A parasitic nematode SPI-I8 protein and its application in preventing and treating inflammatory diseases

Abstract

The present invention relates to a parasitic nematode SPI-I8 protein for preventing or treating inflammatory diseases, which is derived from Nipostrongylus brasiliensis, and the amino acid sequence is shown in SEQ ID No. 2. The present invention finds that the SPI-I8 protein has a good therapeutic effect on localized inflammatory diseases and systemic inflammatory diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a parasitic nematode SPI-I8 protein and an application thereof in preventing and treating inflammatory diseases. Background Art

[0002] Inflammation is the tissue cell reaction of the animal body to pathogenic microorganism infection, trauma, allergy, etc. Normal inflammatory response is a manifestation of the body's immunity and is beneficial, but excessive inflammatory response can also cause body dysfunction such as cytokine storm. Clinically, the high incidence of mammalian inflammation has greatly affected the healthy development of animal husbandry and caused certain economic losses. For example, inflammation of the sow's reproductive system seriously restricts the performance of sows' reproductive performance; and the onset of mastitis in dairy cows leads to a decrease in milk production and reduced nutritional value of milk. At the same time, it also harms human health and causes diseases such as tuberculosis, laryngitis, scarlet fever, and acute enteritis. At present, the prevention, control and treatment of mammalian inflammation in clinical practice are mainly based on drug therapy, and the selection of appropriate anti-inflammatory drugs is supplemented by the selection of sensitive antibiotics for single treatment or combined treatment. Since several commonly used antibiotics are mostly ineffective against widely spread pathogens, they turn into chronic inflammation and cause recurrence of inflammation at any time. Furthermore, in addition to bacteria, the pathogenic microorganisms that cause inflammation include fungi, mycoplasmas and viruses, and antibiotics are ineffective against viruses and many mycoplasmas. These are the fundamental reasons why inflammatory diseases in mammals are not only incurable but also have the tendency to spread.

[0003] Parasites cause considerable morbidity and mortality in animal populations. Among them, serine protease inhibitors (SPIs) exist in the excretion / secretion products of parasitic worms (including flukes, tapeworms, and nematodes), which can inhibit the activity of serine proteases such as elastase, chymotrypsin, trypsin, or coagulation factors, thereby resisting damage to the parasite body by host digestive enzymes and evading the host's killing mechanism; they can also interfere with the function of macrophages, mast cells, or other immune cells, inhibiting the host immune response or achieving immune escape.

[0004] At present, anti-inflammatory drugs are mainly used to relieve inflammatory reactions, which have great side effects and are not effective in severe inflammatory reactions such as inflammatory enteritis and septicemia. The present invention studies the serine protease inhibitors (SPIs) of the Brazilian nematode in order to obtain active substances that can be used for animal inflammatory diseases. Summary of the invention

[0005] The present invention aims to provide a parasitic nematode SPI-I8 protein for preventing or treating inflammatory diseases and its application.

[0006] In one aspect, the present invention provides a parasitic nematode SPI-I8 protein for preventing and / or treating inflammatory diseases, the amino acid sequence of which is shown in SEQ ID No.2.

[0007] In one embodiment, the SPI-I8 protein is derived from Nippostrongylus brasiliensis.

[0008] In one embodiment, the CDS sequence of the SPI-I8 protein is shown as SEQ ID No.1.

[0009] In one embodiment, the SPI-I8 protein can be prepared into a dosage form for intralesional administration, intravascular administration, subcutaneous administration, intranasal administration or intraperitoneal administration.

[0010] In one embodiment, the SPI-I8 protein can be prepared into an injection for intraperitoneal administration and / or intravenous administration.

[0011] On the other hand, the present invention provides an isolated polynucleotide, which is a polynucleotide sequence encoding the above-mentioned SPI-I8 protein.

[0012] In one embodiment, the polynucleotide sequence is as shown in SEQ ID No.1.

[0013] In another aspect, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0014] In one embodiment, the vector is an expression vector. Preferably, the vector is a prokaryotic expression vector. More preferably, the vector is a pET-30a prokaryotic expression vector.

[0015] On the other hand, the present invention provides an engineered host cell, wherein the host cell comprises the above-mentioned SPI-I8 protein, or the above-mentioned polynucleotide, or the above-mentioned vector.

[0016] In one embodiment, the host cell is a prokaryotic cell. Preferably, the host cell is an Escherichia coli cell.

[0017] On the other hand, the present invention provides the use of the above-mentioned SPI-I8 protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned host cell in the preparation of a drug for preventing and / or treating inflammatory diseases.

