CN116286754A - Mannan-binding serine protease homolog and preparation method and application thereof - Google Patents
Mannan-binding serine protease homolog and preparation method and application thereof Download PDFInfo
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- CN116286754A CN116286754A CN202310196396.0A CN202310196396A CN116286754A CN 116286754 A CN116286754 A CN 116286754A CN 202310196396 A CN202310196396 A CN 202310196396A CN 116286754 A CN116286754 A CN 116286754A
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Abstract
The invention discloses a mannan-binding serine protease homolog and a preparation method and application thereof, belonging to the technical field of biological medicine. The invention uses protein separation and purification technology to separate and purify natural mannan combined serine proteinase homolog from tussah, analyzes the primary structure (gene and protein), uses gene engineering technology to realize the expression of mannan combined serine proteinase homolog and derivative or analogue or partial fragment gene in host cell, and purifies to obtain recombinant mannan combined serine proteinase homolog and derivative or analogue or partial fragment thereof, and immunizes animals to obtain corresponding antibody. The natural and recombinant mannans combined serine proteinase homolog, its derivative or analogue or partial fragment and its antibody can be extensively used in the fields of prevention, detection and therapeutic medicine for microbe, etc.
Description
Technical field:
the invention belongs to the technical field of biological medicines, relates to a structure of a mannan-binding serine protease homolog and an obtaining method and application thereof, and in particular relates to a structure of the mannan-binding serine protease homolog and derivatives, analogues and active fragments thereof, a preparation method thereof, and application thereof in detection of microorganisms and related molecular modes thereof, induction of an insect phenol oxidation zymogen activation system, preparation of the mannan-binding serine protease homolog and derivatives, analogues and active fragment antibodies thereof and the like.
The background technology is as follows:
the innate immune system is the most effective and unique defense mechanism of insects against invasion by foreign pathogens, and is an important part of the ecosystem. Proteases in the serine protease cascade pathway are sequentially activated through specific molecular interactions and proteolysis after recognizing abnormal tissues or microorganisms, and form rapid and local amplification reaction of initial signals, thereby exerting immune effects. The active site of the catalytic domain at the carboxy terminus of serine proteases often consists of histidine, aspartic acid and serine residues, i.e. "catalytic triplets". When the catalytic triplet structure is mutated or directly deleted, the serine protease loses catalytic activity, called serine protease homolog (Serine protease homolog, SPH).
Serine proteases and their homologs play a role in the innate immunity of arthropods by participating in the cascade of the pro-phenoloxidase activation system and Toll pathway. In tobacco astronomical moths, after host PRRs recognize invading pathogens, serine proteases HP14, HP21, proHP1, HP6, HP8, PAP1-3 and non-catalytic serine protease homologs (SPH 1 and SPH 2) can constitute extracellular SP-SPH systems that mediate melanin formation and other immune responses. In yellow meal worm, the Toll pathway and PPO are activated simultaneously by a serine protease cascade amplification system that includes three different serine proteases that can be activated sequentially. Only the presence of specific SPH can activate pro-phenoloxidase properly in yellow mealworms triggering melanin synthesis. A highly specific non-catalytic regulatory protein, yellow meal worm SPH1, is essential 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.) A mixed bacterial liquid of silkworm nuclear polyhedrosis virus, beauveria bassiana, bacillus montarvensis and E.coli infects silkworm larvae in a feeding mode, and after induction, 18 SP or SPH gene expression is obviously up-regulated, which indicates that SP and SPH genes possibly participate in the resistance of silkworm to pathogenic microorganisms. Studies by Sakamoto et al (Sakamoto M, ohta M, suzuki A, et al localization of the serine protease homolog BmSPH-1in 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.) in 2011 indicate that silkworm SPH1 and SPH2 may bind to E.coli surface along with lipopolysaccharide binding protein BmLBP from haemolymph into nodules, are important mediators of nodule blackening. Further studies by Lee et al (Lee K S, kim B Y, choo Y M, et al, dual role of the serine protease homolog BmSPH-1in the development and immunity of the silkworm Bombyx mori[J ]. Developmental and Comparative Immunology,2018, 85:170-176.) have found that silkworm SPH-1 can form PPO-activated complexes with silkworm immunoglobulins (B.mori immunoglobulin, bmIML), bmPPAE and BmPPO, localize to blood cells during infection, and promote nodule formation in immune responses. Yang et al (Yang H, ji T, xiong H, et al A try psin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth [ J ]. Developmental and Comparative Immunology,2021, 117:103980.) in 2021 demonstrate that serine protease homolog Mas in procambarus clarkia can participate in crustacean innate immune defenses by activating the pro-phenoloxidase activation system in combination with bacteria.
In summary, serine protease homologs play a critical role in the insect's resistance to invasion and infection-related reactions by pathogenic microorganisms. There is no research on the structure, preparation and biological functions of serine protease homologs of insects of the family Lepidoptera (Lepidotera) and the family Fabaceae, especially of mannan-binding serine protease homologs.
