adjusted to the pH range required by the characteristics of SLP-1 extraction, separation, and purification system with an acidic or basic solution. Neutral salt is added to a concentration of 2-3 mol / L, and then loaded onto a hydrophobic chromatography column previously equilibrated with 2-3 mol / L neutral salt-buffer solution. The unabsorbed impurities are removed by washing with 2-3 mol / L neutral salt-buffer solution. The elution mode can be gradient elution with neutral salt concentration (3.0 mol / L-0.0 mol / L) or stage elution with 2.5 mol / L, 2.0 mol / L, 1.5 mol / L, 1.0 mol / L, 0.5 mol / L, 0.25 mol / L, 0.2 mol / L, 0.1 mol / L, and 0.0 mol / L neutral salt solution, respectively. The presence of target protein is detected by anti-SLP-1 antibody, and the eluate containing SLP-1 is combined and stored for future use. The salt in the combined eluate can be removed by conventional and general dialysis or ultrafiltration, or further dialysis or ultrafiltration with low-concentration buffer required, and the sample solution is stored for future use.

[0026] The characteristics of hydrophobic chromatography separation and purification are: (1) the hydrophobic chromatography medium is selected from phenyl-hydrophobic chromatography additive or n-octane-hydrophobic chromatography additive or hexane-hydrophobic chromatography additive or butane-hydrophobic chromatography additive; (2) the neutral salt is selected from (NH4)2SO4 or Na2SO4 or NaCl; (3) the temperature, buffer and pH of the separation and purification operation are selected according to the characteristics described in the basic conditions of SLP-1 extraction, separation and purification system.

[0027] 4. Gel chromatography separation and purification of SLP-1

[0028] The insect hemolymph obtained by the above method is adjusted to the pH range required by the basic conditions of SLP-1 extraction, separation and purification system with an acidic or basic solution. The sample solution is loaded onto a gel filtration chromatography column equilibrated with a buffer and subjected to separation and purification elution. The presence of the target protein is detected by anti-SLP-1 antibody, and the eluate containing SLP-1 is combined and stored for future use. The salt in the combined eluate can also be removed by conventional and general dialysis or ultrafiltration, or further dialysis or ultrafiltration treatment with a low concentration buffer as needed, and the above sample solution is stored for future use.

[0029] The characteristics of gel chromatography separation and purification are: (1) the chromatography medium can be selected from Sephacryl S-100HR or Sephacryl S-200HR or Sephadex G-50 or Sephadex G-75 or Sephadex G-100 or Sephadex G-150 or Superose 12 prep grade or Superose 6 prep grade or Superdex 30 prep grade or Superdex 75 prep grade or Superose 12HR or Superose 6HR or Superdex Peptide HR or Superdex 75 HR or Superdex Peptide PE and the like gel chromatography additive; (2) the temperature, buffer and pH of the separation and purification operation are selected according to the characteristics described in the basic conditions of SLP-1 extraction, separation and purification system, and in addition, the concentration of the eluate is preferably greater than 0.15M and above.

[0030] 5. Salt precipitation separation and purification of SLP-1

[0031] Take the insect hemolymph obtained by the above method, according to the basic conditions of SLP-1 extraction, separation and purification system characteristics, adjust the pH to the range of the basic conditions of SLP-1 extraction, separation and purification system characteristics with acidic or alkaline solution. Add the conventional and general neutral salt for protein salting-out to the sample solution to a concentration that makes SLP-1 still in a dissolved state, while some impurities are in a precipitated state. Centrifuge to obtain the supernatant solution, and continue to add the conventional and general neutral salt for protein salting-out to a concentration that makes SLP-1 precipitate. Discard the supernatant and dissolve the precipitate in the solution or buffer of the basic conditions of SLP-1 extraction, separation and purification system for storage; the dissolved solution of the precipitate can also be treated by conventional and general dialysis or ultrafiltration to remove the salt or further dialyzed or ultrafiltrated with a low-concentration buffer required for storage of the above sample solution.

[0032] The characteristics of salting-out separation and purification are as follows: (1) the selection of the buffer and pH for separation and purification is based on the characteristics of the basic conditions of SLP-1 extraction, separation and purification system; (2) the operation temperature for separation and purification is 0-45°C, preferably 0°C; (3) the neutral salt used for salting-out is selected from (NH4)2SO4 or Na2SO4 or NaCl, preferably (NH4)2SO4 or Na2SO4; (4) the concentration of the neutral salt when SLP-1 is in a dissolved state is selected from 5%-50%, preferably 25%-40%; (5) the concentration of the neutral salt when SLP-1 is in a salting-out precipitated state is selected from 45%-90%, preferably 55%-70%.

[0033] 6. Ultrafiltration separation and purification of SLP-1 and treatment of SLP-1 solution

[0034] Take the insect hemolymph obtained by the above method, according to the basic conditions of SLP-1 extraction, separation and purification system characteristics, adjust the pH to the range of the basic conditions of SLP-1 extraction, separation and purification system characteristics with acidic or alkaline solution. Utilize the conventional and general ultrafiltration method to separate and purify SLP-1. In one scheme, select an ultrafiltration membrane of a certain specification to make SLP-1 pass through the ultrafiltration membrane, while some impurities are retained by the ultrafiltration membrane, so that SLP-1 is separated and purified; select an ultrafiltration membrane of a certain specification to make SLP-1 be retained, while some impurities pass through the ultrafiltration membrane, so that SLP-1 is separated and purified. In another scheme, select an ultrafiltration membrane of a certain specification to make SLP-1 be retained first, and then select an ultrafiltration membrane of a certain specification to make SLP-1 pass through the ultrafiltration membrane, so that SLP-1 is separated and purified.

