Mannan-bound serine protease homologues, their preparation methods and applications

Abstract

This invention discloses mannan-bound serine protease homologues, their preparation methods, and applications, belonging to the field of biomedical technology. This invention utilizes protein separation and purification technology to isolate and purify natural mannan-bound serine protease homologues from *Silphium tussah*. After resolving their primary structures (genes and proteins), genetic engineering technology is used to achieve the expression of the mannan-bound serine protease homologues, their derivatives or analogs, or partial fragments in host cells. Recombinant mannan-bound serine protease homologues, their derivatives or analogs, or partial fragments are then purified and used to immunize animals to obtain corresponding antibodies. The natural and recombinant mannan-bound serine protease homologues, their derivatives or analogs, or partial fragments, and their antibodies of this invention can be widely used in the fields of prevention, detection, and treatment of microorganisms.

Description

Technical fields:

[0001] This invention belongs to the field of biomedical technology, and relates to the structure, preparation method and application of mannan-bound serine protease homologues. Specifically, this invention relates to the structure and preparation method of mannan-bound serine protease homologues and their derivatives, analogs and active fragments, as well as their applications in the detection of microorganisms and related molecular patterns, induction of insect phenol oxidase activation systems, preparation of mannan-bound serine protease homologues and their derivatives, analogs and active fragment antibodies. Background technology:

[0002] The innate immune system is the most effective and only defense mechanism against invading exogenous pathogens in insects, and it is a crucial part of the ecosystem. In the serine protease cascade pathway, proteases are sequentially activated through specific molecular interactions and proteolysis after recognizing abnormal tissues or microorganisms, forming a rapid, localized amplification response of the initial signal, thereby exerting an immune effect. The active site of the catalytic domain at the carboxyl terminus of serine proteases is often composed of histidine, aspartic acid, and serine residues, i.e., a "catalytic triplet." When this catalytic triplet structure undergoes amino acid mutations or is directly deleted, these serine proteases lose their catalytic activity and are called serine protease homologs (SPH).

[0003] Serine proteases and their homologues play a role in the innate immunity of arthropods by participating in the cascade reactions of the prophenoloxidase activation system and the Toll pathway. In the tobacco hawk moth, after the host PRRs recognize invading pathogens, serine proteases HP14, HP21, proHP1, HP6, HP8, PAP1-3, and non-catalytic serine protease homologues (SPH1 and SPH2) can constitute the extracellular SP-SPH system to mediate melanin formation and other immune responses. In the mealworm, the serine protease cascade amplification system can simultaneously activate the Toll pathway and PPO, and this system includes three different serine proteases that can be activated sequentially. Only the presence of specific SPHs can properly activate prophenoloxidase in the mealworm, triggering melanin synthesis. The highly specific non-catalytic regulatory protein SPH1 in the mealworm is indispensable for regulating melanin production. In 2010, Zhao et al. (Zhao P, Wang G, Dong Z, et al. Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori[J]. BMC Genomics, 2010, 11(1): 405.) infected silkworm larvae with a mixed bacterial solution of silkworm nucleopolyhedrovirus, Beauveria bassiana, Bacillus thuringiensis and E. coli by feeding. After induction, the expression of 18 SP or SPH genes was significantly upregulated, indicating that SP and SPH genes may be involved in the resistance of silkworms to pathogenic microorganisms. In 2011, Sakamoto et al. (Sakamoto M, Ohta M, Suzuki A, et al. Localization of the serine protease homolog BmSPH-1 in nodules of E. coli-injected Bombyx mori larvae and functional analysis of its role in nodule melanization[J]. Developmental and Comparative Immunology, 2011, 35(5): 611-619.) showed that silkworm SPH1 and SPH2 may bind to the surface of E. coli along with lipopolysaccharide-binding protein BmLBP and enter the nodules through hemolymph, and are important regulators of nodule melanization.In 2018, Lee et al. (Lee KS, Kim BY, Choo YM, et al. Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm Bombyx mori[J]. Developmental and Comparative Immunology, 2018, 85: 170-176.) further discovered that silkworm SPH-1 can form a PPO activation complex with silkworm immunoglobulin (B. mori immulectin, BmIML), BmPPAE and BmPPO, which localizes to hemocytes during infection and promotes nodule formation in the immune response. In 2021, Yang et al. (Yang H, Ji T, Xiong H, et al. A trypsin-like serine protease domain of masquerade gene in crayfish Procambarusclarkii could activate prophenoloxidase and inhibit bacterial growth[J]. Developmental and Comparative Immunology, 2021, 117: 103980.) confirmed that the serine protease homologue Mas in Procambarus clarkii can activate the prophenoloxidase activation system by binding with bacteria, thereby participating in the innate immune defense of crustaceans.

