Serine protease inhibitor SPINK8, preparation method and application of serine protease inhibitor SPINK8 in treatment of sepsis acute lung injury

Abstract

The invention discloses a serine protease inhibitor SPINK8 as well as a preparation method and application thereof in acute lung injury caused by sepsis, and belongs to the fields of molecular biology, functional protein technology and biological medicine. The amino acid sequence of the SPINK8 protein is as shown in SEQ ID No. 1. According to the invention, the SPINK8 recombinant vector is constructed through molecular cloning for the first time, and the reSPINK8 recombinant protein is obtained through prokaryotic expression and purification. When the acute lung injury caused by sepsis occurs, the reSPINK8 protein can remarkably inhibit lung inflammatory response and improve the pathological state of the lung, and has immunoregulation and protection effects on the acute lung injury caused by sepsis. In view of the advantages that SPINK8 is small in molecular weight, easy to express and purify and easy to prepare medicines, the SPINK8 has important application value in prevention and treatment of sepsis acute lung injury and targeted medicine screening.

Description

TECHNICAL FIELD

[0001] The present application relates to serine protease inhibitor SPINK8, a preparation method and its application in the treatment of sepsis acute lung injury, belonging to the fields of molecular biology, functional protein technology and biological medicine. BACKGROUND

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, with excessive activation of the inflammatory system as the main pathological feature, and further development can lead to septic shock and multiple organ dysfunction syndrome. Sepsis develops rapidly, has a poor prognosis, and has a high mortality rate, which brings great difficulties to clinical treatment and is a great challenge to intensive care. According to statistics, about 50 million people worldwide develop sepsis every year, and about 11 million cases die. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common complications of sepsis in clinical practice, and the mortality rate of ALI is as high as 30%-40%, which has become a major disease that seriously threatens the health of the Chinese people. The treatment of clinical sepsis lung injury mainly includes antibiotic therapy, intravenous fluid infusion, mechanical ventilation, and removal of the source of infection. However, due to the complex pathogenesis of sepsis lung injury and poor prognosis, it is urgent to discover new potential therapeutic molecules, and in-depth exploration of their safety and effectiveness has important clinical significance for the prevention and treatment of sepsis lung injury.

[0003] Serine protease inhibitor kazal type (SPINK) is a family of conservative serine protease inhibitors containing Kazal domains. Kazal-type serine protease inhibitors are widely present in mammals, birds and invertebrates, and play an important role in regulating inflammatory response, coagulation, fibrinolysis, embryogenesis, individual development, digestion and other physiological and pathological processes. Kazal-type protease inhibitors have one or more Kazal-type domains, and a typical Kazal-type domain generally consists of 40-60 amino acid residues, including 6 cysteine residues and a conserved amino acid sequence. The molecular conformation of Kazal-type protease inhibitor contains three loop structures A, B and C, and the active site P1 is located in the protruding part of the B loop, and the P1 site determines the specificity of the protease.

[0004] SPINK8 belongs to Kazal-type serine protease inhibitors, and human SPINK8 is a secreted protein encoded by 97 amino acids, mainly expressed in bone marrow and placenta. There are few studies on the function of SPINK8, and only research shows that SPINK8 is involved in sperm maturation and serves as a prognostic marker for breast invasive carcinoma. At present, there is no relevant research and report on the role and mechanism of SPINK8 in sepsis acute injury. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a serine protease inhibitor SPINK8, a preparation method thereof and application thereof in sepsis acute lung injury. The prepared reSPINK8 recombinant protein has an inflammation inhibiting function. After treatment of the reSPINK8 protein in vivo during sepsis, the lung tissue inflammation degree is reduced, and the lung injury is significantly reduced. The reSPINK8 recombinant protein involved in the present application provides an important reference value for drug screening for preventing and treating sepsis acute lung injury in clinic.

[0006] To solve the above technical problems, the purpose of the present application is achieved as follows: The serine protease inhibitor SPINK8 involved in the present application has an amino acid sequence as shown in SEQ ID No. 1. SEQ ID No. 1: MKGICSDAILVLATSMWMAFAIDFPLPMASERGQLDKTIVECLKNVNKCWFLSYIKPSEPICGSDQVTYSSDCHLCSKILFEGLNITKLYDGQCENS.