[0035] The purpose of ultrafiltration treatment of SLP-1 solution is to remove salts or small molecular impurities in SLP-1 solution or to replace buffer. In addition, SLP-1 solution is concentrated. The treatment method is as described above, a certain specification of ultrafiltration membrane is selected, so that SLP-1 is retained by the ultrafiltration membrane, and the salt or small molecular impurity or the buffer ion pair of the buffer solution permeates the ultrafiltration membrane, thereby achieving the purpose of removing salt, small molecular impurity or replacing buffer or concentration.

[0036] The characteristics of ultrafiltration separation, purification and treatment are: (1) the ultrafiltration membrane permeable to SLP-1 selects an ultrafiltration membrane with a molecular weight of 20 kDa or 30 kDa or 40 kDa or 50 kDa or 60 kDa, preferably an ultrafiltration membrane with a molecular weight of 20 kDa-50 kDa, and the yield or ultrafiltration efficiency of an ultrafiltration membrane with a molecular weight greater than or less than the preferred specification is affected; (2) the ultrafiltration membrane retaining SLP-1 selects an ultrafiltration membrane with a molecular weight of 3 kDa or 10 kDa or 20 kDa or 30 kDa, preferably an ultrafiltration membrane with a molecular weight of 10 kDa-30 kDa, and the yield or ultrafiltration efficiency of an ultrafiltration membrane with a molecular weight greater than or less than the preferred specification is affected; (3) the operating temperature, buffer and its pH or concentration selection of ultrafiltration separation, purification or treatment are described in the characteristics of the basic conditions of SLP-1 extraction, separation and purification system.

[0037] The purity of SLP-1 obtained by the above method sometimes cannot meet the corresponding needs. The present application is to separate and purify high-purity SLP-1 from insect hemolymph, and can achieve electrophoretic purity or even HPLC purity. The above six separation and purification methods (ion exchange column chromatography, affinity column chromatography, hydrophobic column chromatography, gel filtration column chromatography, salting-out, ultrafiltration) can be freely combined in two separation and purification methods and their order rearranged, or freely combined in three separation and purification methods and their order rearranged, or freely combined in four separation and purification methods and their order rearranged, or freely combined in five separation and purification methods and their order rearranged, or freely combined in six separation and purification methods and their order rearranged, until the purity of the sample meets the expected requirements.

[0038] The present application performs structural analysis on SLP-1 according to the techniques, methods and means of conventional protein chemistry and molecular biology, including: (1) determining the molecular weight of natural SLP-1 by using biological mass spectrometry; (2) degrading SLP-1 obtained according to the present application by using conventional proteolytic enzymes and their hydrolysis conditions, separating the degradation fragments by HPLC, and analyzing the partial amino acid sequences by using biological mass spectrometry or Edman degradation method, so as to obtain the amino acid sequences of multiple fragments within SLP-1; (3) extracting total RNA from insect fat body by using molecular biology techniques and methods, and constructing insect cDNA pool by using RACE technology. According to the amino acid sequences of the degradation fragments of the target protein, primers are designed, and the fragment gene is amplified by PCR. Then, SLP-1 gene-cDNA is obtained by combining RACE technology, the base sequence thereof is obtained by gene sequence determination and analysis, and the full-length primary structure of SLP-1 is deduced from the open reading frame base sequence; (4) extracting the chromosomal gene of the insect from the insect fat body by using molecular biology techniques and methods. The upstream and downstream primers are designed, the chromosomal gene of the insect is used as a template, and the SLP-1 chromosomal gene is amplified by PCR. The intron and exon sequences in the SLP-1 chromosomal gene are obtained by gene sequence determination and analysis; (5) verifying the above structural information by using the molecular weight, partial amino acid sequences within the molecule, cDNA open reading frame sequence, and exon sequences in the chromosomal gene of SLP-1 obtained by the above steps 1-4, so as to obtain the DNA sequence of SLP-1.

[0039] In another aspect, the present application provides a method for preparing a derivative or analog of recombinant SLP-1

[0040] The derivative or analog or active fragment of serine protease homolog SLP-1 according to the present application is selected from Met-SLP-1, Met-His6 tag-SLP-1, Met-SLP-1-His6 tag, Met-His6 tag-thrombin cleavage site-SLP-1, Met-GST tag-thrombin cleavage site-SLP-1, Met-SLP-1-thrombin cleavage site-GST tag, Met-SLP-1-Flag tag, Met-Flag tag-SLP-1, Met-His6 tag-SUMO tag-thrombin cleavage site-SLP-1, Met-SUMO tag-thrombin cleavage site-SLP-1-His6 tag sequence.

[0041] The derivative or analog or active fragment of the serine protease homolog SLP-1 of the present application is achieved by the following technical solution, comprising: 1) recombining the DNA encoding SLP-1 and partial fragments or derivatives or analogs thereof into an expression vector; 2) transforming appropriate host cells (prokaryotic or eukaryotic cells) with the recombinant expression vector of step 1); 3) culturing the transformed host cells of step 2) under suitable conditions for inducing expression; and 4) harvesting and purifying the obtained target protein.