[0004] In summary, serine protease homologues play a crucial role in insect defense against pathogenic microorganisms and in infection-related responses. Currently, there is a lack of research on the structure, preparation, and biological functions of serine protease homologues, especially mannan-bound serine protease homologues, from the Lepidoptera (Bombyx mori) family Bombyx mori. Summary of the Invention:

[0005] This invention focuses on mannan-bound serine protease homologues found in the Bombyx mori (Larger Bombyx mori) family of Lepidoptera. It investigates the preparation methods, primary structures (genes and proteins), biological functions, and applications of natural mannan-bound serine protease homologues. Furthermore, it utilizes genetic engineering techniques to obtain recombinant mannan-bound serine protease homologues, their derivatives or analogs, or active fragments, along with their biological functions and applications. Additionally, it uses natural and recombinant mannan-bound serine protease homologues, their derivatives or analogs, or active fragments as antigens to stimulate antibody production, and studies the applications of these antibodies.

[0006] In this invention, the term "host cell" includes both prokaryotic and eukaryotic cells. Common examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. Common examples of eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

[0007] The technical problem solved by this invention is to provide a method for preparing, defining the structure, biological function, and applications of a mannan-binding serine protease homologue (MSPH) from lepidopteran insects of the family Saturniidae. First, natural mannan-binding serine protease homologues are isolated and purified from lepidopteran insects of the family Saturniidae using protein extraction, separation, and purification techniques. Second, the primary structure (gene and protein) of the mannan-binding serine protease homologue is resolved using protein chemistry and molecular biology techniques, and its gene is obtained. Third, the gene of the mannan-binding serine protease homologue is expressed in host cells using genetic engineering techniques, and recombinant mannan-binding serine protease homologues are obtained using protein extraction, separation, and purification techniques. Simultaneously, derivatives, analogues, or partial fragments of the mannan-binding serine protease homologue are obtained using gene recombination technology. Natural and recombinant mannan-bound serine protease homologues and their derivatives, analogs, or fragments can bind to various microbial-associated molecular patterns, including β-1,3-glucan, peptidoglycan, lipoteichoic acid, and mannan, in addition to lipopolysaccharide. They can specifically activate the phenoloxidase proactivation system induced by various microbial-associated molecular patterns such as lipopolysaccharide, β-1,3-glucan, peptidoglycan, lipoteichoic acid, and mannan, as well as by bacteria, fungi, and other microorganisms, and inhibit the production of antimicrobial peptides mediated by the Toll signaling pathway.

[0008] This invention provides the following technical solution:

[0009] The present invention provides a mannan-bound serine protease homologue, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] Based on the above technical solution, the mannan-bound serine protease homologue is derived from insects of the family Saturniidae in the order Lepidoptera, and is selected from one of the following: tussah silkworm, castor silkworm, stork silkworm, Indian tussah silkworm, amber silkworm, American tussah silkworm, ailanthus silkworm, mountain silkworm, American stork silkworm, camphor silkworm, and maple silkworm.

[0011] In another aspect, the present invention provides a gene encoding the mannan-binding serine protease homologue described above.

[0012] Based on the above technical solution, the nucleotide sequence of the gene of the mannan-binding serine protease homologue is shown in SEQ ID NO: 2.

[0013] Another aspect of the present invention provides derivatives or analogs or active fragments of the mannan-binding serine protease homologue, comprising all or part of the amino acid sequence as shown in SEQ ID NO: 1, and having the biological activity of the mannan-binding serine protease homologue.

[0014] Based on the above technical solution, the derivatives or analogs or active fragments of the mannan-binding serine protease homologues are further selected from Met-MSPH, Met-His6 tag-MSPH, Met-MSPH-His6 tag, Met-His6 tag-thrombin cleavage site-MSPH, Met-GST tag-thrombin cleavage site-MSPH, Met-MSPH-thrombin cleavage site-GST tag, Met-MSPH-Flag tag, Met-Flag tag-MSPH, Met-His6 tag-SUMO tag-thrombin cleavage site-MSPH, and Met-His6 tag-SUMO tag-thrombin cleavage site-MSPH-His6 tag sequences.

[0015] Another aspect of the present invention provides a method for preparing the mannan-bound serine protease homologues, which utilizes one or more of the following as raw material solutions: hemolymph, blood, hemolymphocyte lysate, lymph fluid, and homogenate from the insect family Saturniidae of the order Lepidoptera. The mannan-bound serine protease homologues of electrophoretic purity or even HPLC purity are obtained by one or more of the following methods: ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting out, or ultrafiltration.

[0016] Alternatively, the gene encoding the mannan-binding serine protease homologue can be cloned into a recombinant expression vector, introduced into a host cell, and the recombinant mannan-binding serine protease homologue can be obtained.

[0017] Based on the above technical solutions, further, in the ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting out, or ultrafiltration methods:

[0018] (1) The operating temperature is 0℃-45℃, preferably 0℃-10℃;

[0019] (2) The pH of the solution is between pH 2 and pH 12, preferably between pH 4 and pH 10;

[0020] (3) The reagents for adjusting the acidity or alkalinity of the solution are conventional and commonly used acids, bases, acid solutions or alkaline solutions. The preferred acids or acid solutions are HCl, HAc, phosphoric acid, citric acid, sulfuric acid, boric acid or their mixtures. The preferred bases or alkaline solutions are NaOH, KOH, Tris, sodium or potassium citrate, sodium or potassium phosphate, borax or their mixtures.