The invention comprises the following steps:
the invention relates to a preparation method, a primary structure (genes and proteins), biological functions and application of natural mannan-binding serine protease homologs, and relates to the preparation method, the biological functions and application of the natural mannan-binding serine protease homologs, wherein the recombinant mannan-binding serine protease homologs, derivatives or analogues or active fragments thereof are obtained by utilizing a genetic engineering technology. In addition, natural and recombinant mannans combined serine proteinase homolog and its derivative or analogue or active fragment are used as antigen to stimulate body to produce antibody, and its application is studied.
In the present invention, the term "host cell" includes both prokaryotic cells and eukaryotic cells, and examples of commonly used prokaryotic host cells include E.coli, bacillus subtilis, and the like. Common eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
The technical problem to be solved by the invention is to provide a preparation method, a structure, a biological function and application thereof for obtaining Mannan-binding serine protease homolog (Mannan-binding Serine Protease homologue, MSPH) from lepidoptera (large) silkworm moth insects. Firstly, natural mannans combined serine protease homologs are obtained from lepidoptera (large) silkworm moth insects by separation and purification technology of protein extraction, separation and purification. Next, the primary structure (gene and protein) of the mannan-binding serine protease homolog was analyzed and its gene was obtained using protein chemistry techniques as well as molecular biology techniques. And thirdly, expressing the mannan-binding serine protease homolog gene in host cells by utilizing a genetic engineering technology, and obtaining the recombinant mannan-binding serine protease homolog by using a binding protein extraction, separation and purification technology. Meanwhile, derivatives or analogues or partial fragments of the mannan-binding serine protease homologs are obtained using gene recombination techniques. The natural and recombinant mannans combined serine proteinase homolog, its derivative or analogue or partial fragment can combine various microorganism related molecular modes except lipopolysaccharide, including beta-1, 3-glucan, peptidoglycan, lipoteichoic acid, mannans, etc., and can specifically activate various microorganism related molecular modes of lipopolysaccharide, beta-1, 3-glucan, peptidoglycan, lipoteichoic acid, mannans, etc., and the phenol oxidation zymogen activation system induced by bacteria, fungi, etc., and has inhibiting effect on the production of antibacterial peptide mediated by Toll signal pathway.
The invention provides the following technical scheme:
the invention provides a mannan-binding serine protease homolog, the amino acid sequence of which is shown in SEQ ID NO: 1.
Based on the technical scheme, further, the mannan-binding serine protease homolog is derived from Lepidoptera (Lepidotera) insects of the family Fabricius (Saturn iidae), is selected from one of tussah, castor, wild silkworm, indian tussah, amber silkworm, american tussah, ailanthus, dashan silkworm, american wild silkworm, camphorwood silkworm, and maple silkworm.
In another aspect, the invention provides a gene encoding said mannan-binding serine protease homolog.
Based on the technical scheme, further, the nucleotide sequence of the gene of the mannan-binding serine protease homolog is shown as SEQ ID NO: 2.
In another aspect, the invention provides a derivative or analogue or active fragment of the mannan-binding serine protease homolog comprising the amino acid sequence as set forth in SEQ ID NO:1 and has the biological activity of the mannan-binding serine protease homolog.
Based on the above technical scheme, further, the derivative or analogue or active fragment of the mannans-binding serine protease homolog is selected from Met-MSPH, met-His 6 tag-MSPH, met-MSPH-His 6 Tag, met-His 6 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-His 6 tag-SUMO tag-thrombin cleavage site-MSPH, met-His 6 tag-SUMO tag-thrombin cleavage site-MSPH-His 6 A tag sequence.
The invention also provides a preparation method of the mannan-binding serine protease homolog, which uses one or more than two of haemolymph, blood, hemocyte lysate, lymph fluid and homogenate of lepidoptera silkworm moth insects as raw material liquid, and obtains the mannan-binding serine protease homolog with electrophoretic purity and even HPLC purity through one or more than two of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration methods;
or cloning the gene for encoding the mannan-binding serine protease homolog into a recombinant expression vector, and introducing the recombinant expression vector into a host cell to obtain the recombinant expression mannan-binding serine protease homolog.
Based on the above technical scheme, further, in the ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method:
(1) Operating temperatures in the range from 0 ℃ to 45 ℃, preferably 0 ℃ to 10 ℃;
(2) The pH value of the solution is between pH2 and pH12, preferably between pH4 and pH10;
(3) The reagent for regulating the pH value of the solution is conventional and universal acid, alkali, acid solution or alkali solution, wherein the acid or acid solution is preferably HCl, HAc, phosphoric acid, citric acid, sulfuric acid, boric acid or a mixed solution thereof, and the alkali or alkali solution is preferably NaOH, KOH, tris, sodium citrate or potassium salt, sodium phosphate or potassium salt, borax or a mixed solution thereof;
(4) The buffer is a conventional and universal buffer ion pair buffer, preferably a citrate buffer ion pair, an HCl-Tris buffer ion pair, a citrate-phosphate buffer ion pair, a phosphate buffer ion pair, an acetate buffer ion pair, a borate-Tris buffer ion pair or a combination of the buffer ions;
(5) The ionic strength of the solution or buffer is in the range of 0.001mol/L to 0.5mol/L, preferably 0.01mol/L to 0.1mol/L.
