CN110511268B - Polypeptide capable of preventing or treating sicca syndrome and application thereof - Google Patents
Polypeptide capable of preventing or treating sicca syndrome and application thereof Download PDFInfo
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- CN110511268B CN110511268B CN201910680615.6A CN201910680615A CN110511268B CN 110511268 B CN110511268 B CN 110511268B CN 201910680615 A CN201910680615 A CN 201910680615A CN 110511268 B CN110511268 B CN 110511268B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a polypeptide capable of preventing or treating sicca syndrome, a preparation method and application thereof.
Description
Relates to a sequence table
The present application contains a sequence listing in computer readable form, which is incorporated herein by reference.
Technical Field
The present invention relates to a polypeptide capable of preventing or treating Sjogren's syndrome, polynucleotide encoding the same, and uses thereof.
Technical Field
Sjogren's Syndrome (SS) is a chronic inflammatory autoimmune disease that primarily involves exocrine glands, one of a wide variety of known and unknown etiologies and can cause exocrine disorders, the inflammatory response of which is primarily manifested in the epithelial cells of the exocrine glands. Sjogren's syndrome is characterized by a xerosis condition caused by inflammatory cell infiltration around the acini and ductus of exocrine glands, which results in destruction and atrophy of the epithelial cells of the acini and ductus. Clinically, there are symptoms of multiple system damage caused by involvement of other exocrine glands and organs outside the glands, except for dry mouth and eyes caused by impaired function of salivary glands and lacrimal glands. There are a variety of autoantibodies and hyperimmune globulinemia in the serum of patients. The disease is divided into two types of primary sicca syndrome and secondary sicca syndrome, the primary sicca syndrome belongs to global diseases, and the prevalence rate of Chinese people is 0.3-0.7%.
Disorders caused by external secretion disorders such as "dry eye syndrome" (dry eye disease), "dry mouth syndrome" (dry mouth), "nasal dryness syndrome" (nasal dryness), "skin dryness syndrome" (xeroderma), and "vaginal dryness syndrome" (symptoms are vaginal dryness), and chronic pancreatitis, chronic gastritis, and chronic bronchitis due to inhibition of external secretion, and the like.
Although exocrine disorders can cause severe disease as described above, to date only symptomatic therapies for such disorders, such as artificial hydration, have been effective.
Therefore, an effective method for improving exocrine inhibition is researched, and the method has important significance for clinical application.
Summary of The Invention
The present invention relates to a polypeptide selected from the group consisting of:
(a) A polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO. 1;
(b) A polypeptide derived from (a) by substitution, deletion or addition of one or more amino acids to the amino acid sequence of (a).
The invention also relates to polynucleotides encoding the polypeptides, vectors and cells comprising the polynucleotides, and methods of making the same.
The invention also relates to a pharmaceutical composition containing the polypeptide, the polynucleotide, the vector or the cell and application of the polypeptide, the polynucleotide, the vector or the cell in preparing medicines for preventing or treating exocrine disorder and/or autoimmune diseases, sicca syndrome, and multisystem damage caused by the involvement of dry mouth, dry eyes and other exocrine glands and other organs outside the gland body caused by sicca syndrome.
Drawings
FIG. 1 is a graph showing the effect of the polypeptide of the present invention on infiltration of lymphocytes in the submandibular glands of mice
FIG. 2 influence of the polypeptide of the invention on lymphocyte infiltration area and saliva flow rate in the submandibular glands of mice
Detailed Description
The present invention relates to a polypeptide selected from the group consisting of:
(a) A polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO. 1;
(b) A polypeptide derived from (a) by substitution, deletion or addition of one or more amino acids to the amino acid sequence of (a).
In other embodiments of the invention, the polypeptides provided herein are polypeptides comprising the amino acid sequence shown in SEQ ID NO.1, as well as modified polypeptides or homologous polypeptides thereof.
In other embodiments of the invention, "comprising SEQ ID NO:1 includes, for example, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1, and a polypeptide consisting of a signal peptide sequence added to the amino acid sequence shown in SEQ ID NO:1 and/or a polypeptide comprising an amino acid sequence obtained by adding an appropriate tag sequence to the N-terminus and/or the C-terminus of the amino acid sequence shown in FIG. 1.