[0021] (4) The buffer is a conventional, general-purpose 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, borate buffer ion pair, borate-Tris buffer ion pair or a combination of the above buffer ions.

[0022] (5) The ionic strength of the solution or buffer solution is between 0.001 mol / L and 0.5 mol / L, preferably between 0.01 mol / L and 0.1 mol / L.

[0023] Another aspect of the present invention provides a method for preparing derivatives or analogs or active fragments of the mannan-binding serine protease homologue, wherein the gene encoding the derivative or analog or active fragment of the mannan-binding serine protease homologue is cloned into a recombinant expression vector, introduced into a host cell, and after separation and purification, the recombinantly expressed derivative or analog or active fragment of the mannan-binding serine protease homologue is obtained.

[0024] Based on the above technical solution, the expression system further includes a prokaryotic system and an insect cell system. The host cell of the prokaryotic system is Escherichia coli cell or Bacillus subtilis cell; the host cell of the insect cell system is an insect cell; and the expression form is intracellular expression or secretory expression.

[0025] Another aspect of the present invention provides antibodies against the mannan-bound serine protease homologues or their derivatives or analogs or active fragments thereof, using the natural mannan-bound serine protease homologues or the mannan-bound serine protease derivatives or analogs or active fragments thereof as antigens to stimulate the immune system of mice, rats, rabbits, dogs, sheep, horses or cattle.

[0026] Another aspect of the present invention provides the application of the mannan-bound serine protease homologue, or the mannan-bound serine protease homologue derivative or analog, or active fragment, or the antibody, in the detection of the phenoloxidase activation system, antimicrobial peptide synthesis, microorganisms and their related molecular patterns.

[0027] Based on the above technical solution, the relevant molecular models further include β-1,3-glucan, peptidoglycan, lipoteichoic acid, and mannan.

[0028] The advantages of this invention over the prior art are as follows:

[0029] The method for obtaining natural and recombinant MSPH and its fragments, derivatives, or analogs described in this invention is conventional, simple, and yields high quantities. The natural and recombinant mannan-bound serine protease homologues, their derivatives, analogs, fragments, and antibodies of this invention can be widely used in the fields of prevention, detection, and treatment of microorganisms. Attached image description:

[0030] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0031] Figure 1 For the isolation and purification of natural MSPH, Lane M: Molecular weight markers; Lane 1: Natural MSPH purified by method 1; Lane 2: Natural MSPH purified by method 2; Lane 3: Natural MSPH purified by method 3.

[0032] Figure 2 For MSPH sequence phylogenetic analysis.

[0033] Figure 3 Electrophoresis patterns for the separation and purification of recombinant MSPH (prokaryotic expression system), where Lane M: Molecular weight markers; Lane 1: untagged MSPH; Lane 2: MSPH with an N-terminal histidine tag; Lane 3: MSPH with a C-terminal GST tag; Lane 4: MSPH with a C-terminal histidine tag.

[0034] Figure 4 Electrophoretic patterns for the separation and purification of recombinant MSPH (insect expression system), where Lane M: Molecular weight markers; Lane 1: Recombinant MSPH of pFastBac1-sf9 insect expression system; Lane 2: MSPH of pMIB / V5-His-Sf21 insect expression system.

[0035] Figure 5 To supplement the promoting effect of exogenous MSPH on the phenoloxidase activation system, where HL: silkworm hemolymph; MSPH: recombinant Ap-MSPH protein; MIC: microorganism; the error bar is mean ± standard deviation, and the experiment is repeated 3 times; * represents t-test P<0.05, ** represents t-test P<0.01, *** represents t-test P<0.001, and **** represents t-test P<0.0001.

[0036] Figure 6 To reduce the inhibitory effect of endogenous MSPH on the phenoloxidaseogen activation system, the following groups were selected: NT+buffer: control group injected with physiological saline; dsEGFP: group injected with EGFP double-stranded RNA; dsMSPH: group injected with Ap-MSPH double-stranded RNA. The error bar is mean ± standard deviation, and the experiment is repeated 3 times. ** represents t-test P < 0.01, *** represents t-test P < 0.001, and **** represents t-test P < 0.0001.

[0037] Figure 7 The effect of MSPH on antimicrobial peptide expression.

[0038] Figure 8 The binding affinity of MSPH to PAMPs was analyzed, where A: MSPH binds to Mannan; B: MSPH binds to DAP-PGN; C: MSPH binds to Lys-PGN; D: MSPH binds to Laminarin; E: MSPH binds to LTA; and F: MSPH binds to LPS.