In another aspect, the present invention provides a method for preparing the derivative or analogue or active fragment of the mannans-bound serine protease homolog, cloning the gene encoding the derivative or analogue or active fragment of the mannans-bound serine protease homolog into a recombinant expression vector, introducing the recombinant expression vector into a host cell, and separating and purifying the recombinant expression vector to obtain the derivative or analogue or active fragment of the mannans-bound serine protease homolog.
Based on the technical scheme, the expression system further comprises a prokaryotic system and an insect cell system, wherein host cells of the prokaryotic system are escherichia coli cells or bacillus subtilis cells; the host cells of the insect cell system are insect cells; the expression form is intracellular expression or secretion form expression.
In another aspect, the invention provides antibodies to said mannan-binding serine protease homolog or derivative or analog or active fragment thereof, which stimulate the immune system of mice or rats or rabbits or dogs or sheep or horses or cattle using said natural mannan-binding serine protease homolog or said derivative or analog or active fragment of said mannan-binding serine protease as antigen.
In another aspect, the invention provides the use of said mannan-binding serine protease homolog or said mannan-binding serine protease homolog derivative or analog or active fragment or said antibody to affect a pro-phenoloxidase activation system, antibacterial peptide synthesis, detection of microorganisms and their associated molecular patterns.
Based on the technical scheme, further, the related molecular modes comprise beta-1, 3-glucan, peptidoglycan, lipoteichoic acid and mannans.
Compared with the prior art, the invention has the following beneficial effects:
the natural and recombinant MSPH and the partial fragment, derivative or analogue thereof have conventional, simple and high yield; the natural and recombinant mannans combined serine proteinase homolog, its derivative or analogue or partial fragment and its antibody can be extensively used in the fields of prevention, detection and therapeutic medicine for microbe, etc.
Description of the drawings:
in order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 shows the isolation and purification of native MSPH, wherein Lane M: molecular weight markers; lane 1: natural MSPH purified by method 1; lane 2: natural MSPH purified by method 2; lane3: method 3 purified native MSPH.
FIG. 2 is an MSPH sequence phylogenetic tree analysis.
FIG. 3 is an electropherogram of recombinant MSPH (prokaryotic expression system) separation and purification wherein Lane M: molecular weight markers; lane 1: unlabeled MSPH; lane 2: MSPH with N-terminal pre-fused histidine tag; lane3: MSPH fused with GST tag after C end; lane 4: MSPH fused with histidine tag after C-terminal.
FIG. 4 is an electropherogram of recombinant MSPH (insect expression system) separation and purification wherein Lane M: molecular weight markers; lane 1: recombinant MSPH of the pFastBac1-sf9 insect expression system; lane 2: MSPH of pMIB/V5-His-Sf21 insect expression system.
FIG. 5 is a graph showing the promotion of the pro-phenoloxidase activation system by exogenous MSPH supplementation, in which HL: tussah hemolymph; MSPH: recombinant Ap-MSPH protein; MIC: a microorganism; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.05,/represents t-test P <0.01,/represents t-test P <0.001,/represents t-test P <0.0001.
FIG. 6 is a graph showing the inhibition of the pro-phenoloxidase activation system by reducing endogenous MSPH, wherein NT+ buffer: a control group injected with physiological saline; dsEGFP: injecting EGFP double-stranded RNA group; dsMSPH: injecting Ap-MSPH double-stranded RNA; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.01, t-test P <0.001, t-test P <0.0001.
FIG. 7 shows the effect of MSPH on expression of antimicrobial peptides.
FIG. 8 is an analysis of MSPH binding capacity to PAMPs, wherein A: MSPH binds to Mannan; b: MSPH binds DAP-PGN; c: MSPH binds to Lys-PGN; d: MSPH binds to laminin; e: MSPH binds to LTA; f: MSPH binds LPS.
FIG. 9 is the effect of anti-MSPH antibodies on the pro-phenoloxidase activation system, wherein HL: tussah hemolymph; ab: rabbit Ap-MSPH polyclonal antibody; MIC: a microorganism; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.05,/represents t-test P <0.01,/represents t-test P <0.001,/represents t-test P <0.0001.
The specific embodiment is as follows:
the following examples are intended to enable those skilled in the art to more fully understand the invention and are not intended to limit the scope of the claims in any way.
Example 1: isolation and purification of native MSPH
In this example, tussah is repeatedly washed with distilled water or deionized water, and haemolymph is collected at 10deg.C to-5deg.C by conventional methods such as wax disc method, centrifugation method, back blood vessel blood sampling method, perfusion method, squeezing, homogenizing method, reflection bleeding method, tearing method, cutting method, shearing method, and puncturing method.