[0007] The application also provides a serine protease inhibitor SPINK8 gene, and the nucleotide sequence of the gene is shown in SEQ ID No. 2. SEQ ID No. 2 shows: agaactcactcctgagtgcaaaggcaatgtcaggcacgctggtaaaggaccactagaatccagcagcccagacccctttctttgtggtcaagaagggcaggaaaacaggtgcaggactgctacatcgggaaaggaagaaaatcctactgcctttctggagagactaaggaggcattgaggaagcatacctctctgtcatctggatctattgaaggccaactaatcttaaagggcattggaaccttgaaaagacaaataagctttgccttccttcctagttcattatgaacaaactatcaacaaaaaccatcctgttcagccagtttctcagagcaaccatgggagtcacagcagttcttctgtcaccatgaaggggatctgctcagacgccatccttgttctagctacctccatgtggatggcctttgcaattgacttcccccttcctatggcctctgaaagaggtcagctagacaaaacaatagttgaatgcctcaagaatgtaaataagtgctggtttttatcctacatcaagcccagtgaacctatttgtggcagtgaccaggttacctacagtagtgactgccatctgtgctccaaaattctatttgaagggcttaacataactaaactgtatgatggacaatgtgaaaactcttgaacgtacgacaaagaattataaaacctactgaatctccagattgccaagtgaaacacaatggttgcctcttcaaatatatcccctttaattactaatggttggatcaaactcgttgatttatgtcttcaataaatgattcttagcagaaaa.

[0008] Wherein, the following nucleotide sequence is the CDS region: atgaaggggatctgctcagacgccatccttgttctagctacctccatgtggatggcctttgcaattgacttcccccttcctatggcctctgaaagaggtcagctagacaaaacaatagttgaatgcctcaagaatgtaaataagtgctggtttttatcctacatcaagcccagtgaacctatttgtggcagtgaccaggttacctacagtagtgactgccatctgtgctccaaaattctatttgaagggcttaacataactaaactgtatgatggacaatgtgaaaactcttgaa.

[0009] The present application also provides a recombinant expression vector comprising the gene, wherein the recombinant expression vector is connected with a nucleotide molecule encoding the nucleotide sequence shown in SEQ ID NO. 3. SEQ ID NO. 3 shows: atgattgacttcccccttcctatggcctctgaaagaggtcagctagacaaaacaatagttgaatgcctcaagaatgtaaataagtgctggtttttatcctacatcaagcccagtgaacctatttgtggcagtgaccaggttacctacagtagtgactgccatctgtgctccaaaattctatttgaagggcttaacataactaaactgtatgatggacaatgtgaaaactct.

[0010] The present application also provides a method for preparing a human SPINK8 protein.

[0011] Preferably, the eukaryotic recombinant expression vector comprises a SPINK8 gene.

[0012] Preferably, the prokaryotic recombinant vector comprises a SPINK8 gene.

[0013] Preferably, the recombinant E. coli strain is obtained by transforming the recombinant vector into a host bacterium.

[0014] The eukaryotic expression vector is pCDNA3.1 (+).

[0015] The prokaryotic expression vector is pET-28a (+).

[0016] The host bacterium is E. coli BL21 (DE3).

[0017] Preferably, the preparation method of the SPINK8 protein comprises the following steps: The eukaryotic recombinant expression vector pCDNA3.1(+)-SPINK8 is transformed into a host bacterium, and the pCDNA3.1(+)-SPINK8 recombinant plasmid is extracted in large quantities.

[0018] Preferably, the host bacterium is an Escherichia coli TOP10 strain.

[0019] The pCDNA3.1(+)-SPINK8 recombinant plasmid and the pET-28a(+) plasmid are double-digested.

[0020] The enzyme digestion site is EcoR I / Xho I.

[0021] The recombinant expression plasmid pET-28a(+)-SPINK8 is obtained by using T4 ligase in a 16℃ water bath for 24h.

[0022] The obtained pET-28a(+)-SPINK8 plasmid is transformed into Escherichia coli BL21(DE3) competent cells, and the recombinant strain is obtained by screening.

[0023] The recombinant strain is cultured and induced for expression.

[0024] Preferably, the culture method comprises inoculating the recombinant strain into LB culture medium containing 100μg / mL kanamycin, and the culture condition is 200rpm / min at 37℃ for 3-4h.

