CN111763244A

CN111763244A - Hydrostatin-SN61 as anti-inflammatory active peptide of sea snake, and coding gene and application thereof in pharmacy

Info

Publication number: CN111763244A
Application number: CN202010591277.1A
Authority: CN
Inventors: 陆一鸣; 郭姗姗; 荣旭丽; 王庭芳; 熊礼燕
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-10-13
Anticipated expiration: 2040-06-24
Also published as: CN111763244B

Abstract

The invention relates to Hydrostatin-SN61 as an anti-inflammatory active peptide of a krait, a coding gene thereof and application thereof in pharmacy, belonging to the field of biomedicine. The anti-inflammatory peptide Hydrostatin-SN61 of the blue-loop sea snake is a straight-chain polypeptide, contains 12 amino acid residues, has the molecular weight of 1210.22 daltons, the isoelectric point of 7.4, the gene sequence of the polypeptide is shown as SEQ ID NO. 1, and the primary structure of the whole sequence of the polypeptide is shown as SEQ ID NO. 2. The Hydrostatin-SN61 can obviously inhibit inflammatory reaction caused by TNF-alpha, and can be applied to the preparation of medicaments for treating various complex inflammatory diseases such as colitis, rheumatoid arthritis and the like or autoimmune diseases. The Hydrostatin-SN61 has the advantages of small molecular weight, simple structure, convenient artificial synthesis, and strong anti-inflammatory activity.

Description

Hydrostatin-SN61 as anti-inflammatory active peptide of sea snake, and coding gene and application thereof in pharmacy

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a krait anti-inflammatory active peptide Hydrostatin-SN61, and a coding gene and application thereof in pharmacy.

Background

Tumor necrosis factor (TNF- α) is a cell signaling protein involved in the systemic inflammatory response and is also one of the cytokines that make up the acute phase response. It is produced primarily by activated macrophages, but can also be produced by many other cell types, such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils and neuronal cells. As a pleiotropic cytokine, TNF-alpha is one of the key regulatory factors in biological processes, and has effects in cell regeneration, migration, inflammation, and apoptosis (programmed cell death). TNF- α is an inflammatory mediator of many autoimmune diseases, such as rheumatoid arthritis, Inflammatory Bowel Disease (IBD), and multiple sclerosis, among others. There is evidence that elevated levels of TNF- α are present in intestinal mucosa, stool and blood samples from IBD patients and that levels of TNF- α are associated with clinical disease activity in crohn's disease patients (see document: b.ngo, c.p. farrell, m.barr et al, "Tumor necrosis factor receptor for treatment of inflammatory bone disease: efficacy and safety". Current Molecular Pharmacology, vol.3, No.3, pp.145-152,2010.).

The advent of TNF-alpha monoclonal antibody biological agents radically changes the treatment methods of IBD, rheumatoid arthritis and other complex inflammatory diseases or autoimmune diseases. anti-TNF therapy has been shown to alleviate disease symptoms, heal intestinal mucosal ulcers, reduce hospitalization and surgery, and reduce glucocorticoid use, both during short and long term use. However, not all patients respond equally to their treatment, up to 40% do not respond and even certain adverse reactions such as infection may occur due to indiscriminate blocking of cytokine activity, which may lead to a decrease in host resistance to infection. TNF-alpha exerts multiple biological functions by binding with the corresponding receptor TNFR on cell membranes, including TNFR1 and TNFR2, and TNF-alpha transmits pro-inflammatory and apoptotic signals mainly through TNFR1 to promote the body to generate inflammation or immune response, while TNFR2 has the main function of participating in the body proliferation and regeneration. With the intensive study of disease pathogenesis, more research is currently being conducted to switch the therapeutic target to TNFR. In general, from the viewpoint of anti-inflammatory, the focus of development of this class of drugs is now to block the biological function of TNF- α by blocking the signaling pathway of TNFR1 transmission.