[0039] Figure 9 To investigate the effect of anti-MSPH antibody on the phenoloxidase activation system, where HL: silkworm hemolymph; Ab: rabbit-derived Ap-MSPH polyclonal antibody; MIC: microorganism; the error bar is mean ± standard deviation, and the experiment was repeated 3 times; * represents t-test P<0.05, ** represents t-test P<0.01, *** represents t-test P<0.001, and **** represents t-test P<0.0001. Detailed implementation method:

[0040] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the scope of the claims in any way.

[0041] Example 1: Isolation and purification of natural MSPH

[0042] In this embodiment, the silkworms are repeatedly washed with distilled or deionized water, and hemolymph is collected using conventional methods such as wax plate method, centrifugation method, dorsal blood vessel blood collection method, perfusion method, pressing method, homogenization method, reflective bleeding method, tearing method, cutting method, slit method, puncture method, etc., under conditions of 10℃ to -5℃.

[0043] 1. Method-1

[0044] Hemolymph from silkworms was collected, precipitated with ammonium sulfate, and the precipitate was 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). A small amount of phenylthiourea (2.5 g / L) was added, and the mixture was co-incubated with mannan-agarose affinity chromatography material. Elution was performed using 10 mM imidazole-HCl, pH 7.8, 1.0 M NaCl, and 2 mM EDTA, and the target fraction was collected. The target protein was then obtained by linear gradient elution using Mono S HPLC with 0-0.1M NaCl. After separation and concentration by ultrafiltration, the sample was loaded onto gel filtration chromatography (Toyopearl HW-55S, 1×30cm). The equilibration and elution system was 50mM Tris-HCl, 150mM NaCl, and 3mM EDTA at pH 7.5 to obtain the target protein.

[0045] The test results are as follows Figure 1 Lane 1, the purity of natural MSPH reaches electrophoretic purity.

[0046] 2. Method-2

[0047] A mixture (10 μl) of fungi (Candida albicans), Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Escherichia coli) dissolved in insect physiological saline was injected into the silkworm. After induction for 24–48 hours, the induced hemolymph was collected and diluted 10-fold with 50 mM Tris-HCl buffer (pH 7.5) containing a small amount of phenylthiourea (2.5 g / L). The solution was then passed through a DEAE ion exchange chromatography column and eluted linearly using a 50 mM Tris-HCl buffer (pH 7.5) containing 0.05–1.5 M NaCl. The target fraction was collected, concentrated to 1 mL using ultrafiltration, and then loaded onto a gel filtration chromatography column (Toyopearl HW-55S, 1 × 30 cm). The equilibration and elution system was 50 mM Tris-HCl, 150 mM NaCl, and 3 mM EDTA (pH 7.5). The target protein component was obtained by passing it through hydroxyapatite HPLC with a equilibration buffer of 10 mM glycine / sodium hydroxide buffer at pH 8.5. The protein was then eluted twice with a linear gradient of 10-150 mM glycine / sodium hydroxide at pH 8.5 and 150 mM-1 M glycine / sodium hydroxide at pH 8.5 to obtain the target protein component.

[0048] The test results are as follows Figure 1 Lane 2, the purity of natural MSPH reaches electrophoretic purity.

[0049] 3. Method 3

[0050] After centrifuging to remove blood cells from the silkworm body fluid dissolved in anticoagulant buffer, ammonium sulfate fractionation was performed. 35–45% of the precipitate was dissolved and diluted in borax-sodium hydroxide buffer (0.05 mol / L, pH 9.0) and loaded onto an SP-Sepharose anion exchange column, eluted 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. The fraction containing the target protein was dialyzed and then loaded onto a Mannan-Agarose column under the same conditions, eluted with 10 mM imidazole-HCl, pH 7.8, 1.0 M NaCl, and 2 mM EDTA. The eluted fraction was desalted by ultrafiltration to obtain the target protein.

[0051] The test results are as follows Figure 1 Lane 3, the purity of natural MSPH reaches electrophoretic purity.

[0052] Example 2: MSPH structural analysis and its gene sequence analysis

[0053] The structure of MSPH was determined using conventional protein chemistry and molecular biology techniques, methods, and methods. The primary structure-amino acid sequence of natural MSPH (also known as the mature peptide chain, referred to as MSPH in this patent application) is shown in SEQ ID NO: 1, and the gene sequence encoding natural MSPH is shown in SEQ ID NO: 2.

[0054] The full-length cDNA sequence of MSPH was obtained using molecular biology techniques and methods, as shown in SEQ ID NO: 3. The MSPH gene open reading frame is 1290 bp in length and encodes 429 amino acid residues (its amino acid sequence is shown in SEQ ID NO: 4). Its N-terminus contains a signal peptide region of 18 amino acid residues.

[0055] Homology comparison analysis of the structure of MSPH in this invention showed that MSPH is closely related to serine proteases and their homologues in lepidopteran insects such as the beet armyworm and the silkworm (e.g., ...). Figure 2 (As shown).