The term "modified polypeptide" in the present invention refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:1, and has an activity of preventing or treating exocrine disorder, autoimmune disease and/or Sjogren syndrome.
In a preferred embodiment of the invention, the modification of an amino acid in the modified polypeptide or a homologous polypeptide thereof is a "conservative modification". For example, "conservative substitutions" refer to the substitution of one or more amino acid residues with other amino acids that are chemically similar, without substantially altering the activity of the protein. Examples thereof include a case where a certain hydrophobic residue is substituted with another hydrophobic residue and a case where a certain polar residue is substituted with another polar residue having the same charge.
Functionally similar amino acids for which such conservative substitutions may be made are well known in the art for each amino acid. Specifically, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. In addition, as negative charge (acidity) amino acids, aspartic acid, glutamic acid, and the like can be given.
"homologous polypeptide" in the present invention means a polypeptide comprising a sequence identical to SEQ ID NO:1 has an amino acid sequence having at least 85%, at least preferably 90%, at least preferably 92%, at least preferably 93%, at least preferably 94%, at least preferably 95%, at least preferably 96%, at least preferably 97%, at least preferably 98%, at least preferably 99%, more preferably 100% homology (sequence identity). Preferably, the polypeptide has activity in the prevention or treatment of exocrine disorders, autoimmune diseases and/or sjogren's syndrome.
For the purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.mol. Biol. 48:443-453) as performed in the EMBOSS software package (EMBOSS: the European Molecular Biology Open Software Suite, rice et al 2000,Trends Genet.16:276-277), preferably Needleman-Wunsch program version 3.0.0 or higher. The optional parameters used are gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (emoss version of BLOSUM 62) substitution matrices. The output of Needle labeled "highest identity" (obtained using the-nobrief option) was used as the percent identity and calculated as follows:
(identical residue. Times.100)/(alignment Length-total number of gaps in alignment)
The polypeptides of the invention may be naturally occurring, synthetic, semisynthetic, or recombinantly produced. The polypeptides of the invention may be produced by genetic engineering, by known peptide synthesis or by digestion of the polypeptides of the invention with an appropriate peptidase.
In preferred embodiments of the invention, the polypeptides of the invention may be encoded by recombinant DNA sequences in host cells to produce the polypeptide product by conventional bioengineering methods, or may be synthesized by solid phase or liquid phase synthesis, for example by solid phase chemistry techniques using a Applied Biosystem synthesizer or a PiconeerTM peptide synthesizer as described by Steward and Young (Steward, J.M. and Young, J.D., solid Phase Peptide Synthesis,2nd Ed, pierce Chemical Company, rockford, I11, (1984)). In a preferred embodiment of the present invention, the polypeptide of the present invention may be prepared by Fmoc chemical synthesis using a solid phase synthetic resin as a starting material.
The present invention further provides polynucleotides encoding the above polypeptides, preferably polynucleotides represented by the nucleotide sequences of SEQ ID NO. 2 or SEQ ID NO. 3. The polynucleotides of the invention may be used to produce the polypeptides of the invention as described above in vivo or in vitro, or may be used in gene therapy for diseases attributable to genetic abnormalities in genes encoding the polypeptides of the invention. Any form of the polynucleotides of the invention may be used, provided that it encodes a polypeptide of the invention, including mRNA, RNA, cDNA, genomic DNA, and chemically synthesized polynucleotides. The polynucleotide of the present invention includes a DNA comprising a specified nucleotide sequence and its sequence as long as the resulting DNA encodes the polypeptide of the present invention.
Polynucleotides of the invention may be prepared by methods known to those of skill in the art. For example, polynucleotides of the invention may be prepared as follows: a cDNA library is prepared from cells expressing the polypeptide of the present invention, and hybridization is performed using a partial sequence of its DNA as a probe. cDNA libraries can be prepared, for example, by methods described in molecular cloning, written by Sambrook et al, see Cold spring harbor laboratory Press (1989); alternatively, a commercially available cDNA library may be used.