IL-10 is an immunomodulatory cytokine produced by different cell types, including B lymphocytes, T lymphocytes, macrophages, monocytes, dendritic cells and mast cells IL-10, which simultaneously inhibits T lymphocyte, monocyte function and macrophage activation and effector functions, a cytokine with diverse effects on most hematopoietic cells, the most prominent of which is the limitation and eventual termination of inflammatory responses current research has shown that IL-10 is involved in the pathogenesis of IBD, mice deficient in the IL-10 gene can spontaneously develop colitis (see K ü hn R,

j, Rennick D, Rajewsky K, M ü ler W.Interleukin-10-specific micro deviceto Chronicitis.cell 1993; 75:263 plus 274.) the use of IL-10 knock-out mouse models of idiopathic colitis has been used to study the pathogenesis of IBD and therapeutic drug development.

The green sea snake (Hydrophis cyanocinctus) is a marine-derived traditional Chinese medicine recorded in compendium of materia medica, and the active ingredients of the green sea snake are mainly protein and polypeptide, so that the green sea snake has the effects of resisting inflammation, resisting tumor, resisting thrombus, relieving pain, reducing blood pressure and the like.

Chinese patent CN103030687A discloses an anti-inflammatory peptide Hydrostatin-SN1 of blue-ring sea snake, which contains twenty-two amino acid residues, has a molecular weight of 2483.68 daltons, an isoelectric point of 4.39 and a gene sequence as follows: 5'-GACGAACAAC ACCTAGAGAC CGAACTACAC ACTCTCACCA GCGTGCTGAC AGCCAATGGA TTCCAA-3', the primary structure of the polypeptide complete sequence is: Asp-Glu-Gln-His-Leu-Glu-Thr-Glu-Leu-His-Thr-Leu-Thr-Ser-Val-Leu-Thr-Ala-Asn-Gly-Phe-Gln, has stronger binding capacity with TNFR1 than TNFR2 and TNF-alpha, and has anti-inflammatory activity. However, no literature reports about the biologically active peptide Hydrostatin-SN61 of the Qinghuan sea snake are available.

Disclosure of Invention

The invention takes TNFR1 as a target spot to select a phage display library of the krait venom gland to obtain a new anti-inflammatory active peptide Hydrostatin-SN 61. The anti-inflammatory active peptide is deeply researched, and is found to be capable of effectively inhibiting TNFR1 mediated biological activity, and has great medicinal prospect.

The invention provides a Hydrostatin-SN61 as anti-inflammatory peptide of Qinghuan sea snake, its coding gene and application in pharmacy.

The invention provides a Hydrostatin-SN61 of anti-inflammatory peptide of Qinghuan sea snake, wherein the amino acid sequence of the anti-inflammatory peptide of Qinghuan sea snake is shown as SEQ ID NO. 2.

The anti-inflammatory active peptide of the green-ring sea snake is a straight-chain polypeptide, contains twelve amino acid residues, has the molecular weight of 1210.22 daltons, and the isoelectric point of 7.4.

The invention also provides a coding gene of the anti-inflammatory active peptide Hydrostatin-SN61 of the green-ring sea snake, and the coding gene is a DNA molecule shown as SEQ ID NO. 1.

In a second aspect of the invention, the invention provides a screening method of Hydrostatin-SN61, an anti-inflammatory active peptide of the krait, which comprises the following steps:

(1) biological elutriation: panning a phage display library of snake venom glands of the green-ring sea snake by taking a human recombinant tumor necrosis factor I type receptor (TNFR1) as a target spot, diluting human recombinant TNFR1 to the concentration of 1 mu g/ml by 1 XPBS, and coating a 96-hole ELISA plate by 1 mu l of protein and 99 mu l of Carbonate Buffer Solution (CBS); after 3h, wash 3 times with TBST and block overnight. Coated ELISA plates, each well added 200 u l phage, 37 degrees C were incubated for 3 hours; the plate was washed 4 times with TBST solution, the eluate was added and incubated at 37 ℃ for 20 minutes to elute bound phage from the ELISA plate, and the eluted phage was amplified with the host strain BLT5403(Novagen company) to complete the first round of panning. The biopanning was continued twice as described above and multiple phage clones (containing different inserts, i.e., different target gene fragments) were screened. The phage obtained by the third round of panning of PCR amplification of primers up (SEQ ID NO:3) and down (SEQ ID NO:4), wherein one insert (target gene) is identified as follows by sequencing: 5'-TCGGATCCCCGAGCATCACACCTGACTGGAATACGA-3' (shown as SEQ ID NO: 1), namely SN61 gene sequence; the primary structure of the amino acid sequence is as follows: Ser-Asp-Pro-Gly-Ala-Ser-His-Leu-Thr-Gly-IIe-Arg (SDPGASHLTGIR) as shown in SEQ ID NO 2;

(2) synthesizing: Hydrostatin-SN61 is synthesized by solid phase polypeptide synthesis technology, and purity and molecular weight are analyzed by HPLC and MS, the molecular weight is 1210.22 daltons, and isoelectric point is 7.4.