[0056] Example 3: Obtaining recombinant MSPH and its derivatives, analogs, and active fragments using a prokaryotic expression system

[0057] This embodiment describes the construction strategy and basic method for expressing the MSPH and its derivatives, analogs, and active fragment genes of the present invention in a prokaryotic expression system.

[0058] MSPH and its derivatives, analogs, and active fragments include the following sequences:

[0059] (1) Met-MSPH amino acid sequence

[0060] MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0061] (2) Met-His6 Tag - MSPH Amino Acid Sequence

[0062] M HHHHHH QGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0063] (3) Met-MSPH-His6 Tag Amino Acid Sequence

[0064] MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH

[0065] (4) Met-His6 tag - Thrombin cleavage site - MSPH amino acid sequence

[0066] MHHHHHHLVPRGS QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0067] (5) Met-GST tag - Thrombin cleavage site - MSPH amino acid sequence

[0068] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVP RGSQGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0069] (6)Met-MSPH-Thrombin cleavage site-GST tag amino acid sequence

[0070] MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY LVPRGS ILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

[0071] (7) Met-MSPH-Flag tag amino acid sequence

[0072] MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYDYKDDDDK

[0073] (8) Met-Flag tag - MSPH amino acid sequence

[0074] MDYKDDDDK QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0075] (9) Met-His6 tag - SUMO tag - Thrombin cleavage site - MSPH amino acid sequence

[0076] MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGS QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY

[0077] (10)Met-His6 tag-SUMO tag-Thrombin cleavage site-MSPH-His6 tag amino acid sequence

[0078] MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGS QGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVR AGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKC QDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH.

[0079] The expression vectors, host cells, and expression strategies of prokaryotic expression systems are all conventional and universal expression vectors, host cells, and expression strategies used in gene engineering expression.

[0080] This embodiment is intended to enable those skilled in the art to fully understand the invention, and is not intended to limit the scope of the claims in any way.

[0081] The method for separating and purifying the expressed product is the same as the method, principle, and strategy described in Example 1.

[0082] 1. Construction of expression vector for MSPH gene

[0083] Based on the N-terminal and C-terminal amino acid sequences of MSPH, corresponding oligonucleotide primers were designed. Restriction endonuclease hydrolysis sites were added to the 5′ ends of these primers. PCR amplification was performed using an insect fat body cDNA pool as a template. The products were detected by agarose gel electrophoresis, and the nucleic acid fragments were recovered from the gel. After restriction endonuclease digestion, the resulting plasmid was double-digested and ligated with DNA ligase for recombination. The plasmid was then heat-transformed into competent *E. coli* cells. Positive transformants were obtained through colony PCR and restriction endonuclease digestion verification and submitted to a biotechnology service company for DNA sequencing. Using the above genetic engineering methods, an expression vector for the MSPH gene was constructed.

[0084] The features of the expression vector construction in this embodiment are as follows: 1. Using *E. coli* as the host, the expression vector can be selected from pTYB11, pMAL-C2X, pET-28a, pGEX-2T, pBV220, pQE30, pET20b, etc.; 2. A peptide can be fused to the N-terminus of MSPH as an affinity chromatography tag; 3. A peptide can be fused to the C-terminus of MSPH as an affinity chromatography tag; 4. The tag can be selected from His-Tag (six or more consecutive histidine residues), GST-Tag, Flag-Tag, etc.; 5. An amino acid sequence of a proteolytic enzyme hydrolysis site, such as thrombin, enterokinase, coagulation factor X, etc., can be added between the affinity chromatography tag and MSPH to obtain a recombinant MSPH protein with the same structure as the natural MSPH protein.

[0085] 2. Obtaining recombinant MSPH protein and its derivatives, analogs, and active fragments

[0086] Using genetic engineering techniques, the MSPH gene expression vector was transformed into E. coli. Single colonies were picked and inoculated into LB broiler culture containing antibiotics to induce MSPH gene expression, thereby obtaining a culture medium or bacterial cells containing MSPH. The bacterial cells were first lysed with lysis buffer and sonicated to release the target protein. The supernatant was then collected by centrifugation as the raw material for recombinant MSPH.

[0087] Characteristics of recombinant target gene expression: 1. The expression vector can be transformed into the host by thermal conversion or electroconversion; 2. The induction methods include chemical induction—isopropyl β-D-thiogalactoside (IPTG) induction and heating induction; 3. MSPH gene can be expressed intracellularly or extracellularly; 4. MSPH present in cells needs to be released into solution by means of lysis buffer lysis, sonication, etc.

[0088] Following the methods, principles, and strategies of Example 1, recombinant MSPH and its derivatives, analogs, and active fragments were separated and purified from the MSPH-containing raw material solution to the required purity, until electrophoretic purity or HPLC purity was achieved.

[0089] For example: (1) A tag-free MSPH expression vector was constructed using pTYB11, and the expression vector was transformed into host cells by electroporation. After IPTG induction, MSPH was expressed in the cells. The bacterial cells were resuspended in lysis buffer, sonicated, and centrifuged to obtain the supernatant as the raw material for further separation and purification of MSPH. MSPH was separated and purified to electrophoretic purity according to the methods, principles, and strategies of Example 1. Figure 3 Lane 1).