[0025] The induced expression is adding IPTG with a final concentration of 1mM to the culture solution.

[0026] The bacterial cells are separated and ultrasonically cracked and broken, the recombinant SPINK8 protein (reSPINK8) is harvested, and the recombinant protein is purified.

[0027] Preferably, the purification medium is a nickel ion metal chelate medium.

[0028] The application also provides the application of the serine protease inhibitor reSPINK8 protein in sepsis acute lung injury.

[0029] Preferably, the application comprises at least one of the following effects: Inhibiting lung inflammatory response; Effectively reducing lung tissue damage; Improving the survival rate of sepsis acute lung injury mice.

[0030] Compared with the prior art, the application has the following beneficial effects: (1) The application provides a recombinant E. coli expression transformant capable of effectively expressing a serine protease inhibitor SPINK8 protein. Through detection, the obtained recombinant protein reSPINK8 has a protein size of 12 KDa. Because the molecular weight of the SPINK8 protein is small, the protein is easy to express, so that a large amount of protein can be obtained, and the production cost can be greatly reduced. The affinity chromatography step in the purification process has high purification efficiency and is easy to scale up production.

[0031] (2) The serine protease inhibitor reSPINK8 protein provided by the application has an inflammation inhibition effect.

[0032] In the embodiment of the application, through an inflammatory cell model and an animal model experiment of sepsis acute lung injury, it is confirmed that when sepsis ALI occurs, the transcription level and protein level of SPINK8 are significantly increased. Experiments show that the reSPINK8 protein provided by the application can significantly inhibit the expression of TNF-α, IL-1β and IL-6 inflammatory factors in macrophages under LPS stimulation. Animal level research finds that when sepsis acute lung injury occurs, after the reSPINK8 protein provided by the application is given for intervention, lung inflammation is significantly reduced. It is found through the induction of sepsis acute lung injury models of SPINK8 gene knockout mice and control wild type mice that the lung inflammation and lung tissue damage degree of the SPINK8 gene knockout mice are significantly aggravated, and the survival rate of the mice is significantly reduced. After the sepsis mice are given the reSPINK8 protein treatment, lung inflammation reaction is significantly reduced, and lung tissue damage is obviously improved. The application first confirms that the high expression of SPINK8 in sepsis acute lung injury has an inflammation inhibition effect and can reduce lung damage.

[0033] The application provides a new method for preventing and / or treating sepsis acute lung injury, and because the SPINK8 protein is an endogenous protein in the human body, the possible side effects on the human body are small, the safety as a potential drug is high, and the application has important reference value for developing a new Kazal type inhibitor drug. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 FIG. 1 is a structural diagram of SPINK8 protein. Wherein (A) is a schematic diagram of SPINK8 protein structure, and (B) is specific SPINK8 amino acid sequence information.

[0035] Figure 2 FIG. 2 is an electrophoresis result diagram of reSPINK8 protein after purification. Wherein (A) is a schematic diagram of pET-28a (+)-SPINK8 plasmid construction, and (B) is a Coomassie staining result diagram of reSPINK8 protein after purification.

[0036] Figure 3Figure 8 is a diagram of SPINK8 induced expression up-regulation in sepsis acute lung injury. Wherein (A) mRNA level of SPINK8 after LPS stimulation of macrophages, (B) protein level of SPINK8 after LPS stimulation of macrophages.

[0037] Figure 4 Figure 9 is a diagram of reSPINK8 protein inhibiting inflammatory response of macrophages. Wherein (A) RAW264.7 cells, (B) THP-1 cells, (C) MH-S cells.

[0038] Figure 5 Figure 10 is a diagram of SPINK8 gene knockout aggravating lung injury in sepsis ALI. Wherein (A) mRNA level of SPINK8 in sepsis acute lung injury, (B) protein expression level of SPINK8 in sepsis acute lung injury, (C) H&E lung tissue staining diagram, (D) mRNA level of lung tissue inflammatory factors TNF-α, IL-1β, IL-6 inflammatory factors, (E) mouse survival analysis.