In addition, by sequencing the nucleotides of the obtained cDNA, the translational region encoded by the cDNA can be conventionally determined, and the amino acid sequence of the polypeptide of the present invention can be easily obtained. Further, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA or a part thereof as a probe.
The invention also provides a genetic engineering vector containing the polynucleotide for encoding the polypeptide. The genetic engineering vector can be a common vector or an expression vector. In particular, commercially available expression vectors suitable for prokaryotic cells are typically provided with selectable markers and a cell replication origin, with bacterial promoters such as lacI, T7, λPL and trp, and other genetic elements of the known cloning vector pBR322 (ATCC 37017). Such commercially available vectors include pGEM (Promega) and pKK223-3 (Pharmacia). The appropriate vector derived from pBR322 may be selected according to the appropriate promoter selected and the structural gene sequence to be expressed. GST prokaryotic expression systems can also be used in the present invention. Vectors suitable for eukaryotic cells carry eukaryotic promoters such as CMV, SV40, etc., and include pMT-hIL-3 (Ma Dalong, dichun, pang Jian, et al (1991) high technology communication 11:26-29), pQE-9 (Qiagen), pD10, pNH18A (Stratagene), pKK233-3, pDR540, pRIT5 (Pharmacia), and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL (Pharmacia).
The invention also provides a host cell comprising a vector of the invention, which host cell can be used to express a polypeptide of the invention, including but not limited to: prokaryotic hosts such as E.coli, bacillus, streptomyces, and the like; eukaryotic hosts such as: saccharomyces, aspergillus, insect cells such as Drosophila S2 and Spodoptera Sf 9, animal cells such as CHO, COS (monkey kidney fibroblast Cell line, gluzman (Cell 23:175, 1981) human Cell lines such as PC-3 Cell line, DU145 Cell line and other Cell lines capable of expressing compatible vectors.
Mesenchymal Stem Cells (MSCs) are a class of adult stem cells that can be isolated from a variety of adult tissues and can differentiate into adipocytes, osteoblasts, chondrocytes, and the like. MSC has an important role in preventing or treating autoimmune diseases such as Sjogren syndrome. Our research shows that the polypeptide of the present invention may be used in regulating the immune regulation function of MSCs and has the activity of preventing or treating exocrine dysfunction, autoimmune diseases and/or sicca syndrome.
Differentiation inhibitors, also known as DNA binding inhibitors (Inhibitors of differentiation/DNA binding, ID), belong to one of the helix-loop-helix (HLH) transcription factor family members. The ID protein is a small molecular protein with the molecular weight of 10-20KD, and a high-conservation helical region forms a homo-dimer or a hetero-dimer through interaction of hydrophobic groups on a helix, so that the transcription regulation effect is exerted. Most HLH proteins have a strongly basic region adjacent to the HLH motif, which is necessary for DNA binding, and so such HLH is also known as basic HLH (bHLH) proteins. The ID molecules are a special class of HLH proteins, which, due to their lack of basic amino acid sequences necessary for DNA binding, bind to bHLH to form heterodimers, which inhibit the binding of bHLH to DNA and other tissue-specific bHLH transcription factors (e.g., E proteins), down regulate the activity of the bHLH transcription factors, affect the expression of cell-specific genes, and thus inhibit cell differentiation.
Both the ID and E proteins belong to the bHLH transcription factor family because they have basic helix-loop-helix domains. The E protein family comprises E2A, HEB and E2-2 and is characterized by having a DNA binding domain at the C end besides a bHLH protein interaction domain at the N end, specifically recognizing E box (CANNTG), and activating the expression of a downstream target gene. ID proteins differ from E proteins in that they have only the bHLH protein domain, but lack the DNA binding domain. The function is to combine with E protein, competitively inhibit the transcriptional activity of E protein and regulate the expression of E protein downstream target gene.
ID3 is one of the members of the ID protein family (Id 1-Id 4) and plays an important role in cell differentiation and maturation by binding to E2A. Analysis by molecular dynamics and docking suggests that Lys570, ala595, val598 and Ile599 in E2A/E12, and Glu53, gln63 and Gln66 in ID3 may be key residues for Id3-E2A/E12 interactions, which are hydrogen bonded to each other to maintain the stability of Id3-E2A/E12 complex.