In a third aspect of the invention, the invention provides application of Hydrostatin-SN61 as an anti-inflammatory active peptide of the krait and a coding gene thereof in pharmacy, and the application is application in preparing medicines for treating diseases related to TNF-alpha.

Wherein, the disease related to TNF-alpha comprises a plurality of complex inflammatory diseases such as colitis, rheumatoid arthritis and the like or an application in the medicine of autoimmune diseases.

The invention adopts phage display technology to screen and obtain anti-inflammatory active peptide Hydrostatin-SN61, and Surface Plasmon Resonance (SPR) technology is used for analyzing the interaction between Hydrostatin-SN61 and TNFR1, which proves that the Hydrostatin-SN61 can be directly combined with the TNFR1 and can be combined with TNF-alpha and TNFR2 with relatively weaker capacity; Hydrostatin-SN61 competitively inhibited TNF- α binding to TNFR 1. Further utilizing endotoxin (LPS) to induce a bone marrow-derived macrophage (BMDM) cell model, and detecting the influence of the Hydrostatin-SN61 on MAPKs signal channels at the downstream of TNFR 1; meanwhile, an LPS (lipopolysaccharide) induced mouse acute shock model and an IL-10 gene knockout mouse spontaneous colitis model are established for evaluation

The result shows that the Hydrostatin-SN61 can be directly combined with the TNFR1, can inhibit the biological activity of TNF-alpha at the level of cells and animals, and has good anti-inflammatory activity. Therefore, the compound can be used as an active ingredient for TNF-alpha related anti-inflammatory drugs.

The anti-inflammatory active peptide Hydrostatin-SN61 of the green-ring sea snake contains twelve amino acid residues, has a molecular weight smaller than that of SN1, has a gene sequence and a polypeptide complete sequence primary structure different from that of SN1, has the binding capacity with TNFR1, TNFR2 and TNF-alpha different from that of SN1, and is a novel anti-inflammatory active peptide of the green-ring sea snake.

The Hydrostatin-SN61 has the characteristics of small molecular weight, simple structure, convenient artificial synthesis and strong anti-inflammatory activity. The medicine of the invention can be used for treating TNF-alpha related diseases such as colitis, rheumatoid arthritis and other complex inflammatory diseases or autoimmune diseases, provides a new way for treating the diseases, and has great clinical application value.

Drawings

FIG. 1 shows the HPLC analysis result of Hydrostatin-SN 61.

FIG. 2 shows the MS analysis results of Hydrostatin-SN 61.

FIG. 3 shows the coupling of TNF- α to the chip (final coupling 4200 RU).

FIG. 4 shows TNFR1 coupled to a chip (final coupling 3100 RU).

FIG. 5 shows TNFR2 coupled to a chip (final coupling 2900 RU).

FIG. 6 is a graph showing the binding reaction of Hydrostatin-SN61 directly to TNF- α coupled to a chip.

FIG. 7 is a graph showing the binding reaction of Hydrostatin-SN61 directly to TNFR1 coupled to a chip.

FIG. 8 is a graph showing the binding reaction of Hydrostatin-SN61 directly to TNFR2 coupled to a chip.

FIG. 9 shows TNFR1 coupled to a chip, two strips in sequence from top to bottom: TNF- α alone, TNF- α: Hydrostatin-SN61 is 1:100(n: n).

FIG. 10 shows TNFR2 coupled to a chip, two strips in sequence from top to bottom: TNF- α alone, TNF- α: Hydrostatin-SN61 is 1:50(n: n).

FIG. 11 is the effect of Hydrostatin-SN61 on LPS-induced inflammatory factor expression in mouse BMDM; wherein, A, TNF-alpha expression condition in mouse serum; B. IL-6 expression in mouse serum; C. IL-1 β expression in mouse serum; D. iNOS expression in mouse serum.