[0090] (2) An N-terminal pre-fused histidine-tagged MSPH gene was constructed using pET-28a, and heat-transformed into *E. coli*. After IPTG induction, His-MSPH was expressed intracellularly. The bacterial cells were resuspended in lysis buffer (50 mmol / L PBS, 0.15 mol / L NaCl, 50 mmol / L imidazole), sonicated, and centrifuged to obtain the supernatant, which was used as the raw material for further separation and purification of MSPH. Following the methods, principles, and strategies of Example 1, MSPH was separated and purified to electrophoretic purity (…). Figure 3 Lane 2).

[0091] (3) An MSPH expression vector with a C-terminal fused GST tag was constructed using pGEX-2T, heat-transformed into *E. coli*, and MSPH-GST was expressed intracellularly after induction by heating. The bacterial cells were resuspended in lysis buffer, sonicated, and centrifuged to obtain the supernatant, which was used as the raw material for further separation and purification of MSPH. Following the methods, principles, and strategies of Example 1, MSPH was separated and purified to electrophoretic purity (…). Figure 3 Lane 3).

[0092] (4) An MSPH gene with a C-terminal histidine tag was constructed using pET20b. The expression vector was transformed into host cells by electroporation, and expression was induced by heating, resulting in the extracellular expression of MSPH-His. Following the methods, principles, and strategies of Example 1, MSPH was isolated and purified to electrophoretic purity. Figure 3 Lane 4).

[0093] The above-mentioned tagged purified expression product is subjected to hydrolysis by conventional and common proteolytic enzymes (such as thrombin, enterokinase, coagulation factor X, etc.) to remove the fusion peptide in the expression product, and then separated and purified to obtain MSPH. The structure of the recombinant MSPH is the same as that of the natural MSPH.

[0094] Example 4: Obtaining recombinant MSPH and its derivatives, analogs, and active fragments using an insect cell expression system

[0095] This embodiment describes the construction strategy and basic method for expressing the MSPH and its derivatives, analogs, and active fragment genes of the present invention using an insect cell expression system.

[0096] The expression vectors, host cells, and expression strategies of the insect cell expression system are all conventional and universal expression vectors, host cells, and expression strategies used in gene engineering expression.

[0097] This embodiment is intended to enable those skilled in the art to fully understand the present invention, and is not intended to limit the scope of the claims of the present invention in any way.

[0098] For the separation and purification of the expressed product, the method, principle, and strategy of Example 1 are adopted.

[0099] 1. Using the pFastBac1-sf9 insect expression system, recombinant MSPH and its derivatives, analogs, and active fragments were obtained.

[0100] MSPH and its derivatives, analogs, and active fragment genes were ligated into the pFastBac1 plasmid to construct the pFastBac1-βGRP recombinant expression plasmid. After transposition in E. coli DH10, Bluo-gal, and IPTG induction, the transposition recombinant bacmid was obtained by blue-white screening. The recombinant MSPH was then transfected into insect sf9 cells, and Western blot was used to verify the expression of recombinant MSPH in the cells.

[0101] Cells were collected, resuspended in lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), sonicated, and centrifuged to obtain a raw material solution containing the target protein. Following the methods, principles, and strategies of Example 1, MSPH was purified to electrophoretic purity. The structure of the expression product is shown below. Figure 4 As shown in lane 1.

[0102] 2. Recombinant MSPH and its derivatives, analogs, and active fragments were obtained using the pMIB / V5-His-Sf21 insect expression system.

[0103] MSPH and its derivatives, analogs, and active fragment genes were ligated into the pMIB / V5-His plasmid to construct the pMIB / V5-His-βGRP recombinant expression plasmid. After transposition in E. coli DH5, Bluo-gal, and IPTG induction, the transposition recombinant bacmid was obtained by blue-white screening. The recombinant MSPH was then transfected into insect Sf21 cells, and Western blot was used to verify the expression of recombinant MSPH in the cells.

[0104] Cells were collected, resuspended in lysis buffer (0.05 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0), sonicated, and centrifuged to obtain a raw material solution containing the target protein. This solution was directly loaded onto a pre-equilibrated metal ion chelation chromatography column. After thorough washing with 0.02 mol / L imidazole (pH 8.0) to remove a large amount of contaminating protein, elution was performed with 0.5 mol / L imidazole (pH 8.0). The recombinant protein was efficiently expressed, achieving electrophoretic purity. The electrophoretic identification results after purification are shown below. Figure 4 lane 2.

[0105] Example 5: Obtaining anti-MSPH antibody

[0106] Using conventional and common antibody production techniques, various MSPHs obtained in Examples 1, 3, and 4 were used as antigens to stimulate the immune systems of mice, rats, rabbits, dogs, sheep, horses, or cattle to produce corresponding antibodies.