The polypeptide has the capacity of competitively combining with the ID3 and preventing the combination of the ID3 and the E2A, can prevent the E2A from forming dimer to combine with DNA, finally inhibit the BMP4 expression and reduce the PGE2 secretion, and can be used for regulating and controlling the immune regulation function of MSCs.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide, polynucleotide, vector or cell of the invention; and, if necessary, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are, for example, non-toxic solid, semi-solid or liquid fillers, excipients, diluents, encapsulating materials or other formulation excipients, and the like.
The invention also provides an application of the polypeptide in preparing a medicament for preventing or treating exocrine disorder and/or autoimmune disease.
In a preferred embodiment of the invention, conditions caused by disorders of external secretion such as "dry eye syndrome" (dry eye disease), "xerostomia syndrome" (xerostomia), "nasal xerosis syndrome" (nasal xerosis), "skin xerosis syndrome" (xeroderma), and "vaginal xerosis syndrome" (symptoms are vaginal dryness); and chronic pancreatitis, chronic gastritis, chronic bronchitis, etc. due to inhibition of exocrine secretion.
In a preferred embodiment of the invention, wherein the external secretion disorder is a low dry syndrome, reduced salivary secretion, reduced tear secretion or dry eye syndrome.
The invention also provides application of the polypeptide in preparing a medicament for preventing or treating sicca syndrome, and dry mouth, dry eyes and other exocrine glands and other organs outside the gland caused by sicca syndrome.
In a preferred embodiment of the present invention, wherein the sjogren's syndrome comprises primary sjogren's syndrome, secondary sjogren's syndrome.
In a preferred embodiment of the invention, wherein said involvement of other organs outside the gland is characterized by the occurrence of multisystemic lesions including chronic fatigue due to xerosis syndromes.
Subjects treated with the methods of the invention are those suffering from exocrine disorders including xerosis such as dry eye syndrome (xerophthalmia), xerostomia syndrome (xerostomia), nasal xerosis syndrome (xerophthalmia), cutaneous xeroderma syndrome (xeroderma), vaginal xerosis syndrome (symptoms of vaginal dryness); chronic pancreatitis, chronic gastritis and chronic bronchitis due to inhibition of exocrine secretion. Especially those suffering from conditions that may induce dry eye syndrome, including reduced tear secretion, lacrimation, dry eye, sjogren's syndrome, keratoconjunctivitis sicca, stevens-Johnson syndrome, pemphigoid, blepharitis, diabetes, post cataract surgery, and allergic conjunctivitis. The conditions are also those of dry eye syndrome which may be induced by aging, long-term VDT operation, desiccation of the air-conditioned room. Among them, the external secretion disorder refers to a state in which abnormal external secretion (i.e., reduction or cessation of secretion) that can be caused by any cause is observed, particularly a state having abnormal tear secretion including basal tear secretion and abnormal saliva secretion.
Systemic factors that can cause the low-grade Sjogren syndrome include febrile disease, dehydration, endocrinopathy (myxoedema, barceicosis, diabetes insipidus, etc.), metabolic disorders (diabetes, uremia, cirrhosis, etc.), vitamin A or vitamin B deficiency, autoimmune diseases (Sjogren's syndrome, progressive scleroderma, etc.), anemia, hemorrhage, aging, various drugs (sedatives, parasympathetic blockers, antihistamines, etc.), additionally, local factors can include sialitis, salivary gland atrophy, radiation therapy sequelae, deformity (ectodermal dysplasia, etc.), and the like.
In a preferred embodiment of the present invention, for example, the medicament may be orally administered, such as dragees, capsules, microcapsules, and the like, as desired; or non-orally, e.g., in the form of sterile solvents or suspensions of injectable water or any other pharmaceutically acceptable liquid. For example, the compounds may be admixed with pharmaceutically acceptable carriers or vehicles in unit dosage forms for acceptable pharmaceutical execution, specifically sterile water, physiological saline, vegetable oils, emulsifying agents, suspending agents, surfactants, stabilizers, fragrances, excipients, vehicles, preservatives, binders and the like. The amount of active ingredient in these formulations is in the appropriate dosage within the desired range as indicated.