[0018] In one embodiment, the inflammatory disease is a localized inflammatory disease and / or a systemic inflammatory disease.

[0019] In one embodiment, the localized inflammatory disease comprises enteritis; and the systemic inflammatory disease comprises sepsis or septicemia.

[0020] In one embodiment, the inflammatory disease is enteritis and / or sepsis.

[0021] In one embodiment, the inflammatory disease is inflammatory bowel disease and / or sepsis.

[0022] In one embodiment, administration of the SPI-I8 protein can alleviate the symptom of shortened colon length caused by colitis.

[0023] In one embodiment, administration of the SPI-I8 protein can alleviate the symptom of shortened large intestine length caused by colitis.

[0024] In one embodiment, administration of the SPI-I8 protein can reduce the mortality rate caused by sepsis.

[0025] In one embodiment, the use is for preparing veterinary medicines.

[0026] Beneficial Effects

[0027] The present invention finds that the parasitic nematode SPI-I8 protein can alleviate localized inflammatory responses and systemic inflammatory responses, especially can reduce the mortality caused by sepsis, and has low side effects, and can be prepared into a safe and efficient drug for treating inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Proteomic analysis of EV proteins and EV-depleted ESP proteins of N. brasiliensis.

[0029] Figure 2 .The expression level of SPI-I8 in different developmental stages of N. brasiliensis.

[0030] Figure 3 .Results of the optimal time for SPI-I8 protein induction and protein properties.

[0031] Figure 4 . SPI-I8 protein purified by elution with different concentrations of imidazole.

[0032] Figure 5 .Purified SPI-I8 protein was eluted with 500 mM imidazole.

[0033] Figure 6 .The results of the effect of SPI-I8 protein on colon length in the colitis model.

[0034] Figure 7 .The therapeutic effect of SPI-I8 protein on septic mice.

[0035] Sequence information

[0036] CDS sequence of the SPI-I8 gene of Nippostrongylus brasiliensis (SEQ ID No.1): GGTCCATTCGACTGCGACAAAGACGGTAAATGCAGTCCAGGACTACGATGCGAGGAGGGAACCTGTGTTTTGCGAACAGACTGCCCGATGCTGTCGATGCCTCGAATGAAATCCGGCTGCAAATTGACGATGATGGTCGACGAACGTGACTGTCCGATGCCGAAAATCGAATGCAGCAAGGAGAATCTTAAGTGTGGATCGATCTACTGTGAACCCGGCTACGAATGCGACAAAGACGCCGTCAAGTGCATTCCACGACGTGATTGCCCGAACCTTGTTCTGCCTAAAGTGGACGGATGCACTGACGAAATGACCCTGGATGAATACGACTGTCTGATGCCGGTTCGTACATGCAAACCAACTAAACCACTCCGACAAAAACGGGCAAAGACCACAGATCCATCCGTGAAATGCGCGAAAAACGCCGAGTACCGCGAATGCACCAACTTTTGCCCAGCCAAGTCGTGCAGCAACTATCTAGAGAAGTCGGCATGCTTCTCACTGAGGTGCGGACCACCCGGCTGTATGTGCAAGGAGGGACACGTGCAACTTTCGCATGATATCGAACAGGGATGCGTTTCGAGGGAGACTTGCGTGAAACTGGACTCTCTCAAGAAGAACATCGAACGGAACAAGCCAGCGAACTGA;

[0037] Amino acid sequence encoded by the SPI-I8 gene of Nippostrongylus brasiliensis (SEQ ID No.2): GPFDCDKDGKCSPGLRCEEGTCVLRTDCPMLSMPRMKSGCKLTMMVDERDCPMPKIECSKENLKCGSIYCEPGYECDKDAVKCIPRRDCPNLVLPKVDGCTDEMTLDEYDCLMPVRTCKPTKPLRQKRAKTTDPSVKCAKNAEYRECTNFCPAKSCSNYLEKSACFSLRCGPPGCMCKEGHVQLSHDIEQGCVSRETCVKLDSLKKNIERNKPAN. Detailed implementation manners

[0038] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0039] Example 1: Proteomic analysis of Nipostrongylus brasiliensis

[0040] (1) Cultivation of nematodes in vitro

[0041] The collected adult Japanese strongyloides were washed 5 times with sterile saline, nematode sterilization solution (70mM sodium chloride, 2.5mM potassium chloride, 10mM disodium hydrogen phosphate, 2.5mM sodium dihydrogen phosphate, 5mM glucose, 10U / mL penicillin, 100U / mL streptomycin, 10μg / mL amphotericin B, 80μg / mL ciprofloxacin, 100μg / mL ampicillin, 40μg / mL gentamicin; Rothwell and Sangster, 1993; Kotze, 2003) and DMEM containing 1% triple antibody (penicillin-streptomycin-gentamicin), and washed 5 times each time with inversion on a shaker for 5 minutes. After removing the contamination, every 1000 nematodes were transferred to a T75 cell culture flask containing 50 mL of DMEM and 1% triple antibody, and then cultured in an incubator at 38°C containing 10% CO2 for 24 h. The culture supernatant was collected and used for ESP (excretory secretory protein) separation.