[0042] The present application also provides a method for separating and purifying the expression product of SLP-1 and partial fragments or derivatives or analogs thereof. The desired expression product can be separated and purified from the lysate or culture solution of the genetically engineered cells by using methods such as salt precipitation, ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration, as well as various combinations of the above methods. During the separation and purification of the expression product, the presence and corresponding molecular size of the expression product can be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), or Western blotting.

[0043] The present application also provides the biological functions of SLP-1 and partial fragments or derivatives or analogs thereof, and applications thereof

[0044] Another object of the present application is to determine the biological activity of natural, recombinant SLP-1 and partial fragments or derivatives or analogs thereof, in terms of the activation of the prophenoloxidase activation system induced by pathogen-associated molecular patterns and microorganisms. At the same time, the expression of SLP-1 in the process of immune response of the body is investigated, and the applications of natural, recombinant SLP-1 and partial fragments or derivatives or analogs thereof are also studied. In addition, the present application also studies the preparation of antibodies stimulated by natural, recombinant SLP-1 and partial fragments or derivatives or analogs thereof as antigens, and the applications thereof.

[0045] The present application has the following beneficial effects relative to the prior art:

[0046] The method for obtaining natural, recombinant SLP-1 and partial fragments or derivatives or analogs thereof is conventional, simple, and high-yield. The natural, recombinant serine protease homolog SLP-1 and partial fragments or derivatives or analogs thereof, as well as antibodies thereof, can be widely applied in the fields of prevention, detection, and therapeutic drugs for microorganisms. At the same time, the antibodies prepared by using natural, recombinant serine protease homolog SLP-1 and partial fragments or derivatives or analogs thereof as antigens can be applied in the fields of prevention, detection, and therapeutic drugs for microorganisms. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced as follows.

[0048] Figure 1 Electrophoretogram of isolation and purification of native SLP-1, wherein, Lane M: Molecular weight markers; lane 1: native SLP-1 purified by method 1; lane 2: native SLP-1 purified by method 2; lane 3: native SLP-1 purified by method 3.

[0049] Figure 2 Electrophoretogram of isolation and purification of recombinant SLP-1 (prokaryotic expression system), wherein, Lane M: Molecular weight markers; lane 1: SLP-1 without tag; lane 2: SLP-1 with N-terminal pre-fusion histidine tag; lane 3: SLP-1 with N-terminal pre-fusion histidine tag-SUMO tag and thrombin cleavage site; lane 4: SLP-1 with C-terminal post-fusion histidine tag.

[0050] Figure 3 Electrophoretogram of isolation and purification of recombinant SLP-1 (insect expression system), wherein, Lane M: Molecular weight markers; lane 1: recombinant SLP-1 expressed by pFastBac1-sf9 insect expression system; lane 2: SLP-1 expressed by pMIB / V5-His-Sf21 insect expression system.

[0051] Figure 4 Relationship between SLP-1 mRNA expression and immunity.

[0052] Figure 5 Effect of exogenous recombinant SLP-1 and its polyclonal antibody on prophenoloxidase activating system, wherein, HL: hemolymph of Antheraea pernyi, SLP-1: recombinant SLP-1 protein; Ab: recombinant SLP-1 rabbit polyclonal antibody; error bar is mean ± standard deviation, and the experiment was repeated for 3 times; * represents significant difference in t test, P<0.1, ** represents significant difference in t test, P<0.05, **** represents significant difference in t test, P<0.001.

[0053] Figure 6To reduce the inhibitory effect of endogenous SLP-1 on phenol oxidase proactivating system, wherein, Buffer: injection buffer solution control group; dsEGFP: injection of EGFP double-stranded RNA 36h after blood lymph; dsSLP-1: injection of SLP-1 double-stranded RNA 36h after blood lymph; error line is mean ± standard deviation, experimental repeat 3 times; ** represents t test with significant difference, P<0.05; *** represents t test with significant difference, P<0.01.

[0054] Figure 7 To analyze the binding ability of SLP-1 and pathogen-associated molecular patterns, wherein, A: SLP-1 and LPS binding; B: SLP-1 and DAP-PGN binding; C: SLP-1 and Lys-PGN binding; D: SLP-1 and Laminarin binding; E: SLP-1 and LTA binding; F: SLP-1 and Mannan binding. DETAILED DESCRIPTION

[0055] The following examples can make the professional technical personnel more fully understand the present application, and not in any way limit the scope of the present application claims.

[0056] Example 1: isolation and purification of natural SLP-1

[0057] In this example, the tussah silk moth is repeatedly washed with distilled water or deionized water, and blood lymph is collected at 10℃ to-5℃ by using conventional methods such as wax disc method, centrifugation method, dorsal blood vessel blood collection method, perfusion method, squeezing, homogenization method, reflex flow blood collection method, tearing method, cutting method, cutting open method, puncture method, etc.