Examples of additives which can be formulated into tablets or capsules are binders such as gelatin, corn starch and acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint. When the unit dosage form is a microcapsule, the above ingredients may also include a liquid carrier, such as an oil. The sterile composition for injection may be formulated following conventional pharmaceutical vehicles such as distilled water for injection. Physiological saline, dextrose, and other isotonic liquids, including adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as the aqueous solution for injection. These may be used in combination with suitable solubilisers such as alcohols, in particular such as ethanol, polyols such as propylene glycol and polyethylene glycol, nonionic surfactants such as polysorbate 80. Sesame oil or soybean oil may be used as an oily liquid, and may be used in combination with methyl benzoate, benzyl alcohol as a solubilizer, and may be used in combination with a buffer such as a phosphate buffer, sodium acetate buffer; analgesics, such as procaine hydrochloride; stabilizers such as benzyl alcohol; formulated with an antioxidant. The prepared injection may be filled into a suitable ampoule.
The pharmaceutical compounds of the present invention may be administered to a patient using methods well known to those skilled in the art, such as intra-arterial, intravenous, transdermal injection, and intranasal, transbronchial, intramuscular, or oral administration. The dosage and method of administration will vary depending on the weight and age of the patient and the method of administration; and may be routinely selected by those skilled in the art. If the compound is encoded by DNA, the DNA may be inserted into a vector for gene therapy and the vector administered for treatment. The dosage and method of administration will vary with the weight, age, and symptoms of the patient, but can be appropriately selected by one skilled in the art.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, which are intended as illustrations of several aspects of the invention.
Example 1: preparation of the polypeptide of the invention
S1, adding a eukaryotic translation initiation sequence kozak sequence gccac at the 5' end of a gene of a polypeptide coding sequence SEQ ID NO. 2, and then continuing adding a restriction enzyme Not1 recognition sequence (gcggccgc) at the 5' end and a restriction enzyme BamH1 recognition sequence (ggattc) at the 3' end of the gene, namely synthesizing a polypeptide coding whole gene, wherein the sequences are shown in SEQ ID NO. 3;
s2, recombining the expressed gene into a plasmid vector of pQCIN through a molecular cloning technology, carrying out enzyme digestion on restriction enzymes Not1 and BamH1 (purchased from New England Biolabs company) at 37 ℃ for three hours, simultaneously carrying out enzyme digestion on a pQCIN blank plasmid (purchased from Clone tech company) at 37 ℃ overnight with the same enzyme digestion, recycling the insert and the vector fragment after enzyme digestion by using a gel recycling kit, connecting the insert and the vector with T4 ligase at room temperature for 1 hour, converting the connection product into an escherichia coli strain DH5 alpha, culturing at 37 ℃ for overnight, carrying out plasmid extraction by using a standard plasmid miniextraction kit after bacteria shake, adding a small amount of Not1 and BamH1 into a part of the extracted plasmid, and carrying out enzyme digestion at 37 ℃ for half an hour, and analyzing whether the size of the insert is correct according to the result of agarose gel;
s3, expressing the pQCIN vector in escherichia coli;
s4, collecting cells, crushing the cells, and collecting a supernatant;
s5, removing nonspecifically bound hybrid protein by utilizing a Ni-NTA affinity column;
and S6, concentrating the protein after enzyme digestion to prepare the polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
Example 2: activity assay of the polypeptide of the present invention
Step S1, experimental grouping
24 female NOD/Ltj mice (purchased from Beijing Fukang biotechnology Co., ltd.) of 6 weeks old are selected and divided into an experimental group and a control group, each group comprising 12 mice;
step S2, preparation of injection preparation
The control group was subcutaneously injected with 100 ul/body weight of physiological saline, and the treatment group was subcutaneously injected with polypeptide at 50ng/g body weight of each. Polypeptide concentration was quantified using a 2D Quant protein quantification kit (purchased from GE Healthcare company, usa);