[0042] (2) Separation of EVs (extracellular vesicles) and ESPs without EVs

[0043] First, impurities such as eggs and dead cells in the culture medium were removed by centrifugation at 2,000×g for 30 min at 4°C, and the supernatant was collected and centrifuged at 10,000×g for 45 min to remove the precipitate (medium and large vesicles). The remaining supernatant was filtered through a 0.45 μm filter and centrifuged at 100,000×g and 4°C in an ultracentrifuge for 70 min, and the supernatant and precipitate were collected separately.

[0044] 1) The obtained precipitate was resuspended in 10 mL of pre-cooled PBS, and then the previous centrifugation step (100,000 × g, 4 ° C, 70 min) was repeated, and finally resuspended with 400 μL of pre-cooled PBS; 2% sodium deoxycholate lysis buffer containing protease inhibitors was added thereto, mixed and dried at 95 ° C for 10 min, cooled to room temperature, and centrifuged at 4 ° C for 15 min at 20,000 × g; the collected supernatant contained the Brazilian nematode EV protein.

[0045] 2) The collected supernatant was added to an ultrafiltration tube for concentration until the liquid was about 500 μL; then it was washed twice with 5 mL of pre-cooled PBS, and the volume was finally concentrated to 250 μL, which was the ESP with EV removed; then a corresponding volume of lysis buffer containing 8 M urea / 50 mM Tris-HCL and protease inhibitors was added to obtain the ESP protein of the Brazilian nematode with EV removed.

[0046] 3) Add dithiothreitol (DTT; final concentration 10mM) to the two proteins (EV protein and ESP protein without EV) and place them in a 37°C water bath for 1h, then add an appropriate amount of iodoacetamide (IAA; final concentration 20mM) in the dark for 30min. According to the steps described in the BCA kit manual, the concentrations of the two proteins were determined, and 10μg of each sample was also taken for the preparation of SDS-PAGE samples.

[0047] (3) Protein digestion and separation by high performance liquid chromatography (HPLC)

[0048] 150 μg of each sample was mixed with 3 μg of pancreatin and incubated at 37°C for 16 h to digest the protein into peptides. The enzymatic peptides were desalted, dried, and then redissolved in pure water and stored at -20°C. After equal amounts of peptides from all samples were mixed, they were diluted with mobile phase A (5% ACN, pH = 9.8) and injected using the Thermo Scientific UltiMate TM The samples were separated by using a 3000 binary rapid separation system, and the separation column was a 3.5 μm 4.6×150 mm Agilent ZORBAX 300 Extend-C18 column. The samples were subjected to liquid phase separation. The gradient elution was performed at a flow rate of 0.3 mL / min: 5% mobile phase B (5% ACN, pH=9.8) to 21.5% mobile phase B for 38 min, 21.5% to 40% mobile phase B for 20 min, 40% to 90% mobile phase B for 2 min, 90% mobile phase B for 3 min, and 5% mobile phase B for 10 min. The elution peak was monitored at a wavelength of 214 nm and one component was collected every minute. The samples were combined in combination with the chromatographic elution peaks to obtain 10 components, which were then freeze-dried.

[0049] (4) Nano-LC-MS / MS proteomics analysis

[0050] EV proteins and ESP proteins after EV removal were identified by standardized Nano-LC-MS / MS. The drained peptide samples were reconstituted with 0.1% FA, centrifuged at 20,000×g for 10 min, and the supernatant was injected. TM The sample was separated by a 1200 system. The sample entered a self-packed C18 column (100 μm inner diameter, 1.8 μm column particle size, about 35 cm column length) at a flow rate of 300 nL / min.

[0051] The results after analysis are as follows Figure 1 As shown, after removing the ESP of EV ( Figure 1 There are about 600 proteins in ESP*, EV( Figure 1 There are about 700 proteins in the exosome, of which about 300 are shared proteins. SPI-I8 is one of the shared proteins.