[0107] The production of MSPH antibodies in the serum of immunized mice, rats, rabbits, dogs, sheep, horses, or cattle was detected using conventional and universal antibody detection methods.

[0108] Once immunized mice, rats, rabbits, dogs, sheep, horses, or cattle develop MSPH antibodies, serum from these immunized mice, rats, rabbits, dogs, sheep, horses, or cattle is collected and stored using standard and universal animal serum collection and storage methods. This serum can be used directly.

[0109] Using conventional and universal antibody separation and purification techniques, such as salting out, various types of chromatography media, and antibody affinity chromatography media, MSPH antibodies of different purities can be separated and purified from stored serum containing MSPH antibodies until electrophoretic or HPLC-pure MSPH antibodies are obtained to suit different applications.

[0110] Example 6: Bioactivity of recombinant and natural MSPH and its derivatives, analogs, and active fragments

[0111] In this embodiment, recombinant, natural MSPH and its derivatives, analogs, and active fragments exhibit the same biological activity. The tussah silkworm is used as a representative lepidopteran insect in the description of its biological activity. Those skilled in the art can use the biological activity of MSPH and its derivatives, analogs, and active fragments as a core foundation to further expand the application scope of MSPH and its derivatives, analogs, and active fragments.

[0112] 1. Effects of MSPH on the phenoloxidaseogen activation system

[0113] (1) The promoting effect of supplementing exogenous MSPH on the phenoloxidase activation system

[0114] The effects of recombinant and natural MSPH on the phenoloxidaseogen activation system were investigated using six soluble pathogen-associated molecular models and three pathogenic microorganisms. The results are as follows: Figure 5 As shown, compared with the buffer solution control group, all six soluble pathogen-associated molecular patterns and three pathogenic microorganisms significantly activated the phenol oxidase-associated system (PPO-AS); and after the addition of exogenous recombinant MSPH or natural MSPH, all experimental groups showed a significant increase in phenol oxidase activity.

[0115] (2) Reduce the inhibitory effect of endogenous MSPH on the phenoloxidase-activating system

[0116] RNAi technology was used to reduce the expression of endogenous MSPH, and the effect of endogenous MSPH on PPO-AS was further investigated. Results are as follows: Figure 6 As shown, compared with the saline injection control group and the dsEGFP control group, the PO activity in each experimental group after MSPH interference was significantly reduced.

[0117] 2. Effects of MSPH on antimicrobial peptide expression

[0118] In this experiment, after downregulating endogenous Ap-MSPH expression 24 hours after injection of dsMSPH, E. coli, S. aureus, and C. albicans were injected into tussah silkworm larvae to investigate six antimicrobial peptides and key proteins in the Toll pathway within the silkworm. Changes in mRNA levels. Results as follows: Figure 7 As shown, compared with the control group, the MSPH-interference experimental group, after injection of E. coli, showed increases in antimicrobial peptides Defensin, Gloverin, Attacin, Lysozyme, Lebocin, Moricin, and key proteins of the Toll pathway. The mRNA levels were significantly upregulated. The same results were also observed under S. aureus-induced immunization conditions.

[0119] 3. Binding specificity of MSPH and its analogues, active fragments, and microbial-associated molecular patterns.

[0120] Microthermophoresis (MST) was used to detect the recognition ability of MSPH and its analogues, and active fragments, for six soluble typical molecular patterns (PAMPs) from different microbial species. Lys-PGN and LTA are Gram-positive bacteria-specific PAMPs, DAP-PGN and LPS are Gram-negative bacteria-specific PAMPs, and laminarin (soluble β-1,3-glucan) and Mannan are fungal-specific PAMPs. PAMPs of the same concentration were coated onto the probe. MSPH or its analogues, and active fragments were co-incubated with the probe. The binding of recombinant PGRP-SA or its analogues, and active fragments to soluble microbial-related molecular patterns was detected by measuring the directional movement of molecules in the sample within a microscopic temperature gradient field. Figure 8 As shown, recombinant His6-MSPH exhibits broad-spectrum binding activity to PAMPs other than LPS. The same experimental results can be obtained using the natural MSPH or recombinant MSPH analogues and active fragments described in Examples 1, 3, and 4.

[0121] The above experimental results show that the natural and recombinant MSPH, as well as its derivatives, analogs, and active fragments of this invention, have broad-spectrum binding properties to PAMPs other than LPS. Furthermore, on the one hand, it is a component of the prophenoloxidase-activating system in insects, significantly activating the prophenoloxidase-activating system; on the other hand, it has a significant inhibitory effect on the production of antimicrobial peptides in insects.

[0122] Example 7: Applications of natural and recombinant MSPH and its derivatives, analogs, active fragments and antibodies

[0123] This embodiment uses the biological activity of MSPH as a representative example for description. MSPH derivatives, analogs, and active fragments also possess the same biological activity. The tussah silkworm is also used as a representative experimental insect for biological activity among lepidopteran insects. Those skilled in the art can further expand the application scope of MSPH and its derivatives, analogs, active fragments, and antibodies based on the biological activity of MSPH and its derivatives, analogs, active fragments, and antibodies.