[0039] Figure 6 Figure 11 is a diagram of reSPINK8 protein treatment effect on sepsis acute lung injury. Wherein (A) schematic diagram of different doses of reSPINK8 protein treating sepsis acute lung injury, (B) H&E lung tissue staining diagram, (C) mRNA level of lung tissue inflammatory factors TNF-α, IL-1β, IL-6, (D) mouse survival analysis. DETAILED DESCRIPTION

[0040] The present application is further illustrated by the following description and examples with reference to the accompanying drawings. The examples given are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0041] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0042] Example 1: Construction of pET-28a(+)-SPINK8 recombinant plasmid 1.1 Experimental materials Plasmid extraction kit, EcoR I restriction endonuclease, Xho I restriction endonuclease, T4 ligase, TOP10 competent cells, LB agar plate (kanamycin resistance), DNA gel recovery kit 1.2 Experimental method (1) pCDNA3.1(+)-SPINK8 and pET-28a(+) plasmids were extracted using a plasmid extraction kit.

[0043] (2) Take 2.5 μg of pCDNA3.1(+)-SPINK8 plasmid and 2.5 μg of pET-28a(+) plasmid respectively, and then add 1 μL EcoR I restriction endonuclease (15 U / μL) and 1.5 μl Xho I restriction endonuclease (10 U / μL) for double digestion.

[0044] (3) Perform agarose gel electrophoresis on the enzyme digestion products and recover the target fragment by gel cutting.

[0045] (4) Take 6 μg of pET-28a(+) vector and 2 μg of target fragment after double digestion with EcoRI and XhoI, and ligate them with T4 ligase in a 16℃ water bath for 24 h to obtain recombinant expression plasmid pET-28a(+)-SPINK8.

[0046] (5) Add the recombinant expression plasmid to TOP10 competent cells, place on ice for 30 min, heat shock at 42℃ for 60 s for transformation, then add LB medium without antibiotics, culture at 37℃ and shake at 200 rpm for 60 min, spread on LB agar plates containing kanamycin, culture at 37℃ overnight, pick 3-5 single clones and shake.

[0047] (6) Identification was performed by colony PCR, and the specific method is as follows: SPINK8 primers: Upstream 5'-3': ACTGCCATCTGTGCTCCAAA Downstream 5'-3': TTGAAGAGGCAACCATTGTGT Gently mix the above reaction system, and then perform the following PCR reaction: 95℃ for 5 min; 95℃ for 15 s, 64℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 6 min, and finally cool down to 4℃.

[0048] (7) Take bacterial culture for sequencing verification.

[0049] 1.3 Experimental Results Sequencing results showed that the SPINK8 gene was successfully inserted into the pET-28a(+) expression vector and maintained the correct reading frame. Subsequently, the correctly sequenced strains were preserved and plasmids were extracted.

[0050] Example 2: Prokaryotic expression and purification of the serine protease inhibitor reSPINK8 protein 2.1 Experimental Materials Kanamycin, LB medium, BL21(DE3) strain, IPTG, nickel agarose, cell lysis broth, imidazole, EDTA, glycerol, dialysis bag 2.2 Experimental Methods (1) Take 0.5 μL of pET-28a(+)-SPINK8 recombinant plasmid (200 ng / μL) and add it to 100 μL of BL21(DE3) competent cells. After placing on ice for 30 min, heat shock at 42℃ for 60 s to transform the cells. Then add LB medium without antibiotics and culture at 37℃ and 200 rpm for 60 min. Spread the culture onto LB agar plates containing kanamycin and culture at 37℃ overnight.

[0051] (2) Select a single clone and shake it in a shaker at 37°C for 10-12 hours.

[0052] (3) Transfer and expand culture at a ratio of 1:100, shake in a shaker at 200 rpm / min and 37℃ for 2-3 hours. When the OD value reaches about 0.6, add the inducing agent IPTG to a final concentration of 1 mM and shake for 3-4 hours.

[0053] (4) Centrifuge and collect the bacterial cells.

[0054] (5) Use the bacterial lysis buffer to suspend the collected bacterial precipitate and gently blow it evenly.

[0055] (6) Repeat the freeze-thaw cycle of the bacterial cells 3 to 5 times.

[0056] (7) The bacterial cells were ultrasonically disrupted. The ultrasonic conditions were: 300W power, 45min, 10s ultrasonic, 10s pause as one cycle.

[0057] (8) After the ultrasound is completed, centrifuge at 12,000 rpm and 4°C for 20 min and collect the supernatant.