[0052] Example 2: Analysis of the transcription level of SPI-I8 gene in different developmental stages of N. brasiliensis

[0053] (1) Collecting nematodes

[0054] The eggs were enriched by the saturated saline flotation method, resuspended in D-Hank's culture medium, placed on a 2% agarose gel culture plate and incubated at 28°C, and the L1 larvae were collected after 24 hours, the L2 larvae were collected after 72 hours, and the L3 (infectious) larvae were collected after 168 hours. The infective larvae infect animals, and the L4 larvae and adults were collected.

[0055] (2) Total RNA extraction (TRIzol method) and reverse transcription reaction

[0056] Take the worm bodies at different developmental stages and put them into 1.5mL EP tubes, add 1mL TRIzol and 5 magnetic beads, use a tissue cell grinder, 60HZ, 120s, to fully break up the worm bodies, and let it stand at room temperature for 10min to fully lyse. Add 0.2mL chloroform, shake vigorously for 15s, and let it stand at room temperature for 10min; centrifuge at 12000rpm for 15min at 4℃; aspirate the upper aqueous phase liquid into another centrifuge tube, add 0.4mL isopropanol, mix well, and let it stand at room temperature for 10min; centrifuge at 12000rpm for 10min at 4℃, and discard the supernatant liquid; add 1mL 75% ethanol solution (prepared with 1‰ DEPC water), wash the precipitate thoroughly, and centrifuge at 12000rpm for 15min at 4℃; open the lid and dry for 5-10min until the white precipitate becomes transparent; add an appropriate amount of RNase-free water to dissolve the precipitate, and perform quality inspection using a nano micro-nucleic acid analyzer. IIQ RT SuperMix for qPCR (+gDNAwiper) kit was used to reverse transcribe sample RNA.

[0057] (3) Fluorescence quantitative PCR

[0058] The reaction system was configured in a Roche LC480 fluorescence quantitative PCR instrument, with pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and 40 cycles; denaturation at 95°C for 15 s, annealing at 60°C for 60 s, and denaturation at 95°C for 15 s to collect Ct value information; the obtained CT values ​​were analyzed by relative quantitative analysis using the 2-ΔCT value comparison method using Microsoft Excel, and significance analysis was performed using the t-test method and one-way analysis of variance. The software GraphPad Prism 8.0 was used to draw graphs to analyze the transcription level of the SPI-I8 gene at different developmental stages of the insect body. The results are shown in Figure 2. Figure 2 As shown. SPI-I8 gene in the egg stage ( Figure 2 egg), no expression of transcription in L1 and L2 stages, low-level transcription in L3, and low-level transcription in L4 and adult stages ( Figure 2 Ad) in has high levels of transcription.

[0059] Example 3: Induced expression and purification of SPI-I8 protein

[0060] (1) Select BamHI and HindⅢ restriction sites, insert the CDS sequence of SPI-I8 gene (the CDS sequence of SPI-I8 gene is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.2) into the pET-30a prokaryotic expression vector, transform the E. coli BL21 expression strain, and perform PCR and double restriction digestion identification. The positive bacterial solution was inoculated into 50 mL LB liquid medium containing kanamycin (50 ng / μL) at a ratio of 1:100, cultured at 37℃ with shaking until OD600 reached 0.6, added IPTG inducer to a final concentration of 1 mM, induced at 37℃, and collected bacterial samples at 0h, 2h, 4h, 6h, and 8h of induction (the 8h bacterial solution was centrifuged and added with PBS buffer, ultrasonically disrupted for 10min, and the supernatant and precipitate were collected respectively) for SDS-PAGE and Western blot analysis to determine the optimal induction time and the solubility characteristics of the recombinant protein.

[0061] (2) The bacterial solution was inoculated into 300 mL of LB medium containing kanamycin at a ratio of 1:100 and expanded to the logarithmic phase, and IPTG was added for induction of expression; the induced bacterial solution was collected into a 50 mL centrifuge tube and centrifuged at 8000 rpm for 5 min; the supernatant was discarded, and 7 mL of PBS buffer (50 mM, pH 7.4) was added to resuspend the bacterial precipitate, which was thoroughly mixed by blowing and then ultrasonically disrupted (working for 3.5 s and resting for 4 s for a total of 30 min); centrifuged at 8000 rpm for 10 min, the supernatant was collected, purified by Ni-NTA agarose resin, and eluted with different concentrations (250 mM and 500 mM) of imidazole; the eluate of different concentrations was collected for SDS-PAGE electrophoresis to detect the purification effect.