step S3, saliva flow rate detection of mice
1) Anesthetic preparation: 100mg/ml ketamine and 20mg/ml calazine are put into physiological saline for injection, and the injection is sealed for standby after high-temperature high-pressure disinfection;
2) Saliva flow rate detection
Saliva flow rate = saliva weight per 10 minutes
3 minutes after the mouse is injected with anesthetic intraperitoneally, 50mg/ml pilocarpine 0.1ml/kg is injected intraperitoneally; after the mice are completely anesthetized, placing a capillary glass tube into the mouth of the mice, and placing a pre-weighed 0.75ml centrifuge tube at the other end of the capillary; starting timing from the time when the first drop of saliva flows into the tube, stopping collecting saliva after 10 minutes; weighing saliva;
step S4, histological observation of the submaxillary gland of the mouse
The cervical vertebrae of the mice are killed, free submandibular glands are dissected and taken out, the mice are placed in 4% paraformaldehyde for fixation for 24 hours at the temperature of 4 ℃, dehydrated, embedded, sliced and HE stained, observed under a light microscope, photographed, 10 fields of view with 200 times magnification are randomly taken, and the inflammatory cell infiltration area is calculated by using Image Pro Plus 6.0 software;
as shown in FIG. 1, physiological saline and polypeptide were subcutaneously injected into 6 weeks NOD/Ltj mice, and after 2 weeks it was found that the infiltration of lymphocytes in the submandibular glands of NOD/Ltj mice in the polypeptide-treated group was significantly reduced, as shown in FIG. 2, physiological saline and polypeptide were subcutaneously injected into 6 weeks NOD/Ltj mice, and after 2 weeks it was found that the saliva flow rate in the submandibular glands of NOD/Ltj mice in the polypeptide-treated group was significantly increased.
Lymphocyte infiltration and secretory dysfunction in salivary glands are common exocrine disorders or pathological and clinical manifestations of autoimmune diseases. Thus, the polypeptide of the present invention can inhibit lymphocyte function and restore gland function, so as to prevent or treat exocrine disorder and/or autoimmune diseases, sicca syndrome, and the damage of multiple systems caused by the dry mouth, dry eyes, other exocrine glands and other organs outside the gland.
Sequence listing
<110> capital medical university Hui Beidou (Beijing) technology Co., ltd
<120> a polypeptide for preventing or treating Sjogren's syndrome and use thereof
<141> 2019-07-25
<150> 2018108585100
<151> 2018-07-31
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> Escherichia coli
<400> 1
Leu Arg Glu Leu Val Pro Gly Val Pro Arg Gly Thr Gln Leu Ser Gln
1 5 10 15
Val Glu Ile Leu
20
<210> 2
<211> 60
<212> DNA
<213> Synthesis
<400> 2
ctgcgggaac tggtgccggg agtcccgcga ggcactcagc ttagccaggt ggaaatcctg 60
<210> 3
<211> 80
<212> DNA
<213> Synthesis
<400> 3
gcggccgcgc caccctgcgg gaactggtgc cgggagtccc gcgaggcact cagcttagcc 60
aggtggaaat cctgggattc 80
Claims (4)
1. Use of a polypeptide having an amino acid sequence as set forth in SEQ ID No.1 for the manufacture of a medicament for the treatment of an exocrine disorder, wherein the exocrine disorder is a low-grade dry syndrome, a hyposalivation, a hypolacrimation, or a dry eye syndrome.
2. The application of the polypeptide with the amino acid sequence shown as SEQ ID NO.1 in preparing medicines for treating sicca syndrome, xerostomia, xerophthalmia and other exocrine glands and other organs outside the gland affected and causing multisystem damage caused by sicca syndrome.
3. The use of claim 2, wherein the sjogren's syndrome is primary sjogren's syndrome or secondary sjogren's syndrome.
4. The use according to claim 2, wherein the involvement of other organs outside the gland is associated with multiple systemic lesions including chronic fatigue due to xerosis syndrome.
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| US7629131B2 (en) * | 2006-01-27 | 2009-12-08 | Biocheck, Inc. | Rabbit monoclonal antibodies against mouse/human Id3 proteins |
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| "BC145873.1";Strausberg,R.L., et al.;《NCBI Genbank》;20070712;序列 * |
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| "Q02535.2";Ellmeier, W., et al.;《NCBI Genbank》;20160706;第1-5页 * |
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| CN110511268A (en) | 2019-11-29 |
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