FIG. 12 is a graph of the effect of Hydrostatin-SN61 on the level of phosphorylation of MAPKs signaling pathways downstream of TNFR1 in LPS-induced mouse BMDM.

FIG. 13 is the effect of Hydrostatin-SN61 on an animal model of mouse LPS-induced acute shock.

FIG. 14 is the effect of Hydrostatin-SN61 on weight changes in mice in the IL-10-/-mouse model of idiopathic colitis.

FIG. 15 is the effect of Hydrostatin-SN61 on the disease Activity index in a model of IL-10-/-idiopathic colitis in mice.

FIG. 16 is the effect of Hydrostatin-SN61 on mouse spleen index in a model of IL-10-/-mouse idiopathic colitis.

FIG. 17 is the effect of Hydrostatin-SN61 on the expression of the inflammatory factor IL-1. beta. in colon tissue of IL-10-/-mice as a model of spontaneous colitis in mice.

FIG. 18 is a graph of the effect of Hydrostatin-SN61 on the phosphorylation level of the MAPKs signaling pathway downstream of TNFR1 in colon tissue of mice in a model of IL-10-/-spontaneous colitis in mice.

FIG. 19 is a 100-fold (100-fold) light-micrograph of histological sections showing the effect of Hydrostatin-SN61 on histopathological lesions of the colon of mice in the IL-10-/-mouse model of idiopathic colitis.

FIG. 20 is a photomicrograph (100-fold) of tissue sections showing the effect of Hydrostatin-SN61 on TNF- α expression levels in mouse colon tissue in IL-10-/-mouse model of idiopathic colitis.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but the following examples should not be construed as limiting the present invention. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Example 1: the source and synthesis of Hydrostatin-SN61 as anti-inflammatory peptide of Hydrostatin.

1. Construction of phage display library of venom glands of krait

Taking freshly isolated Qinghuan sea snake venom glands (0.18-0.2mg), extracting total RNA by a trizol method, separating mRNA by a Straight A's mRNA Isolation system kit (Novagen company) magnetic bead method, reverse transcribing Orient Express Oligo (dT) (Novagen company) to obtain a first strand of cDNA, and synthesizing a second strand of cDNA by Primer cDNAsynthesis Kits (Novagen company). The cDNA obtained above was ligated into T7 vector using T7 Select Cloning Kit (Novagen company), and packaged with T7 Select packaging Extract Kit (Novagen company) to finally obtain the phage display library of the venom glands of the green-ring sea snake venom. Measuring the titer of the library by a plate counting method, amplifying the library, adding 0.1% of 80% glycerol into the amplified library, mixing uniformly, and storing at-80 ℃.

2. Screening and obtaining of Hydrostatin-SN61

TNFR1 was used as a target for panning the phage display library of the venom glands of the green-ring sea snake venom: human recombinant TNFR1 was diluted to a concentration of 1. mu.g/ml with 1 XPBS and 96-well ELISA plates were coated with 1. mu.l protein + 99. mu.l CBS; after 3h, wash 3 times with TBST and block overnight. Coated ELISA plates, each well added 200 u l phage, 37 degrees C were incubated for 3 hours; the plate was washed 4 times with TBST solution, the eluate was added and incubated at 37 ℃ for 20 minutes to elute bound phage from the ELISA plate, and the eluted phage was amplified with the host strain BLT5403(Novagen company) to complete the first round of panning. The biopanning was continued twice as described above and multiple phage clones (containing different inserts, i.e., different target gene fragments) were screened.

Monoclonal phages were isolated from the phages obtained in the third panning run with primers up: 5'-GGAGCTGTCGTATTCCAGTC-3' (shown in SEQ ID NO:3), primer down: 5'-TTGGGGAGTTCTGGGCAAAT-3' (shown in SEQ ID NO:4), PCR amplification of the third round of panning, wherein one insert (target gene) was sequenced as follows: 5'-TCGGATCCCCGAGCATCACACCTG ACTGGAATACGA-3' (shown as SEQ ID NO: 1), namely SN61 gene sequence. The primary structure of the amino acid sequence is as follows: Ser-Asp-Pro-Gly-Ala-Ser-His-Leu-Thr-Gly-IIe-Arg (SDPGASHLTGIR) as shown in SEQ ID NO. 2.