[0062] The results are as follows Figure 3-5 As shown, where:

[0063] Figure 3 The expression of SPI-I8 at different induction times (0h, 2h, 4h, 6h, 8h). Figure 3 In the figure, S is the supernatant, P is the precipitate, M is the marker, and the box is the location of SPI-I8. Figure 3 The results showed that 8h was the best induction time and the induced recombinant protein SPI-I8 was expressed in the supernatant as a soluble protein.

[0064] Figure 4 The elution of SPI-I8 was performed with different concentrations of imidazole. Figure 4 In the figure, P is the flow-through before column hanging, A is the flow-through after column hanging, M is a marker, 5, 10, 20 refer to the components eluted with 5mM, 10mM, and 20mM imidazole eluents, respectively, 125(2-8) refers to the components eluted 2-8 times with 125mM imidazole eluent, 250(2,4) refers to the components eluted 2nd and 4th times with 250mM imidazole eluent, and the box indicates the location of SPI-I8. Figure 4 The results showed that 250 mM imidazole concentration could elute and purify the recombinant protein SPI-I8 in large quantities.

[0065] Figure 5 Elution was performed with 500 mM imidazole in the case of SPI-I8. Figure 5 In the figure, P is the flow-through before column hanging, A is the flow-through after column hanging, M is a marker, 500 (1-10) refers to the components obtained by eluting 1-10 times with 500 mM imidazole eluent, and the position of SPI-I8 is shown in the box. Figure 5 The results showed that 500 mM imidazole concentration could elute and purify the recombinant protein SPI-I8 in large quantities.

[0066] Example 4: Therapeutic effect of SPI-I8 protein on enteritis

[0067] Inflammatory bowel disease was tested in mice using DSS (dextran sulfate sodium). C57BL / 6 mice (n=20) were randomly divided into 4 groups, one of which was a blank control group and was given fresh water ( Figure 6 Group 2 was the positive control group, which was given fresh water containing 2.5% DSS (MP Biomedicals) Figure 6 3 groups were intraperitoneally injected with 50 μg SPI-I8 protein ( Figure 6 4 groups were given fresh water containing 2.5% DSS and injected with 50 μg SPI-I8 protein ( Figure 6 After 7 days of treatment, the colon length of each mouse was measured at autopsy.

[0068] The results are as follows Figure 6 As shown, the colon length of the positive control group mice given DSS was significantly shorter than that of the blank control group; the colon length of the mice given DSS and injected with SPI-I8 protein was significantly longer than that of the positive control group; there was no significant difference in colon length between the mice injected with SPI-I8 protein alone and the blank control group. This shows that DSS successfully induced a small amount of colon inflammation and shortened the colon length; SPI-I8 protein can significantly reduce the effect of colitis on colon length without obvious side effects.

[0069] Example 5. Therapeutic effect of SPI-I8 protein on sepsis

[0070] Sepsis was induced in mice by LPS injection. C57BL / 6 (n=20) were divided into two groups. The first group was injected with 50μg SPI-I8 protein intraperitoneally every day for 3 consecutive days, and 150μg SPI-I8 protein and 50mg LPS (Sigma-Aldrich) were injected through the tail vein on the 4th day. The second group was not pretreated, and the same volume of PBS and 50mg LPS were injected through the tail vein on the 4th day. The death of the mice treated with the drug was observed every 24h until all the mice in the control group died.

[0071] The results are as follows Figure 7 As shown, most of the SPI-I8 protein-treated mice ( Figure 7 Nb-SPI-I8+LPS) survived during the observation period, but mice injected with PBS and LPS ( Figure 7 All mice induced by LPS had died on day 3. This indicates that SPI-I8 protein can significantly improve the survival rate of mice with lethal sepsis.

[0072] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of a parasitic nematode SPI-I8 protein in the preparation of a drug for preventing and / or treating inflammatory diseases, wherein the amino acid sequence of the SPI-I8 protein is shown in SEQ ID No. 2; the inflammatory disease is colitis and / or sepsis.

2. The use according to claim 1, characterized in that: The SPI-I8 protein is prepared into an injection for intraperitoneal administration and / or intravenous administration.

3. The use according to claim 1, characterized in that: Administration of the SPI-I8 protein can alleviate the symptom of shortened colon length caused by colitis.

4. The use according to claim 1, characterized in that: Administration of the SPI-I8 protein can reduce the mortality rate caused by sepsis.

5. The use according to claim 1, characterized in that: The application is application in preparing veterinary medicines.

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