[0058] 1. Method-1

[0059] The blood lymph was collected, and the precipitate was collected after ammonium sulfate precipitation, dissolved in 10 mM Tris-HCl, 150 mM NaCl, pH 7.4 buffer solution, and flowed through LPS modified Sepharose 4B, and eluted under the following conditions: 10 mM Tris-HCl, 500 mM NaCl, pH 7.4; 10 mM Tris-HCl, 150 mM NaCl, 4 M Urea, pH 7.4; 10 mM Tris-HCl, 150 mM NaCl, 8 M Urea, pH 7.4, and the target component was collected. The target component was dialyzed against 10 mM glycine / NaOH pH 8.5, and flowed through hydroxyapatite HPLC, and the equilibrium buffer was 10 mM glycine / NaOH pH 8.5, and the linear gradient elution was performed twice with 10-150 mM glycine / NaOH pH 8.5 and 150 mM-1 M glycine / NaOH pH 8.5. The collected component was dialyzed against 10 mM Tris-HCl pH 7.4, and flowed through Mono S HPLC, and the linear gradient elution was performed using 0-0.1 M NaCl, and the target protein component was obtained.

[0060] The test results are shown in Figure 1, lane 1, and the purity of the natural SLP-1 reached electrophoretic purity. Figure 1

[0061] 2. Method-2

[0062] A mixture of fungi (Candida albicans), gram-positive bacteria (Staphylococcus aureus) 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) was injected into the body of the tussah silkworm, and the bacteria-induced blood lymph was collected after induction for 24-48 hours, diluted 10 times with 50 mM Tris-HCl buffer pH 5.5, and flowed through a Mono-Q ion exchange chromatography column, and linear gradient elution was performed using 0.05-1.5 M NaCl in 50 mM Tris-HCl buffer pH 5.5. The target component was collected, concentrated to 1 mL using an ultrafiltration membrane, and then loaded onto a gel filtration chromatography column (Toyopearl HW-55S, 1 x 30 cm), and the equilibrium and elution systems were both 50 mM Tris-HCl 150 mM NaCl 3 mM EDTA pH 7.5, and the target protein component was obtained.

[0063] The test results are shown in Figure 2, lane 2, and the purity of the natural SLP-1 reached electrophoretic purity. Figure 1

[0064] 3. Method-3 ​​

[0065] After centrifugation to remove blood cells from the silkworm body fluid dissolved in an anticoagulant buffer solution, ammonium sulfate fractionation was performed, and the 30-45% precipitate fraction was dissolved and diluted with borax-sodium hydroxide buffer (0.05 mol / L, pH 9.0), and then loaded onto an SP-Sepharose anion exchange column, and eluted by washing with 50 mM-1.5 M NaCl; the fraction containing the target protein was loaded onto a Phenyl-sepharose 6-Fast Flow column, and eluted by washing; the fraction containing the target protein was dialyzed and then loaded onto a heparin chromatography column under the same conditions, and eluted with 20 mM Tris-HCl, 0.3 M NaCl, pH 5.0; the eluted fraction was desalted by ultrafiltration, and the fraction containing the target fragment was loaded onto an LPS affinity chromatography column Sepharose CL-6B column for further separation and purification, thereby obtaining the target protein.

[0066] The test results are as follows Figure 1 , lane 3, the purity of natural SLP-1 reached electrophoretic purity.

[0067] Example 2: SLP-1 structure analysis and gene sequence analysis

[0068] According to conventional protein chemistry and molecular biology techniques, methods and means, the structure of SLP-1 was analyzed. The primary structure-amino acid sequence of natural SLP-1 (also known as mature peptide chain, referred to as SLP-1 in this patent) is shown in SEQ ID NO: 1, and the gene sequence encoding natural SLP-1 is shown in SEQ ID NO: 2.

[0069] Using molecular biology techniques and methods, the full-length cDNA sequence of SLP-1 was obtained, as shown in SEQ ID NO: 3, and the SLP-1 gene open reading frame is 1266 bp in length, which can encode 421 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 4), and the N-terminal contains a signal peptide region of 16 amino acid residues.

[0070] The homology alignment results for the structure of SLP-1 according to the present application show that SLP-1 has 58% identity with Manduca sexta serine proteinase-like protein 4, 55% identity with Manduca sexta serine proteinase-like protein 1b, 55% identity with Bombyx mori clip domain serine protease 11, and 54% identity with Helicoverpa armigera serine proteinase-like protein 1.

[0071] Example 3: Obtainment of recombinant SLP-1 and its derivatives, analogs, active fragments by using prokaryotic expression system

[0072] This example illustrates the construction strategy and basic method for expressing the gene of SLP-1 and its derivatives, analogs, active fragments of the present application by using prokaryotic expression system.

[0073] Structure of SLP-1 derivatives, analogs, active fragments

[0074] (1) Amino acid sequence of Met-SLP-1

[0075] MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY

[0076] (2) Amino acid sequence of Met-His6 tag-SLP-1

[0077] M HHHHHHQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY

[0078] (3) Met-SLP-1-His6 tag amino acid sequence

[0079] MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY HHHHHH

[0080] (4) Met-His6 tag-thrombin cleavage site-SLP-1 amino acid sequence

[0081] M HHHHHHLVPRGSQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY

[0082] (5) Met-GST tag - Thrombin cleavage site - SLP-1 amino acid sequence

[0083] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVP RGSQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY

[0084] (6) Met-SLP-1-thrombin cleavage site-GST tag amino acid sequence

[0085] MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY LVPRGSSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