[0124] 1. MSPH and its derivatives, analogs, and active fragments are used for the detection of microorganisms.

[0125] As described in Example 6, MSPH and its derivatives, analogs, and active fragments can be used as effective activating components for the phenol oxidase proactivation system.

[0126] Any pathogenic microorganism to be tested was added to silkworm hemolymph supplemented with MSPH. The control group consisted of the same dose of the sample containing the pathogenic microorganism to silkworm hemolymph without MSPH supplementation. The activation of the PPO system in the hemolymph was observed at the same time points. Significant differences were observed between the experimental and control groups, indicating that the tested samples contained the microorganism or its related molecular pattern.

[0127] 2. Applications of MSPH and its derivatives, analogs, and active fragment antibodies

[0128] Antibodies against MSPH and its derivatives, analogs, and active fragments obtained in Example 5 were used to perform MSPH immunoassays on lepidopteran insect samples using conventional and universal techniques and methods in immunology and molecular biology. Similarly, this method is also applicable to immunoassay tracking analysis and qualitative and quantitative analysis of samples during the isolation, purification, and preparation of MSPH from lepidopteran insects. Experiments in this area have already been applied in the examples described above regarding the isolation, purification, and preparation of natural and recombinant MSPH and its derivatives, analogs, and active fragments.

[0129] Endogenous MSPH protein was blocked using anti-MSPH antibody. The effects of endogenous MSPH on the phenoloxidaseogen activation system were investigated using six soluble pathogen-associated molecular patterns (PAMPs) and three pathogenic microorganisms (MICs). Results are as follows: Figure 9 As shown, compared with the control group, PAMPs and MICs significantly activated PPO-AS; simultaneously, the addition of anti-MSPH antibody resulted in a significant decrease in PO activity in all experimental groups. This indicates that MSPH antibody can inhibit the activation of the phenoloxidaseogen activation system induced by microorganisms and their related molecular patterns.

[0130] A sufficient dose of MSPH antibody was added to any sample of microorganisms to be tested. Microbial detection of the sample was performed according to the method described in this embodiment for the detection of MSPH and its derivatives, analogs, and active fragments. As before, even if the sample contained a detectable amount of microorganisms, they could not be detected (negative result). This experiment was designed as a negative control group for microbial detection of the sample.

[0131] The above results indicate that antibodies against MSPH and its derivatives, analogs, and active fragments shield the binding bioactivity of MSPH and its related molecular patterns by binding to them, thereby rendering MSPH and its derivatives, analogs, and active fragments inactive. This binding shielding principle has broad applicability.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

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

1.

2. A gene encoding the mannose-binding serine protease homolog according to claim 1.

3. The gene of a mannan-binding serine protease homolog according to claim 2, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO:

2.

4. A recombinant mannan-binding serine protease homologue, characterized in that, The recombinant mannose-binding serine protease homolog is selected from Met-MSPH, Met-Histidine tag-MSPH, Met-MSPH-His6 tag, Met-His6 tag-Thrombin cleavage site-MSPH, Met-GST tag-Thrombin cleavage site-MSPH, Met-MSPH-Thrombin cleavage site-GST tag, Met-MSPH-Flag tag, Met-Flag tag-MSPH, Met-His6 tag-SUMO tag-Thrombin cleavage site-MSPH, Met-His6 tag-SUMO tag-Thrombin cleavage site-MSPH-His6 tag sequence; The amino acid sequence of Met-MSPH is as follows: MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY; The amino acid sequence of Met-Histidine tag-MSPH is as follows: M HHHHHH QGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY; The amino acid sequence of Met-MSPH-His6 tag is as follows: MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH Amino acid sequence of Met-His6 tag-thrombin cleavage site-MSPH is as follows: MHHHHHHHLVPRGS QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY; Amino acid sequence of Met-GST tag-thrombin cleavage site-MSPH is as follows: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD LVPR GSQGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY; Amino acid sequence of Met-MSPH-thrombin cleavage site-GST tag is as follows: MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY LVPRGS ILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD; Amino acid sequence of Met-MSPH-Flag tag is as follows: MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYDYKDDDDK The amino acid sequence of Met-Flag tag-MSPH is as follows: MDYKDDDDK QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY The amino acid sequence of Met-His6 tag-SUMO tag-thrombin cleavage site-MSPH is as follows: MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGS QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY; The amino acid sequence of Met-His6 tag-SUMO tag-thrombin cleavage site-MSPH-His6 tag is as follows: MHHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGS QGDV MGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH 5. The method of producing a mannan-binding serine protease homologue according to claim 1, characterized in that, The gene encoding the mannose-binding serine protease homolog is cloned into a recombinant expression vector, introduced into a host cell, and a recombinantly expressed mannose-binding serine protease homolog is obtained.

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