1. Method-1
Collecting Xylosmae haemolymph, precipitating with ammonium sulfate, collecting precipitate, and dissolving in insect physiological saline (120 mM NaCl, 0.9mM CaCl) 2 、2.7mM KCl、0.5mM MgCl 2 、1.8mM NaHCO 3 、1mM NaH 2 PO 4 A small amount of phenylthiourea (2.5 g/L) was added to 38.8mM glucose, incubated with a mannan-agaros affinity chromatography packing, eluted with 10mM imidozole-HCl, pH 7.8,1.0M NaCl,2mM EDTA, and the objective fraction was collected. Subjecting to Mono S HPLC, linear gradient eluting with 0-0.1M NaCl to obtain target protein component, separating and concentrating with ultrafiltration membrane, and loading onto gel filtration chromatography (Toyopearl HW-55S,1×30)cm), the balance and elution systems were 50mM Tris-HCl, 150mM NaCl, 3mM EDTA pH7.5, to obtain the objective protein component.
As a result of the test, as shown in FIG. 1, lane1, the purity of the native MSPH reached electrophoretic purity.
2. Method-2
A mixture (10. Mu.l) of fungus (Candida albicans), gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli) dissolved in insect physiological saline was injected into tussah body, induced for 24 to 48 hours, and the induced haemolymph was collected, diluted 10-fold with 50mM Tris-HCl buffer pH7.5 containing a small amount of phenylthiourea (2.5 g/L), passed through DEAE ion exchange chromatography column, and subjected to linear gradient elution with 50mM Tris-HCl buffer pH7.5 of 0.05 to 1.5M NaCl. The target fractions were collected, concentrated to 1mL by ultrafiltration membrane separation, and then loaded onto gel filtration chromatography (Toyopearl HW-55S, 1X 30 cm), with an equilibration and elution system of 50mM Tris-HCl, 150mM NaCl, 3mM EDTA pH 7.5. The obtained target protein component is subjected to hydroxyapatite HPLC, the balance buffer solution is 10mM, the pH value is 8.5 glycine/sodium hydroxide buffer solution, and 10-150mM glycine/sodium hydroxide pH value is 8.5, and 150mM-1M glycine/sodium hydroxide pH value is 8.5, and linear gradient elution is carried out twice, so that the target protein component is obtained.
The test results are shown in FIG. 1, lane2, and the purity of the native MSPH reached electrophoretic purity.
3. Method-3
Centrifuging the tussah body fluid dissolved in the anticoagulation buffer solution to remove blood cells, carrying out ammonium sulfate fractional precipitation, taking 35-45% of precipitation components, dissolving and diluting with borax-sodium hydroxide buffer solution (0.05 mol/L, pH 9.0), loading the solution on an SP-Sepharose anion exchange column, and washing and eluting with 50mM-1.5 MNaCl; loading the component containing the target protein into a Phenyl-sepharose6-Fast Flow for washing and eluting; after dialysis of the fractions containing the object, they were loaded onto a mann-agarsose column under the same conditions, 10mM imidozole-HCl, pH 7.8,1.0M NaCl,2mM EDTA eluted; the eluting component is subjected to ultrafiltration to remove salt, so that the target protein is obtained.
As a result of the test, as shown in FIG. 1, lane3, the purity of the native MSPH reached electrophoretic purity.
Example 2: MSPH structural analysis and gene sequence analysis thereof
MSPH is subjected to structural analysis according to the conventional protein chemistry and molecular biology techniques, methods and means. Obtaining the primary structure-amino acid sequence of natural MSPH (also called mature peptide chain, MSPH for short in the application) as SEQ ID NO:1, the gene sequence of the coding natural MSPH is shown as SEQ ID NO: 2.
Obtaining the full-length cDNA sequence of MSPH by using molecular biology technology and method, as shown in SEQ ID NO:3, the total length of the open reading frame of the MSPH gene is 1290bp, which can code 429 amino acid residues (the amino acid sequence of the open reading frame is shown as SEQ ID NO: 4), and the N end of the open reading frame comprises a signal peptide region of 18 amino acid residues.
The structure of MSPH of the invention is subjected to homology comparison analysis, and the result shows that: MSPH has a relatively close relationship with lepidopteran serine protease such as spodoptera litura and silkworm and homologs thereof (as shown in figure 2).
Example 3: recombinant MSPH and its derivatives, analogues and active fragments obtained by using prokaryotic expression system
This example illustrates the strategy and basic methodology for the construction of prokaryotic expression systems for the expression of the MSPH and its derivatives, analogs, active fragment genes of the present invention.