[0086] (7) Met-SLP-1-Flag tag amino acid sequence

[0087] MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY DYKDDDDK

[0088] (8) Met-Flag tag-SLP-1 amino acid sequence

[0089] M DYKDDDDKQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWA NPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAA HIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAP NVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSIL SYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVY VDVSKVRNWIDDEVRGKGYMSEVYTY

[0090] (9) Met-His6 tag-SUMO tag-thrombin cleavage site-SLP-1 amino acid sequence

[0091] M HHHHHH SASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNF LVPRGSQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWA NPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAA HIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAP NVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSIL SYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVY VDVSKVRNWIDDEVRGKGYMSEVYTY

[0092] (10) Met-SUMO tag-thrombin cleavage site-SLP-1-His6 tag amino acid sequence

[0093] MSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNF LVPRGSQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWAN PEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHI IKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGV VCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQ LHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVR NWIDDEVRGKGYMSEVYTYHHHHHH.

[0094] 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.

[0095] The method for separating and purifying the expression product is the method, principle and strategy of Example 1.

[0096] 1. Construction of SLP-1 gene expression vector

[0097] According to the N-terminal and C-terminal amino acid sequences of SLP-1, 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, a biotechnology service company is submitted for DNA sequence determination. Through the above genetic engineering method, the expression vector of SLP-1 gene is constructed.

[0098] The features of the expression vector construction in this embodiment are as follows: 1. The expression vector 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 to the N-terminal of SLP-1 as the affinity chromatography tag; 3. A peptide segment can be fused to the C-terminal of SLP-1 as the affinity chromatography tag; 4. The tag can be selected from His-Tag (containing six or more consecutive histidines), GST-Tag, Flag-Tag, etc.; 5. The amino acid sequence of the proteolytic enzyme cleavage site, such as thrombin, enterokinase, blood coagulation factor X, etc. can be added between the affinity chromatography tag and SLP-1 to obtain the recombinant SLP-1 protein consistent with the structure of the native SLP-1 protein.

[0099] 2. Obtaining of the recombinant SLP-1 protein and its derivatives, analogs, active fragments

[0100] The SLP-1 gene expression vector is transformed into E. coli by genetic engineering technology, and the single colony is inoculated into LB medium containing antibiotics to induce the expression of the SLP-1 gene, thereby obtaining the culture solution or bacterial body containing SLP-1. The bacterial body is first lysed by lysis buffer and ultrasonically broken to release the target protein, and then the supernatant is collected by centrifugation as the raw material solution of the recombinant SLP-1 for standby use.

[0101] The features of the expression of the recombinant target gene are as follows: 1. The transformation of the expression vector into the host can be selected from heat transformation and electroporation; 2. The induction of the expression includes chemical induction-isopropyl β-D-thiogalactoside (IPTG) induction and warming induction; 3. The SLP-1 gene can be expressed in the cell or outside the cell; 4. The SLP-1 present in the cell needs to be released into the solution by lysis buffer lysis, ultrasonic breaking, etc.

[0102] According to the methods, principles, strategies, etc. in Example 1, the recombinant SLP-1 and its derivatives, analogs, active fragments are separated and purified from the above-mentioned raw material solution containing SLP-1 to the required purity until reaching electrophoretic purity or HPLC purity.

[0103] For example: (1) The pTYB11 is used to construct the SLP-1 expression vector without a tag, the expression vector is transformed into the host cell by electroporation, and SLP-1 is expressed in the cell after IPTG induction. The bacterial body is resuspended by lysis buffer, ultrasonically broken, and centrifuged to obtain the supernatant as the raw material solution for further separation and purification of SLP-1. According to the methods, principles, strategies, etc. in Example 1, SLP-1 is separated and purified to electrophoretic purity (Figure 1, lane 1). Figure 2

[0104] ​(2) Using pET-28a to construct SLP-1 gene with N-terminal pre-fusion histidine tag, heat transforming E. coli, and inducing with IPTG, His-SLP-1 is expressed in the cell. Resuspend the bacterial body with lysis buffer (50 mmol / L PBS, 0.15 mol / L NaCl, 50 mmol / L imidazole), and perform ultrasonic disruption. Centrifugation obtains supernatant as raw material liquid for further separation and purification of SLP-1. According to the method, principle, strategy, etc. of Example 1, separate and purify SLP-1 to electrophoretic purity (Figure 2, lane 2). Figure 2

[0105] (3) Using pET-28a-SUMO to construct SLP-1 expression vector with N-terminal pre-fusion histidine-SUMO tag and thrombin cleavage site, heat transforming E. coli, and inducing expression by warming, His-SUMO-thrombin cleavage site-SLP-1 is expressed in the cell. Resuspend the bacterial body with lysis buffer, and perform ultrasonic disruption. Centrifugation obtains supernatant and thrombin hydrolysis sample for removing tag as raw material liquid for further separation and purification of SLP-1. According to the method, principle, strategy, etc. of Example 1, separate and purify SLP-1 to electrophoretic purity (Figure 2, lane 3). Figure 2

[0106] (4) Using pET20b to construct SLP-1 gene with C-terminal post-fusion histidine tag, and using electroporation method to transform the expression vector into host cell, SLP-1-His is expressed in the cell. According to the method, principle, strategy, etc. of Example 1, separate and purify SLP-1 to electrophoretic purity (Figure 2, lane 4). Figure 2

[0107] The above purified expression product with tag is subjected to hydrolysis of the above conventional and general proteolytic enzyme (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 SLP-1. The structure of the recombinant SLP-1 is the same as that of natural SLP-1.