Hydrostatin-SN61 was synthesized by solid phase peptide synthesis technique, and its purity and molecular weight were analyzed by HPLC (FIG. 1) and MS (FIG. 2), and the results showed that its purity was > 98.22%, molecular weight was 1210.22 daltons, and isoelectric point was 7.4.

Hydrostatin-SN61 obtained in example 1 was used in the following experiment.

Example 2: the biomacromolecule interaction analyzer BIACORE T200 based on Surface Plasmon Resonance (SPR) is used for detecting the interaction between SN61 and TNF-alpha, TNFR1 and TNFR2 coupled on the chip.

1. PBS-P running buffer was flowed through the channels set in the two CM-5 sensor chips at a flow rate of 10. mu.l/min, respectively, until a baseline level was reached.

2. PBS-buffer is used for activating surface reaction groups of each channel of the chip, simultaneously, the pre-enrichment of ligands is carried out, 1 XPBS buffer is used for dissolving TNF-alpha, TNFR1 and TNFR2 freeze-dried powder, and the optimum pH value of the coating protein is determined to be 4.5.

3. TNF-alpha, TNFR1 and TNFR2 proteins were covalently coupled to the sensor chip surface by amino coupling using a pH4.5 injection at a protein concentration of 50ug/ml, and the amounts of TNF-alpha, TNFR1 and TNFR2 were about 4200 (FIG. 3), 3100 (FIG. 4) and 2900 (FIG. 5) Reaction Units (RU), respectively.

4. Hydrostatin-SN61 is dissolved in HBS-EP Buffer, and is injected after being diluted in a gradient way, the response value of each concentration is recorded, and the binding capacity between the drug with different concentrations and TNF-alpha, TNFR1 and TNFR2 is detected. As shown in FIG. 6, the concentration of Hydrostatin-SN61 was 15.625. mu.M, 31.25. mu.M, 62.5. mu.M, 125. mu.M, 250. mu.M, and the binding ability to TNF- α was about 100. mu.M; as shown in FIG. 7, Hydrostatin-SN61 was able to interact directly with TNFR1, with the concentration of Hydrostatin-SN61 being 31.25. mu.M, 62.5. mu.M, 125. mu.M, 250. mu.M, 500. mu.M, and the binding capacity to TNFR1 being about 77.4. mu.M; as shown in FIG. 8, Hydrostatin-SN61 bound TNFR2, and Hydrostatin-SN61 was at concentrations of 3.90625. mu.M, 7.8125. mu.M, 15.625. mu.M, 31.25. mu.M, 62.5. mu.M, and 125. mu.M, and had a binding ability to TNFR2 of about 200. mu.M.

5. Further testing the competitive inhibition effect of Hydrostatin-SN61 on the binding of TNF-alpha and TNFR1 and TNFR 2: preparing a TNF-alpha protein solution with a concentration of 250nM by using EP buffer, mixing the TNF-alpha protein solution with Hydrostatin-SN61, performing on-machine detection, and comparing changes of the saturated concentration and the response value of TNF-alpha before and after adding Hydrostatin-SN61, wherein as shown in figure 9, the Hydrostatin-SN61 (TNF-alpha: Hydrostatin-SN61 is 1:100, n: n) can competitively inhibit the interaction of TNF-alpha and TNFR 1; as shown in FIG. 10, and Hydrostatin-SN61 (TNF-. alpha.: Hydrostatin-SN61 is 1:50, n: n) can competitively inhibit the interaction of TNF-. alpha.with TNFR 2.

Example 3: inhibition effect of Hydrostatin-SN61 on LPS-induced mouse bone marrow-derived BMDM inflammatory factor

LPS is further adopted to induce mouse BMDM, and the inhibition effect of the Hydrostatin-SN61 on LPS-induced inflammatory reaction is detected.