MSPH and its derivatives, analogues, active fragments include the following sequences:
(1) Met-MSPH amino acid sequence
MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(2)Met-His 6 tag-MSPH amino acid sequence
MHHHHHHQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(3) Met-MSPH-His6 tag amino acid sequence
MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH
(4) Met-His6 tag-thrombin cleavage site-MSPH amino acid sequence
MHHHHHHLVPRGSQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(5) Met-GST tag-thrombin cleavage site-MSPH amino acid sequence
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVP RGSQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(6) Met-MSPH-thrombin cleavage site-GST tag amino acid sequence
MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYLVPRGSILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD
(7) Met-MSPH-Flag tag amino acid sequence
MQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYDYKDDDDK
(8) Met-Flag tag-MSPH amino acid sequence
MDYKDDDDKQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(9)Met-His 6 tag-SUMO tag-thrombinCleavage site-MSPH amino acid sequence
MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTY
(10)Met-His 6 tag-SUMO tag-thrombin cleavage site-MSPH-His 6 Tag amino acid sequence
MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSQGDVMGGDLDSIINQIFTPSTVVAPVTTTTTTTTTTTTVKPVIDDRAPSTLVPPNDPKDKSCVMNNKQGECVTYYLCNKSNNTVITDGIGLLDIRAEGPCVSYMDVCCFLSDTRPPTDPITPKPEIVKPQREGCGWLNPEGVGMRTKGETDGETKFGEFPWMVAILKIELVNNDDPNGQKLNVYVGGGSLIHPSAVLTAAHYVADRPELRVRAGEWDTQNNKEIYPYQDREVESIEVHKDFNGGNLFYDVAILFLKTPMDLAPNVGLACLPPPEEQPNPGSRCFATGWGKDKFEKEGRYQVILKKVEVPVVDRQKCQDSLRTTRLGRFFQLHSSFMCAGGEPGKDTCKGDGGSPLVCPIEFEKERYVQNGIVAWGIGCGEMGVPGVYVDVSKVRNWIDDKIKGKRYQTDVYTYHHHHHH。
The expression vector, the expression host cell and the expression strategy of the prokaryotic expression system are conventional and universal expression vector, expression host cell and expression strategy of genetic engineering expression.
This implementation is intended to provide a more complete understanding of the invention to those skilled in the art, and is not intended to limit the scope of the claims in any way.
The method, principle, strategy, etc. of example 1 were used for the isolation and purification of the expression product.
Construction of expression vector for MSPH Gene
Respectively designing corresponding oligonucleotide primers according to the N-terminal and C-terminal amino acid sequences of MSPH, and simultaneously respectively adding restriction endonuclease hydrolysis site sequences at the 5' ends of the two oligonucleotide primers; performing PCR amplification by using insect fat body cDNA pool as a template, detecting a product by agarose gel electrophoresis, and performing gel recovery of a nucleic acid fragment; carrying out restriction endonuclease digestion, carrying out double digestion on the plasmid, carrying out recombination connection under the action of DNA ligase, and thermally converting competent cells of the escherichia coli; and (3) performing colony PCR and restriction endonuclease digestion verification, screening to obtain positive transformants, and then submitting the positive transformants to a biotechnology service company for DNA sequence determination. By the genetic engineering method, an expression vector of the MSPH gene is constructed.
The expression vector of this example was constructed as follows: 1. the escherichia coli is taken as a host, and the expression vectors can be selected from pTYB11, pMAL-C2X, pET-28a, pGEX-2T, pBV, pQE30, pET20b and the like; 2. a peptide fragment can be fused in front of the N end of MSPH to be used as a Tag (Tag) of affinity chromatography; 3. a peptide fragment can be fused after the C fragment of MSPH as a Tag (Tag) for affinity chromatography; 4. the Tag may be His-Tag (six or more histidines in succession), GST-Tag, flag-Tag, etc.; 5. amino acid sequences of proteolytic enzyme hydrolysis sites, such as thrombin, enterokinase, factor X, etc., may be added between the affinity chromatography tag and the MSPH to obtain recombinant MSPH proteins consistent with the native MSPH protein structure.
2. Recombinant MSPH protein and its derivative, analogue and active fragment obtaining
And (3) transforming the MSPH gene expression vector into escherichia coli by utilizing a genetic engineering technology, picking single bacterial colonies, inoculating the single bacterial colonies into LB containing antibiotics, and inducing the expression of the MSPH gene, thereby obtaining culture solution or thalli containing the MSPH. The thalli is firstly cracked by a cracking solution and is broken by ultrasonic, and after target protein is released, supernatant fluid is collected by a centrifugal method and is used as raw material liquid of recombinant MSPH for standby.
Characteristics of recombinant gene expression of interest: 1. the mode of transforming the expression vector into the host may be selected from a thermal transformation method and an electrotransformation method; 2. the mode of induction expression comprises chemical induction-isopropyl beta-D-thiogalactoside (IPTG) induction and heating induction; the MSPH gene can be expressed in cells or outside cells; 4. MSPH existing in cells needs to be released into the solution by means of lysis of lysate, ultrasonication and the like.
Recombinant MSPH, its derivatives, analogues, active fragments are isolated and purified from the above MSPH-containing stock solution to the desired purity by the method, principle, strategy, etc. of example 1 until electrophoretic purity or HPLC purity is achieved.
For example: (1) Constructing a non-tag MSPH expression vector by pTYB11, transferring the expression vector into a host cell by adopting an electrotransformation method, and expressing the MSPH in the cell by IPTG induction. And (3) re-suspending the thalli by using a lysis buffer solution, performing ultrasonic disruption, and centrifuging to obtain a supernatant serving as a raw material liquid for further separating and purifying MSPH. MSPH was isolated and purified to electrophoretically pure according to the method, principle, strategy, etc. of example 1 (FIG. 3, lane 1).