[0108] Example 4: Obtaining recombinant SLP-1 and its derivatives, analogs, active fragments by using eukaryotic expression system

[0109] This example lists and describes the construction strategy and basic method of expressing SLP-1 and its analogs, active fragments of the application by using insect cell expression system.

[0110] The expression vector, expression host cell, and expression strategy of the insect cell expression system are all conventional and general expression vector, expression host cell, and expression strategy for genetic engineering expression.

[0111] ​​​This example is to make the professional technical personnel more comprehensive understanding of the present application, and is not in any way limit the scope of the present application.

[0112] For the separation and purification method of the expression product, the method, principle, strategy, etc. of Example 1 are adopted.

[0113] 1. Obtain recombinant SLP-1 and its derivatives, analogs, active fragments by using pFastBacl-sf9 insect expression system

[0114] Link the SLP-1 and its derivatives, analogs, active fragment genes to the pFastBacl plasmid, construct the pFastBacl-SLP recombinant expression plasmid, transfect E. coli DH10, and after Bluo-gal and IPTG induction, obtain the transfection recombinant bacmid by blue-white screening, transfect insect cells sf9, and verify the expression of recombinant SLP-1 in cells by Western blot.

[0115] Collect the cells, resuspend with lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), centrifuge after ultrasonic crushing to obtain the raw material liquid containing the target protein. According to the method, principle, strategy, etc. of Example 1, separate and purify SLP-1 to electrophoretic purity. The structure of the expression product is as shown in Figure 4 , and the electrophoretic identification result after purification is as shown in Figure 3 , lane 1.

[0116] 2. Obtain recombinant SLP-1 and its derivatives, analogs, active fragments by using pMIB / V5-His-Sf21 insect expression system

[0117] Link the SLP-1 and its derivatives, analogs, active fragment genes to the pMIB / V5-His plasmid, construct the pMIB / V5-His-SLP-1 recombinant expression plasmid, transfect E. coli DH5, and after Bluo-gal and IPTG induction, obtain the transfection recombinant bacmid by blue-white screening, transfect insect cells Sf21, and verify the expression of recombinant SLP-1 in cells by Western blot.

[0118] Collect the cells, resuspend with lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), centrifuge after ultrasonic crushing to obtain the raw material liquid containing the target protein. Directly load on the pre-equilibrated metal ion chelation chromatography column, after 0.02 mol / L imidazole (pH 8.0) is fully washed to remove a large amount of impurities, elute with 0.5 mol / L imidazole (pH 8.0), and high-efficiency expression of the recombinant protein is obtained, reaching electrophoretic purity. The electrophoretic identification result after purification is as shown inFigure 3 lane 2.

[0119] Example 5: Obtaining Anti-SLP-1 Antibodies

[0120] According to conventional and general antibody production techniques, the immune systems of mice or rats or rabbits or dogs or sheep or horses or cattle are stimulated to produce corresponding antibodies using the various SLP-1 obtained in Examples 1, 3, and 4 as antigens.

[0121] Using conventional and general antibody detection methods, the production of SLP-1 antibodies in the sera of immunized mice or rats or rabbits or dogs or sheep or horses or cattle is detected.

[0122] After the immunized mice or rats or rabbits or dogs or sheep or horses or cattle produce SLP-1 antibodies, the sera of the immunized mice or rats or rabbits or dogs or sheep or horses or cattle are collected and stored using conventional and general animal serum collection and storage methods. The sera can be used directly.

[0123] Using conventional and general antibody separation and purification techniques, such as salting-out, various types of chromatographic media, antibody affinity chromatographic media, and the like, SLP-1 antibodies of different purities are separated and purified from the stored sera containing SLP-1 antibodies until electrophoretically pure or HPLC pure SLP-1 antibodies are obtained to suit different application requirements.

[0124] Example 6: Biological Activity of Recombinant and Natural SLP-1, Derivatives, Analogs, Active Fragments, and Antibodies Thereof

[0125] The recombinant, natural SLP-1, and derivatives, analogs, and active fragments thereof in this example have the same biological activity. Descriptions are made using the tussock moth as a representative of the biological activity experimental insects of Lepidoptera. Professional technical personnel can further expand the application range of SLP-1 and its derivatives, analogs, and active fragments based on the biological activity of SLP-1 and its derivatives, analogs, and active fragments.

[0126] 1. Analysis of the correlation between SLP-1 and microbial infection

[0127] E. coli, Staphylococcus aureus, and Candida albicans were injected into tussock moth larvae using insect saline. The tussock moth larvae were collected at 0 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, and 48 h, and total RNA was extracted from the whole worms. After reverse transcription, the expression level of SLP-1 at the mRNA level was detected by real-time quantitative PCR. The results are shown in Figure 4 After induction by the three pathogenic bacteria, the expression of SLP-1 gradually increased and reached a peak at 18 h, and then gradually decreased. The above experimental results show that SLP-1 has certain correlation with the innate immune defense system of insects.

