Hydrostatin-SN61 as anti-inflammatory active peptide of sea snake, and coding gene and application thereof in pharmacy
阅读说明：本技术 青环海蛇抗炎活性肽Hydrostatin-SN61及其编码基因和在制药中的应用 (Hydrostatin-SN61 as anti-inflammatory active peptide of sea snake, and coding gene and application thereof in pharmacy ) 是由 陆一鸣 郭姗姗 荣旭丽 王庭芳 熊礼燕 于 2020-06-24 设计创作，主要内容包括：本发明涉及青环海蛇抗炎活性肽Hydrostatin-SN61及其编码基因和在制药中的应用,属于生物医学领域。青环海蛇抗炎活性肽Hydrostatin-SN61是一种直链多肽,含有12个氨基酸残基,分子量为1210.22道尔顿,等电点7.4,其基因序列如SEQ ID NO:1所示,多肽全序列一级结构如SEQ ID NO:2所示。本发明的Hydrostatin-SN61能够明显抑制TNF-α引起的炎症反应,可以作为制备如结肠炎、类风湿性关节炎等多种复杂炎症疾病或自身免疫性疾病的治疗药物被应用。青环海蛇活性肽Hydrostatin-SN61具有分子量小、结构简单、人工合成方便、抗炎活性强的特点。(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.)
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;
(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.
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.
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,
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-
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
(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
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-
FIG. 2 shows the MS analysis results of Hydrostatin-
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.
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