[0058] (9) Add 1 mL of nickel agarose gel to the protein purification column and wash and equilibrate the column with 10 times the column bed volume of buffer A (10 mM imidazole) at a flow rate of 2 mL / min.

[0059] (10) Slowly add the collected supernatant into a nickel agarose gel and bind at 4°C for 2 hours. Load the column at a flow rate of 1 mL / min.

[0060] (11) Elution: Add buffer B (20mM imidazole), buffer C (40mM imidazole), buffer D (100mM imidazole), and buffer E (250mM imidazole) to the column in sequence to elute the sample at a flow rate of 2mL / min and collect the eluent.

[0061] (12) Add the collected eluent to the dialysis bag and gently place it in the dialysis solution. Dialyze at 4°C for 12 hours.

[0062] (13) Collect the protein solution after dialysis and use the affinity purification method to bind the target protein with the affinity filler for 4 hours to specifically adsorb endotoxin, thereby removing the endotoxin in the target protein.

[0063] (14) The collected eluent was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining to collect the target protein.

[0064] (15) Specific preparation methods for relevant reagents: Cell lysis buffer: Weigh 6.05g Tris into a beaker and add 800mL of sterile water to adjust the pH to 7.8. Then weigh 17.532g NaCl into the beaker and bring the volume to 1L.

[0065] Buffer A: Weigh 0.136g imidazole into a beaker and add 150mL of bacterial lysis buffer. Stir well, then add an appropriate amount of bacterial lysis buffer to bring the volume to 200mL.

[0066] Buffer B: Weigh 0.272g imidazole into a beaker and add 150mL of bacterial lysis buffer. Stir well, then add an appropriate amount of bacterial lysis buffer to bring the volume to 200mL.

[0067] Buffer C: Weigh 0.544 g imidazole into a beaker and add 150 mL of bacterial lysis buffer. Stir well, then add an appropriate amount of bacterial lysis buffer to bring the volume to 200 mL.

[0068] Buffer D: Weigh 1.36g imidazole into a beaker and add 150mL of bacterial lysis buffer. Stir well, then add an appropriate amount of bacterial lysis buffer to bring the volume to 200mL.

[0069] Buffer E: Weigh 3.404 g imidazole into a beaker and add 150 mL of cell lysis buffer. Stir well, then add an appropriate amount of cell lysis buffer to bring the volume to 200 mL.

[0070] 2.3 Experimental Results Coomassie Brilliant Blue staining results indicated that the recombinant reSPINK8 protein was successfully expressed and purified.

[0071] Example 3: Detection of the inhibitory effect of reSPINK8 recombinant protein on inflammatory response 3.1 Experimental Materials THP-1 (human monocytic leukemia cells), RAW 264.7 (mouse monocytic macrophage leukemia cells), MH-S (mouse alveolar macrophages), reSPINK8 protein, inflammatory factor ELISA kit (TNF-α, IL-1β, IL-6), lipopolysaccharide (LPS), phorbol ester (PMA) 3.2 Experimental Methods 3.2.1 Detection of the anti-inflammatory effect of reSPINK8 protein in THP-1 macrophages (1) Resuscitate THP-1 cells and culture them in suspension in 1640 complete medium.

[0072] (2) Culture in suspension for 1.5 to 2 days, and passage THP-1 cells once.

[0073] (3) After passage, THP-1 cells were seeded into 6-well plates and PMA (final induction concentration 100 ng / mL) was added to induce macrophages.

[0074] (4) After 48 hours of induction, THP-1 cells changed from suspension growth to adherent growth, from round to irregular shape, and their volume further increased. The cytoplasm became loose, the cell nucleus enlarged significantly, and a large number of obvious organelles were visible. A small number of protrusions were visible around the cell membrane, indicating that macrophage induction was successful.

[0075] (5) Replace with fresh 1640 complete medium and add lipopolysaccharide (LPS) (final induction concentration 100 ng / mL) and IFN-γ (final induction concentration 20 ng / mL) for 36 h. Then, add reSPINK8 protein (final concentration 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL) to the experimental group and add an equal volume of 1640 basal medium for 12 h.

[0076] (6) Collect cell supernatant and use ELISA to detect the protein levels of TNF-α, IL-1β and IL-6.

[0077] 3.2.2 Detection of the anti-inflammatory effect of reSPINK8 protein in RAW264.7 macrophages (1) Resuscitate RAW264.7 cells and culture them in DMEM complete medium.