[0128] 2. Effects of SLP-1 and its antibodies on prophenoloxidase-activating system

[0129] (1) Effects of exogenous recombinant SLP-1 and its polyclonal antibodies on prophenoloxidase-activating system

[0130] Representative PAMPs of Gram-negative bacteria, Gram-positive bacteria and fungi, DAP-PGN, Lys-PGN and Laminarin, were used to activate the blood plasma of the silkworm, Bombyx mori, respectively. Subsequently, the effects of exogenous recombinant SLP-1 on the above-mentioned activation were investigated. On the other hand, the effects of endogenous SLP-1 on the prophenoloxidase-activating system were investigated by blocking the function of endogenous SLP-1 with the addition of SLP-1 polyclonal antibodies. The native blood plasma was used as a control group. The results, as shown in Figure 5 , indicated that PAMPs could significantly activate the prophenoloxidase cascade activation system compared with the control group. Exogenous recombinant SLP-1 could significantly enhance this process, while anti-SLP-1 polyclonal antibodies could significantly inhibit this process. The above-mentioned experimental phenomena indicated that SLP-1 in the blood plasma was involved in the activation of the PAMPs-mediated prophenoloxidase cascade system and induced the production of PO.

[0131] (2) Reduction of the inhibitory effect of endogenous SLP-1 on the prophenoloxidase-activating system

[0132] To further investigate the effects of endogenous SLP-1 on the PPO-AS, the expression level of SLP-1 was down-regulated by injecting double-stranded RNA of SLP-1 into the silkworm larvae using RNAi technology. The blood plasma of the silkworm collected 36 h after the interference was used to investigate the activation of PPO in each group under the induction of six PAMPs. The results, as shown in Figure 6 , indicated that the PO activity of the SLP-1 interference group was significantly lower than that of the groups injected with dsEGFP and buffer solution, which inversely indicated that SLP-1 in the blood plasma was involved in the activation of the PAMPs-mediated prophenoloxidase cascade system.

[0133] 3. Binding specificity of SLP-1 and its analogues and active fragments to microbial associated molecular patterns

[0134] The microscale thermophoresis (MST) was used to detect the recognition ability of SLP-1 and its analogs, active fragments to six kinds of soluble typical molecular patterns (PAMPs) from different kinds of microorganisms. Lys-PGN and LTA belong to specific PAMPs of Gram-positive bacteria, DAP-PGN and LPS belong to specific PAMPs of Gram-negative bacteria, laminarin (soluble β-1, 3 glucan) and mannan belong to specific PAMPs of fungi. The same concentration of PAMPs was coated on the probe, and SLP-1 or its analogs, active fragments were incubated with the probe respectively. The binding of recombinant PGRP-SA or its analogs, active fragments to soluble microorganism-related molecular patterns was detected by measuring the directional movement of molecules in the micro temperature gradient field. As shown in Figure 7 The same experimental results were obtained by using natural SLP-1 or recombinant SLP-1 analogs, active fragments described in examples 1, 3 and 4.

[0135] The above experimental results show that the natural, recombinant SLP-1 and its derivatives, analogs, active fragments of the present application can specifically recognize typical molecular patterns from different kinds of microorganisms and significantly activate the phenol oxidase proactivator system, and the anti-SLP-1 antibody can inhibit the phenol oxidase proactivator system.

[0136] Example 7: Application of natural, recombinant SLP-1 and its derivatives, analogs, active fragments and antibodies

[0137] This example is described by taking the biological activity of SLP-1 as a representative, and SLP-1 derivatives, analogs, active fragments also have the same biological activity. At the same time, tussah silkworm is also taken as a representative of the biological activity of insects in Lepidoptera. Based on the biological activity of SLP-1 and its derivatives, analogs, active fragments and antibodies, the application range of SLP-1 and its derivatives, analogs, active fragments and antibodies can be further expanded.

[0138] 1. SLP-1 and its derivatives, analogs, active fragments for detecting microorganisms

[0139] As described in example 6, SLP-1 and its derivatives, analogs, active fragments can specifically bind to various microorganism-related molecular patterns such as lipopolysaccharide, β-1, 3-glucan, peptidoglycan, lipoteichoic acid, mannan, etc., and act as effective activating components of the phenol oxidase proactivator system, and the anti-SLP-1 antibody can act as an inhibitor of the process by blocking the activity of SLP-1.

[0140] Any pathogenic microorganism to be detected, using natural SLP-1 or recombinant SLP-1 and its derivatives, analogs, active fragments, through the recognition of the typical molecular patterns contained in it, i.e. lipopolysaccharide or β-1,3-glucan or peptidoglycan or lipoteichoic acid or mannan, can be used for microbial examination; in addition, the microorganism to be detected can also be added to the additional SLP-1 supplemented tussah silkworm hemolymph, and the control group is to add the same dose of sample of the microorganism to be detected to the tussah silkworm hemolymph without SLP-1 supplement. At the same time, the activation of PPO system in hemolymph is observed, and there is a significant difference between the experimental group and the control group, indicating that the sample contains microorganisms or related molecular patterns.

[0141] 2. Application of SLP-1 and its analogs, active fragments and antibodies

[0142] Antibodies against SLP-1 and its derivatives, analogs, active fragments obtained in Example 5 are used for immunological and molecular biological techniques and methods to detect SLP-1 in lepidopteran insect samples. Similarly, it is also suitable for immunodetection and tracking analysis during the isolation and purification of SLP-1 from lepidopteran insects, as well as qualitative and quantitative detection and analysis of samples. The experiments in this aspect have been applied in the above-mentioned natural, recombinant SLP-1 and its derivatives, analogs, active fragments isolation and purification process.