The specific implementation steps are that male C57BL/6 mice of 4 weeks old are taken, after being killed by dislocation of neck, soaked in 75% ethanol solution, the skin of hind limb of the mice is cut in a clean bench, and peeled to ankle, the tail end and ankle of the femur are separated by an ophthalmic scissors, muscle is removed, the femur and tibia of the mice are taken out and placed in RMPI 1640 culture medium, the RMPI 1640 culture medium is sucked by a sterile injector, bone marrow tissue is flushed out, the RMPI culture medium containing the bone marrow tissue is collected into a 50mL sterile centrifuge tube 1640, centrifuged for 5min at 25 ℃, 1000rpm and supernatant is discarded, erythrocyte lysate is added into the precipitate, heavy suspension precipitation is carried out, the precipitate is kept still for 3min, RMPI 1640 complete culture medium with twice volume of the erythrocyte lysate is added, uniformly blown, centrifuged for 5min at 25 ℃, 1000rpm is discarded, 1mL RMPI 1640 complete culture medium is added, counting and dilution is carried out to 1 × 10 heavy suspension, and heavy suspension is carried out⁶Each cell/mL was inoculated into a 12-well cell culture well plate, and M-CSF was added to the medium to a final concentration of 20ng/mL to induce differentiation. The liquid is changed at the third day,and observing adherent cells extending out of pseudopodia on the sixth day, namely BMDM which is completely differentiated, mixing LPS with the final concentration of 1 mu g/mL and Hydrostatin-SN61 with the final concentrations of 50 mu mol/L, 100 mu mol/L and 200 mu mol/L respectively, adding the mixture into a 12-well plate, incubating the mixture in a cell incubator for 6 hours, extracting total RNA of each group of cells by using a total RNA rapid extraction kit, and detecting the expression level of the inflammatory factors of the cells by an RT-PCR experiment (figure 11), wherein the result shows that the 100 mu mol/L and 200 mu mol/L Hydrostatin-SN61 can obviously inhibit the mRNA expression levels of the inflammatory factors TNF- α, IL-1 β, iNOS and IL-6.

Example 4: effect of Hydrostatin-SN61 on LPS Induction of phosphorylation levels of MAPKs signaling pathways downstream of TNFR1 in BMDM derived from mouse bone marrow

The specific experimental steps are as follows: BMDM single cell suspensions were obtained according to example 3, diluted to a cell concentration of 2 × 106 cells/mL, and seeded into 6-well cell culture well plates. After differentiation was complete, LPS at a final concentration of 1. mu.g/mL was mixed with Hydrostatin-SN61 at final concentrations of 50. mu. mol/L, 100. mu. mol/L, and 200. mu. mol/L, respectively, and added to a 6-well plate. After incubation for 6h in a cell culture box, extracting total cell protein, and carrying out Western Blot experiment to detect the change of MAPKs signal channels (figure 12), wherein the result shows that the Hydrostatin-SN61 can effectively inhibit the activation of MAPKs signal channels downstream of TNFR1 of BMDM induced by LPS.

Example 5: effect of Hydrostatin-SN61 on mouse LPS-induced acute shock animal model

The specific experimental steps are as follows: selecting 6-8 weeks C57BL/6 male mice (8 mice per group), carrying out intraperitoneal injection of LPS (15mg/kg) for induction and establishment of an acute shock model, carrying out intervention of 10 mug/kg, 50 mug/kg and 250 mug/kg of Hydrostatin-SN61, counting the survival rate of the mice and drawing a survival curve (figure 13). The results show that: Hydrostatin-SN 6150 mu g/kg and 250 mu g/kg can effectively relieve acute shock induced by LPS and improve the survival rate of mice.