(2) Constructing an MSPH gene with a histidine tag fused before the N end by adopting pET-28a, thermally converting escherichia coli, inducing the escherichia coli by IPTG, expressing His-MSPH in cells, re-suspending thalli by adopting a lysis buffer (50 mmol/LPBS,0.15mol/L NaCl,50mmol/L imidazole), performing ultrasonic crushing, and centrifuging to obtain a supernatant which is used as a raw material liquid for further separating and purifying the MSPH. MSPH was isolated and purified to electrophoretically pure according to the method, principle, strategy, etc. of example 1 (FIG. 3, lane 2).
(3) Constructing an MSPH expression vector fused with GST tag after the C end by pGEX-2T, thermally converting escherichia coli, and expressing the MSPH-GST in cells by heating induction. And (3) re-suspending the thalli by using a lysis buffer solution, performing ultrasonic disruption, and centrifuging to obtain a supernatant serving as a raw material liquid for further separating and purifying MSPH. MSPH was isolated and purified to electrophoretically pure according to the method, principle, strategy, etc. of example 1 (FIG. 3, lane 3).
(4) Constructing an MSPH gene fused with a histidine tag after the C end of pET20b, transferring an expression vector into a host cell by adopting an electrotransformation method, and expressing the MSPH-His outside cells by heating and inducing. MSPH was isolated and purified to electrophoretically pure according to the method, principle, strategy, etc. of example 1 (FIG. 3, lane 4).
The purified expression product containing the tag is hydrolyzed by the conventional and universal proteolytic enzyme (such as thrombin, enterokinase, coagulation X factor and the like) to remove fusion peptide fragments in the expression product, and then the fusion peptide fragments are separated and purified to obtain the MSPH, wherein the structure of the recombinant MSPH is the same as that of the natural MSPH.
Example 4: recombinant MSPH and its derivatives, analogues and active fragments obtained by using insect cell expression system
This example illustrates the strategy and basic methodology for constructing an insect cell expression system for expressing the MSPH and its derivatives, analogs, active fragment genes of the present invention.
The expression vector, the expression host cell and the expression strategy of the insect cell expression system are conventional and universal expression vectors, expression host cells and expression strategies of genetic engineering expression.
The present examples are intended to provide a more complete understanding of the present invention to those skilled in the art, and are not intended to limit the scope of the claims in any way.
The method, principle, strategy, etc. of example 1 were used for the isolation and purification of the expression product.
1. Recombinant MSPH, derivatives, analogues and active fragments thereof obtained by using pFastBac1-sf9 insect expression system
MSPH, derivatives, analogues and active fragment genes thereof are connected into pFastBac1 plasmid to construct pFastBac 1-beta GRP recombinant expression plasmid, after induction of transpositional escherichia coli DH10, bluo-gal and IPTG, blue-white screening is carried out to obtain transpositional recombinant bacmid, insect cells sf9 are transfected, and Western blot verifies that the recombinant MSPH is expressed in cells.
The cells were collected, resuspended in lysis buffer (0.05 mol/L Tris-HCl,0.5mol/L NaCl, pH 8.0), sonicated and centrifuged to give a stock solution containing the target protein. MSPH was isolated and purified to electrophoretically pure according to the method, principle, strategy, etc. of example 1. The structure of the expression product is shown in FIG. 4, lane 1.
2. Recombinant MSPH, derivatives, analogues and active fragments thereof are obtained by using pMIB/V5-His-Sf21 insect expression system
MSPH, derivatives, analogues and active fragment genes thereof are connected into pMIB/V5-His plasmid to construct pMIB/V5-His-beta GRP recombinant expression plasmid, after induction of transpositional escherichia coli DH5, bluo-gal and IPTG, blue-white screening is carried out to obtain transpositional recombinant bacmid, insect cells Sf21 are transfected, and Western blot verifies that the recombinant MSPH is expressed in cells.
Collecting cells, re-suspending with lysis buffer (0.05 mol/L Tris-HCl,0.5mol/L NaCl, pH 8.0), ultrasonically crushing, centrifuging to obtain a raw material liquid containing target protein, directly loading the raw material liquid on a pre-balanced metal ion chelating chromatographic column, fully washing with 0.02mol/L imidazole (pH 8.0) to remove a large amount of impurity proteins, eluting with 0.5mol/L imidazole (pH 8.0), efficiently expressing recombinant protein, reaching electrophoresis purity, and purifying to obtain electrophoresis identification results as shown in figure 4 and Lane 2.
Example 5: acquisition of anti-MSPH antibodies
The immune system of mice or rats or rabbits or dogs or sheep or horses or cattle was stimulated to produce the corresponding antibodies using the various MSPHs obtained in examples 1,3, 4 as antigens according to conventional, general antibody production techniques.
The production of MSPH antibodies in the serum of immunized mice or rats or rabbits or dogs or sheep or horses or cattle is detected using conventional, universal antibody detection methods.
After the MSPH antibody is produced by the immunized mice or rats or rabbits or dogs or sheep or horses or cattle, the serum of the immunized mice or rats or rabbits or dogs or sheep or horses or cattle is collected and stored by adopting a conventional and universal animal serum collection and storage method, and the serum can be directly applied.