[0143] In any sample of microorganism to be detected, a sufficient dose of SLP-1 antibody is added, and the method for detecting microorganisms using SLP-1 and its derivatives, analogs, active fragments in this embodiment is used for the detection of microorganisms in the sample to be detected. As above, even if the amount of microorganism that can be detected in the sample cannot be detected (negative result), this experiment is designed as a negative control group for the detection of microorganisms in the sample.

Claims

1. A serine protease homolog, SLP-1, characterized in that, The amino acid sequence of which is shown as SEQ ID NO:

1.

2. A gene encoding the serine protease homolog SLP-1 of claim 1.

3. The gene of serine protease homolog SLP-1 according to claim 2, characterized by, The nucleotide sequence of the gene is shown as SEQ ID NO:

2.

4. A derivative or analogue or active fragment of the serine protease homologue SLP-1 according to claim 1, characterised in that, The derivative or analog or active fragment of the serine protease homolog SLP-1 is selected from Met-SLP-1, Met-His6 tag-SLP-1, Met-SLP-1-His6 tag, Met-His6 tag-thrombin cleavage site-SLP-1, Met-GST tag-thrombin cleavage site-SLP-1, Met-SLP-1-thrombin cleavage site-GST tag, Met-SLP-1-Flag tag, Met-Flag tag-SLP-1, Met-His6 tag-SUMO tag-thrombin cleavage site-SLP-1, Met-SUMO tag-thrombin cleavage site-SLP-1-His6 tag sequence; The amino acid sequence of Met-SLP-1 is as follows: MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-His6 tag-SLP-1 is as follows: M HHHHHH QGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWA NPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAA HIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAP NVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSIL SYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVY VDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-SLP-1-His6 tag is as follows: MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY HHHHHH The amino acid sequence of Met-His6 tag-thrombin cleavage site-SLP-1 is as follows: M HHHHHH LVPRGSQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGV GMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVR AGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTR CFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDG GSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-GST tag-thrombin cleavage site-SLP-1 is as follows: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPRGS QGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-SLP-1-thrombin cleavage site-GST tag is as follows: MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWAN PEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHI IKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVG VVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYF QLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSK VRNWIDDEVRGKGYMSEVYTY LVPRGS SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGD VKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSK LPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIP QIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD The amino acid sequence of Met-SLP-1-Flag tag is as follows: MQGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY DYKDDDDK The amino acid sequence of Met-Flag tag-SLP-1 is as follows: M DYKDDDDK QGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVP YYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWA NPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAA HIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAP NVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSIL SYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVY VDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-His6 tag-SUMO tag-thrombin cleavage site-SLP-1 is as follows: M HHHHHH SASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNF LVPRGS QGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTY The amino acid sequence of Met-SUMO tag-thrombin cleavage site-SLP-1-His6 tag is as follows: MSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNF LVPRGS QGMKLLNEIIKRIFPLPSIETSTTRTTTSSTAKTAMNEIKPTAFVPPLNTNETSCTLNGKDGICVPYYLCDSNNKINVGGEGLVESRSFGPCLSNLDVCCFRPDQISSTEPNIKKMEPLKLQREGCGWANPEGVGMRTTDETDGKTKFGEFPWMVAIVKTESLIDNYPNGPNGTVYVGGGSLIHPSVVLTAAHIIKDRLNLKVRAGEWDTRTTKEIYRHQERDVESIVIHKEFNEETNYYDVAVLFLKSPMDMAPNVGVVCLPSHDDMAYPDTRCFASGWGKDKSTIQGRYSTTLKKVEVQVVAHDTCQASLRTSILSYYFQLHSTFMCASGKPGKDTCKGDGGSPLVCPIQFEKDRYVQNGIVSWGIRCGETGIPGVYVDVSKVRNWIDDEVRGKGYMSEVYTYHHHHHH.

5. A process for the preparation of the serine protease homologue SLP-1 according to claim 1, characterized in that, Using one or more than two kinds of combination of blood lymph, blood, blood cell lysate, lymph, homogenate of the silkworm as raw material liquid, electrophoretically pure or even HPLC pure serine protease homolog SLP-1 is obtained by one or more than two kinds of combination of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method; Or the gene encoding the serine protease homolog SLP-1 is cloned into a recombinant expression vector, introduced into a host cell, and the recombinant expressed serine protease homolog SLP-1 is obtained.

6. A method for the preparation of a derivative or analogue or active fragment of the serine protease homologue SLP-1 according to claim 4, characterised in that, The gene encoding the derivative or analog or active fragment of the serine protease homolog SLP-1 is cloned into a recombinant expression vector, introduced into a host cell, and the recombinant expressed derivative or analog or active fragment of the serine protease homolog SLP-1 is obtained after isolation and purification.

7. The use of the serine protease homolog SLP-1 of claim 1 or the derivative or analog or active fragment of the serine protease homolog SLP-1 of claim 4 in the preparation of a reagent for activating the phenoloxidase system or in the preparation of a microbial associated molecular pattern binding reagent; The microbial associated molecular pattern is Lys-PGN, LTA, DAP-PGN, LPS, laminarin or Mannan.

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