Example 6: effect of Hydrostatin-SN61 on IL-10 gene knockout of mouse colitis

The specific implementation steps are as follows: male IL-10 knockout mice of 6-8 weeks of age were selected and randomized into 5 groups, each group containing 6 mice. The administration of piroxicam at a dose of 10mg/kg/d by intragastric administration accelerates the occurrence of spontaneous colitis, and the weight and state of the mice are recorded every day until the mice show obvious colitis symptoms (weight is obviously reduced, rectal prolapse, diarrhea, hematochezia, and the like) and the administration is started. The normal group (normal) was not treated and the remaining groups were administered intraperitoneally. The model group (model) mice were injected with physiological saline at a dose of 100. mu.l/20 g, the positive control group with 5mg/kg/d of Infliximab (IFX), the Hydrostatin-SN61 group with 400. mu.g/kg/d, and the negative control group with 400. mu.g/kg/d of random peptide. The weight change of each group of mice was recorded daily (fig. 14), and Hydrostatin-SN61 was effective in alleviating the weight loss caused by colitis. Disease Activity Index (DAI) scoring according to Table 1, results are shown in FIG. 15, and Hydrostatin-SN61 can effectively relieve symptoms of diarrhea and hematochezia in colitis mice, and reduce DAI scoring. After 40 days of the administration treatment, mice were sacrificed by dislocation of cervical vertebrae, spleens of the mice were removed, spleen weights were measured and spleen indexes were calculated, and the results showed that the Hydrostatin-SN61 could effectively inhibit spleen swelling caused by colitis (FIG. 16); extracting total RNA in colon tissue, carrying out reverse transcription to obtain cDNA, detecting the expression level of inflammatory factors by using an RT-PCR technology, wherein the result shows that the Hydrostatin-SN61 can reduce the mRNA expression level of proinflammatory factor IL-1 beta (figure 17); extracting total protein of colon tissue, carrying out Western blot to detect the change of MAPKs signal channels, and finding that Hydrostatin-SN61 can inhibit the phosphorylation activation of ERK (figure 18); the colon terminal tissue is fixed by 10 percent formalin, sliced, and the change of the colon is observed after HE staining, and the observation shows that the Hydrostatin-SN61 can effectively inhibit the colon lesion of the spontaneous colitis of the IL-10-/-mice (figure 19); the TNF-alpha expression level in the colon tissue section of the mouse is observed, and the fact that the Hydrostatin-SN61 can reduce the expression of the inflammatory factor of the colon tissue of the mouse is also verified (figure 20).

TABLE 1 DAI Scoring criteria

The results show that the Hydrostatin-SN61 can effectively treat the IL-10-/-mice spontaneous colitis animal model.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

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Claims

1. The anti-inflammatory active peptide of the green-ring sea snake is characterized in that the amino acid sequence of the anti-inflammatory active peptide of the green-ring sea snake is shown as SEQ ID NO. 2.

2. An anti-inflammatory active peptide of the green-ring sea snake of claim 1, which is characterized in that: the anti-inflammatory active peptide of the green-ring sea snake is a straight-chain polypeptide, contains twelve amino acid residues, has the molecular weight of 1210.22 daltons, and the isoelectric point of 7.4.

3. The gene encoding the anti-inflammatory peptide of the sea cucumber as claimed in claim 1 or 2, wherein the encoding gene is a DNA molecule as shown in SEQ ID NO. 1.

4. A method of screening for anti-inflammatory peptides of the green-ring sea snake of claim 1 or 2, comprising the steps of:

(1) biological elutriation: panning a phage display library of snake venom glands of the green-ring sea snake by taking a human recombinant tumor necrosis factor I type receptor TNFR1 as a target spot, diluting the human recombinant TNFR1 to the concentration of 1 mu g/ml by using 1 XPBS, and coating a 96-hole ELISA plate by using 1 mu l of protein and 99 mu l of carbonate buffer CBS; washing with TBST for 3 times after 3h, and sealing with blocking solution overnight; coated ELISA plates, each well added 200 u l phage, 37 degrees C were incubated for 3 hours; washing the plate for 4 times by TBST, adding eluent, incubating for 20 min at 37 ℃, eluting the combined phage from the ELISA plate, amplifying the eluted phage by host bacteria BLT5403, and completing the first round of panning;

performing biopanning twice according to the method, screening to obtain multiple phage clones, performing PCR amplification on phage obtained by third panning with primer up shown in SEQ ID NO.3 and primer down shown in SEQ ID NO. 4, and sequencing to obtain Hydrostatin-SN61 with gene sequence shown in SEQ ID NO. 1 and amino acid sequence shown in SEQ ID NO. 2;

5. Use of an anti-inflammatory active peptide of Serpentis cuphea as claimed in claim 1 or 2 in the manufacture of a medicament for the treatment of a TNF- α related disorder, wherein said TNF- α related disorder is colitis or rheumatoid arthritis.

6. Use of a gene encoding an anti-inflammatory active peptide of Serpentis as claimed in claim 3, in the manufacture of a medicament for the treatment of a TNF- α related disorder, wherein said TNF- α related disorder is colitis or rheumatoid arthritis.