[0078] (2) Culture for 1.5 to 2 days, and passage RAW264.7 cells once.

[0079] (3) After passage, RAW264.7 cells were seeded into 6-well plates and lipopolysaccharide (LPS) (final induction concentration 1 μg / mL) was added for 36 h.

[0080] (4) Add reSPINK8 protein (experimental group) (final concentration 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL) to the induced RAW264.7 cells, and add an equal volume of DMEM basal medium to the control group and incubate for 12 h.

[0081] (5) Collect cell supernatant and use ELISA to detect the protein levels of TNF-α, IL-1β and IL-6.

[0082] 3.2.3 Detection of the anti-inflammatory effect of reSPINK8 protein in MH-S alveolar macrophages (1) Resuscitate MH-S alveolar macrophages and culture them in 1640 complete medium.

[0083] (2) Culture for 1.5 to 2 days, and passage MH-S cells once.

[0084] (3) After passage, MH-S cells were seeded into 6-well plates and lipopolysaccharide (LPS) (final induction concentration 1 μg / mL) was added for 12 h.

[0085] (4) Add reSPINK8 protein (experimental group) (final concentration 0.5 μg / mL, 2.5 μg / mL, 5 μg / mL) to the induced MH-S cells, and add an equal volume of DMEM basal medium to the control group and incubate for 12 h.

[0086] (5) Collect cell supernatant and use ELISA to detect the protein levels of TNF-α, IL-1β and IL-6.

[0087] 3.3 Experimental Results Compared with the control group, reSPINK8 protein significantly inhibited the expression of TNF-α, IL-1β and IL-6 in LPS-induced THP-1, RAW264.7 and MH-S cells, indicating that reSPINK8 protein has an inhibitory effect on inflammatory response.

[0088] Example 4: Expression of SPINK8 in macrophages during acute lung injury due to sepsis 4.1 Experimental Materials THP-1 (human monocytic leukemia cells), RAW 264.7 (mouse monocytic macrophage leukemia cells), MH-S (mouse alveolar macrophages), Trizol, reverse transcription kit, TB Green® Fast qPCR Mix kit, anti-SPINK8 antibody, horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) 4.2 Experimental Methods 4.2.1 RNA extraction using the Trizol method (1) LPS-induced macrophage (THP-1, RAW 264.7, MH-S) inflammation model, collected inflammatory macrophages and transferred them to 1.5ml centrifuge tubes.

[0089] (2) Add 1 ml of Trizol to macrophages and homogenize them thoroughly. Let them stand at room temperature for 5 min to allow them to lyse completely.

[0090] (3) Add 200 μl of chloroform, gently shake for 15 s, and let stand at room temperature for 5 min.

[0091] (4) Centrifuge at 4℃, 12000g, for 15min. A clear liquid layering can be seen. Carefully aspirate the upper liquid into a new sterile 1.5ml centrifuge tube.

[0092] (5) Add 500 μl of isopropanol, gently mix the liquid in the centrifuge tube, and let it stand at room temperature for 10 min.

[0093] (6) Centrifuge at 4℃, 12000g, for 10 min, discard the supernatant. At this time, a translucent white solid can be seen at the bottom of the centrifuge tube, which is the total RNA.

[0094] (7) Add 1 ml of 75% ethanol, gently wash the precipitate, centrifuge at 7500g for 5 min at 4℃, and discard the supernatant.

[0095] (8) Dry at room temperature or under vacuum for 6-7 minutes, add 20-30 μl of DEPC water to dissolve the RNA sample, and measure the OD value to quantify the RNA concentration.

[0096] 4.2.2 RNA Reverse Transcription (1) The RNA reverse transcription procedure was performed on ice according to the instructions of the BeyoRT™ II cDNA First Strand Synthesis Kit from Shanghai Beyotime Biotechnology Co., Ltd. Reverse transcription reaction system (2) Mix gently and centrifuge until sedimentation reaches the bottom of the tube.

[0097] (3) Bathe in a 65℃ water bath for 5 minutes, then immediately ice bath for 2 minutes.

[0098] (4) Reverse transcription reaction system (5) Incubate at 42℃ for 60 minutes, then incubate at 80℃ for 10 minutes.