The MSPH antibody with different purity is separated and purified from the stored serum containing the MSPH antibody by using the conventional and general antibody separation and purification technology, such as salting out, various types of chromatography media, antibody affinity chromatography media and the like, until the MSPH antibody with electrophoretic purity or HPLC purity is obtained, so as to be suitable for the application with different requirements.
Example 6: recombinant and natural MSPH and its derivatives, analogues and biological activity of active fragments
In this example recombinant, native MSPH and its derivatives, analogues, active fragments have the same biological activity. Tussah is described as representative of the biological activity assay of lepidopteran insects. The biological activity of MSPH, derivatives, analogues and active fragments thereof can be used as a core and a basis by the skilled person, so that the application range of the MSPH, the derivatives, the analogues and the active fragments thereof is further widened.
Effect of msph on pro-phenoloxidase activation system
(1) Supplementing exogenous MSPH to promote the pro-phenoloxidase activation system
The effect of recombinant and native MSPH on the pro-phenoloxidase activation system was examined using six soluble pathogen-associated molecular patterns with three pathogenic microorganisms, and the results are shown in fig. 5, where the six soluble pathogen-associated molecular patterns and three pathogenic microorganisms each significantly activated the pro-phenoloxidase activation system (PPO-AS) AS compared to the buffer control group; after exogenous recombinant MSPH or natural MSPH is added, each experimental group shows the phenomenon that the activity of phenol oxidase is obviously increased.
(2) Reducing the inhibition of the pro-phenoloxidase activation system by endogenous MSPH
RNAi technology is adopted to reduce the expression of endogenous MSPH, and the influence of the endogenous MSPH on PPO-AS is further examined. The results are shown in fig. 6, where PO viability was significantly reduced in each experimental group after MSPH interference compared to the saline injected control group and dsEGFP control group.
Effect of MSPH on antibacterial peptide expression
In the experiment, after the expression of endogenous Ap-MSPH is down-regulated by injecting dsMSPH for 24h, E.coli, S.aureus and C.albicans are respectively injected into the tussah larvae to examine six antibacterial peptides and Toll pathway key proteins in the tussah larvae
Variation in mRNA levels. As shown in FIG. 7, the results show that the experimental group interfered with MSPH was injected with E.coli, and the antibacterial peptide Defensin, gloverin, attacin, lysozyme, lebocin, moricin and Toll pathway key protein +.>
Is significantly up-regulated. The same experimental results also appear under s.aureus induced immunization conditions.
Binding specificity of MSPH and its analogues, active fragments and microorganism-related molecular patterns
The recognition ability of MSPH and its analogues, active fragments, to 6 soluble, typical molecular patterns (PAMP) from different species of microorganisms, respectively, was examined using a micro-thermophoresis instrument (MST). 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. Beta. -1,3 glucan) and Mannan belong to specific PAMP of fungi. PAMP with the same concentration is coated on a probe, MSPH or an analogue and an active fragment thereof are respectively incubated with the probe, and the binding condition of the recombinant PGRP-SA or the analogue and the active fragment thereof and a molecular mode related to soluble microorganisms is detected by measuring the directional movement of molecules of a sample in a microcosmic temperature gradient field. As shown in FIG. 8, recombinant His 6 MSPH has broad-spectrum binding properties to other PAMPs except LPS. The same experimental results can be obtained with the native MSPH or recombinant MSPH analogues and active fragments described in examples 1,3, 4.
The experimental results show that: the natural and recombinant MSPH, the derivatives, analogues and active fragments thereof of the invention have broad-spectrum binding performance on other PAMPs except LPS. Furthermore, on the one hand, the components of the in-vivo phenoloxidase activating system of the insects can obviously activate the phenoloxidase activating system; on the one hand, the polypeptide has obvious inhibiting effect on the generation of the antibacterial peptide in the insect body.
Example 7: natural and recombinant MSPH and derivatives, analogues, active fragments and application of antibodies thereof
This example describes the biological activity of MSPH as representative, and MSPH derivatives, analogues, and active fragments also have the same biological activity. Meanwhile, the biological activity test insect of tussah as lepidoptera insect is also described as representative. The application range of MSPH, derivatives, analogues, active fragments and antibodies can be further expanded by the skilled in the art based on the biological activities of MSPH and derivatives, analogues, active fragments and antibodies.
MSPH and derivatives, analogues, active fragments thereof for detection of microorganisms
MSPH and its derivatives, analogues, active fragments can be used as the active activating component of the pro-phenol oxidation enzyme activating system as described in example 6.
Any pathogenic microorganism to be detected is taken and added into tussah hemolymph additionally supplemented with MSPH, and the control group is that the same dosage of sample of the microorganism to be detected is added into tussah hemolymph not supplemented with MSPH. The activation of the PPO system in the haemolymph was observed at the same time, and the experimental group and the control group were significantly different, indicating that the detection sample contained microorganisms or related molecular patterns.