[0099] (6) After reverse transcription is completed, place the product at -80℃.

[0100] 4.2.3 Real-time quantitative PCR (qPCR) detection of SPINK8 mRNA levels in LPS-induced inflammatory macrophages (1) Real-time quantitative PCR was performed in accordance with the instructions of the TB Green® Fast qPCR Mix kit. All sample addition steps were performed on ice.

[0101] (2) qPCR reaction system (3) Reaction conditions: Pre-denaturation at 95°C for 30 seconds PCR reaction at 95°C for 5 seconds 60℃ for 15 seconds; The PCR reaction cycle number was 40.

[0102] (4) Data processing: qPCR data results were processed using 2... -△△Ct Law.

[0103] 4.2.4 Western blot analysis of SPINK8 protein levels in LPS-induced inflammatory macrophages (1) LPS-induced macrophage (THP-1, RAW264.7, MH-S) inflammation model, collected inflammatory macrophages and extracted total cell protein.

[0104] (2) SDS-PAGE electrophoresis, 80V for 1.5h, stop electrophoresis when the bromophenol blue gel is placed at 2 / 3 of the gel.

[0105] (3) Transfer: 0.2µm pore size PVDF membrane, 200mA, transfer for 20 minutes.

[0106] (4) Blocking: After the transfer is completed, carefully remove the PVDF membrane and place it in Western blot washing buffer for 1-2 minutes to remove the transfer buffer on the PVDF membrane. Then place it in blocking buffer (5% skim milk powder) and block at room temperature for 1 hour.

[0107] (5) Primary antibody incubation: Dilute the antibody according to the antibody titer instructions, put the blocked PVDF membrane into the anti-SPINK8 antibody, and incubate overnight on a shaker at 4°C.

[0108] (6) Washing the membrane: Wash the membrane 3-4 times with 1×TBST, 15 minutes each time.

[0109] (7) Secondary antibody incubation: Dilute the antibody according to the antibody titer instructions, put the PVDF membrane into horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) secondary antibody, and incubate at room temperature for 1 hour.

[0110] (8) Washing the membrane: Wash the membrane 3-4 times with 1×TBST, 15 minutes each time.

[0111] (9) Development: Prepare ECL working solution according to ECL luminescent solution A : ECL luminescent solution B = 1 : 1, and drop it onto PVDF membrane for development.

[0112] 4.3 Experimental Results In LPS-induced inflammatory macrophages, both the mRNA and protein levels of SPINK8 were significantly upregulated.

[0113] Example 5: The therapeutic effect of reSPINK8 protein in acute lung injury of sepsis. 5.1 Experimental Materials Male C57BL / 6 mice (6-7 weeks old) (purchased from Nanjing Model Animal Research Center, China, and housed in the SPF-grade animal facility of the First Affiliated Hospital of Huzhou University), SPINK8 gene knockout mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and housed in the SPF-grade animal facility of the First Affiliated Hospital of Huzhou University), sodium pentobarbital, 21G sterile injection needles, absorbable surgical sutures, physiological saline, xylene, hematoxylin staining solution, and eosin staining solution.

[0114] 5.2 Experimental Methods 5.2.1 Mouse cecal ligation and puncture model SPF-grade mice were randomly divided into two groups: a sham operation group and a sepsis group (CLP). Mice were fasted for 24 hours prior to surgery but allowed free access to water. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and placed on a flat plate. The abdominal hair was gently shaved with an electric razor and disinfected with 75% alcohol. In the sham operation group, a longitudinal incision of approximately 2 cm was made along the right side of the linea alba. The cecum was carefully located, lifted, and returned to the abdomen without further treatment. The wound was then carefully sutured. In the sepsis group, a longitudinal incision of approximately 2 cm was made along the right side of the linea alba. The cecum was carefully located and moderately ligated from the blind end upwards to half its total length. A 21G needle was inserted transversely through the cecum, puncturing it twice. A small amount of intestinal contents was gently squeezed out through the puncture site using sterilized forceps. The treated cecum was then returned to the abdominal cavity, and the wound was carefully sutured. After the surgery, each mouse was injected with 1 mL of preheated saline at 37°C to replenish fluids, and then returned to its cage. The mice were allowed to wake up naturally and drink and eat freely.

[0115] 5.2.2 The therapeutic effect of reSPINK8 protein on acute lung injury in sepsis Therapeutic effect: SPF-grade C57BL / 6 mice were fasted for 24 hours but given free access to water. Following this, the mice underwent sham surgery and cecal ligation and puncture. Sepsis-associated ALI mice were treated with the medication 12 hours post-surgery. The experimental group mice received different doses of reSPINK8 protein (5µg and 10µg) via nasal drops, while the control group mice received an equal volume of sterile saline via nasal drops.

[0116] 5.2.3 H&E staining (1) Dewaxing: Dewaxing paraffin sections with xylene in two separate steps, each lasting 20 minutes.

[0117] (2) Rehydration: Soak the slices in 100%, 95%, 85% and 75% ethanol for 5 minutes each, and then wash with distilled water.

[0118] (3) Hematoxylin staining of cell nuclei: Place the slide in hematoxylin staining solution for 5 minutes and rinse with running water for 2 minutes.

[0119] (4) Hydrochloric acid alcohol differentiation: wash with water for 3 minutes, bluing for a few seconds, and then wash with water for 8 minutes.

[0120] (5) Eosin staining of cytoplasm: Place the slide in eosin staining solution for 2 minutes and rinse with running water for 2 minutes.

[0121] (6) Dehydrate and dry sections with gradient alcohol, clear with xylene, and mount with neutral resin.

[0122] 5.2.4 Mouse survival analysis After surgical modeling, mice were housed in separate cages according to their groups, and the survival status of each group was observed and recorded daily. The survival status of each group of mice was observed over 72 hours.

[0123] 5.2.5 Effects of reSPINK8 protein on pro-inflammatory factors in lung tissue of septic ALI mice As described in section 5.2.2 above, after in vivo intervention with reSPINK8 protein, mouse lung tissue was collected, and total RNA was extracted using the Trizol method. qPCR was used to detect the mRNA levels of TNF-α, IL-1β, and IL-6 in the lung tissue.

[0124] 5.2.6 Data Statistics and Analysis Graphpad Prism 9 software was used for statistical analysis of the experimental data. The Unpaired Student's t-test was used to compare the data between the two groups. The Log-rank (Mantel-Cox) test was used for survival statistics. All experiments were repeated at least three times. Statistical standards are expressed as Mean ± SEM, where ns represents no statistical significance, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0125] 5.3 Experimental Results In acute lung injury (ALI) of sepsis, the mRNA and protein levels of SPINK8 in mouse lung tissue were significantly upregulated. Intervention with reSPINK8 protein in inflammatory macrophages significantly reduced the levels of pro-inflammatory factors in macrophages and alleviated the inflammatory response. SPINK8 gene knockout significantly aggravated lung tissue damage and inflammation in mice, and significantly reduced survival rates. Treatment of septic mice with different doses of reSPINK8 protein significantly reduced lung tissue damage, significantly decreased the mRNA levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in lung tissue, and significantly improved survival rates. These results indicate that reSPINK8 protein has an inhibitory effect on the inflammatory response and plays a protective role against ALI in sepsis. Therefore, this recombinant serine protease inhibitor has potential application value as a clinical drug for the prevention and treatment of ALI in sepsis.

[0126] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A serine protease inhibitor SPINK8, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The gene encoding SPINK8, the serine protease inhibitor as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. A recombinant expression vector comprising the encoding gene as described in claim 2, characterized in that, The recombinant expression vector is linked to a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.

3.

4. Recombinant expression cells comprising the recombinant expression vector as described in claim 3.

5. The recombinant expression cell according to claim 4 is characterized in that, The host cell was BL21(DE3) Escherichia coli.

6. A method for preparing the serine protease inhibitor SPINK8, characterized in that, The recombinant expression vector as described in claim 3 is introduced into host cells to induce expression.

7. The preparation method according to claim 7, characterized in that, The conditions for inducing expression include: a final IPTG concentration of 1 mM in the host cell culture system, an induction temperature of 37°C, and an induction time of 3-4 h.

8. Application of the serine protease inhibitor SPINK8 in acute lung injury of sepsis.

9. The application according to claim 8, characterized in that, The reSPINK8 protein improves the treatment efficacy of acute lung injury in sepsis by inhibiting the inflammatory response in lung macrophages, thus alleviating lung tissue damage and reducing the degree of inflammation.

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