MSPH and its derivatives, analogues and application of active fragment antibody
The antibodies against MSPH, derivatives, analogues and active fragments thereof obtained in example 5 were used for MSPH immunodetection of lepidopteran insect samples by conventional and general techniques and methods such as immunology and molecular biology. The method is also suitable for the immunodetection tracking analysis and the qualitative and quantitative detection analysis of samples in the process of preparing MSPH by separating and purifying lepidopteran insects. The experiments in this regard have been applied to the examples of the preparation of the above-described isolated and purified natural and recombinant MSPH and its derivatives, analogs and active fragments.
Endogenous MSPH proteins were blocked using anti-MSPH antibodies and the effect of endogenous MSPH on the pro-phenoloxidase activation system was examined by six soluble pathogen-associated molecular patterns (PAMPs) with three pathogenic Microorganisms (MIC). The results are shown in FIG. 9, which shows that PAMPs and MICs significantly activated PPO-AS compared to the control group; after the anti-MSPH antibody is added, each experimental group shows the phenomenon that the PO activity is obviously reduced. It was demonstrated that MSPH antibodies can inhibit activation of the pro-phenoloxidase activation system induced by microorganisms and their related molecular patterns.
A sufficient dose of MSPH antibody is added to any sample of the microorganism to be detected. According to the method for detecting microorganisms by using MSPH, derivatives, analogues and active fragments thereof in the embodiment, the detection of microorganisms in the sample to be detected is performed. As a result, even if the amount of the microorganism detected in the sample is not detected (negative result), the experimental design was applied as a negative control group for detecting the microorganism in the sample.
The above results indicate that: the antibodies of MSPH and derivatives, analogues and active fragments thereof shield the binding biological activity of the antibodies with the microorganisms and related molecular modes through the binding with the MSPH and the derivatives, analogues and active fragments thereof, so that the MSPH and the derivatives, analogues and active fragments thereof lose the original biological activity. The shielding principle based on the combination can be widely applied.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A mannan-binding serine protease homolog comprising the amino acid sequence set forth in SEQ id no: 1.
2. The mannans-bound serine protease homologue according to claim 1, wherein the mannans-bound serine protease homologue is derived from Lepidoptera (Lepidoptera) insects of the family sarcinidae (sarcinidae) and is selected from one of tussah, castor silkworm, tussah, amber silkworm, us tussah, ailanthus altissima, wild silkworm, american tussah, camphorwood silkworm, maple silkworm.
3. A gene encoding the mannan-binding serine protease homolog of claim 1 or 2.
4. A gene of a mannan-binding serine protease homolog according to claim 3, wherein the nucleotide sequence of the gene is set forth in SEQ ID NO: 2.
5. The derivative or analogue or active fragment of a mannan-binding serine protease homolog according to claim 1 or 2, comprising the amino acid sequence set forth in SEQ ID NO:1 and has the biological activity of the mannan-binding serine protease homolog.
6. The derivative or analogue or active fragment of a mannan-binding serine protease homolog according to claim 5, wherein the derivative or analogue or active fragment of a mannan-binding serine protease homolog is selected from the group consisting of Met-MSPH, met-histidine tag-MSPH, met-MSPH-His 6 Tag, met-His 6 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-His 6 tag-SUMO tag-thrombin cleavage site-MSPH, met-His 6 tag-SUMO tag-thrombin cleavage site-MSPH-His 6 A tag sequence.
7. The method for producing a mannan-binding serine protease homolog according to claim 1 or 2, wherein the mannan-binding serine protease homolog having an electrophoretic purity or an HPLC purity is obtained by using one or a combination of two or more of hemolymph, blood, hemocyte lysate, lymph fluid, and homogenate of a lepidopteran silkworm moth insect as a raw material liquid by one or a combination of two or more of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out, and ultrafiltration;
or cloning the gene for encoding the mannan-binding serine protease homolog into a recombinant expression vector, and introducing the recombinant expression vector into a host cell to obtain the recombinant expression mannan-binding serine protease homolog.
8. The method for producing a derivative or analogue or active fragment of a mannan-binding serine protease homolog according to claim 5 or 6, wherein the gene encoding the derivative or analogue or active fragment of the mannan-binding serine protease homolog is cloned into a recombinant expression vector, introduced into a host cell, and isolated and purified to obtain the derivative or analogue or active fragment of the mannan-binding serine protease homolog expressed recombinantly.
9. The antibody of the mannan-binding serine protease homolog according to claim 1 or 2 or the derivative or analog or active fragment of the mannan-binding serine protease homolog according to claim 5 or 6, characterized in that the natural mannan-binding serine protease homolog or the derivative or analog or active fragment of the mannan-binding serine protease homolog is used as antigen for stimulating the immune system production in mice or rats or rabbits or dogs or sheep or horses or cattle.
10. Use of a mannan-binding serine protease homolog according to claim 1 or 2 or a derivative or analog or active fragment of a mannan-binding serine protease homolog according to claim 5 or 6 or an antibody according to claim 9 for affecting a pro-phenol oxidizing enzyme activation system, antimicrobial peptide synthesis, microorganism and related molecular pattern detection.
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