WO2013004069A1 - 沙门氏菌鞭毛蛋白衍生物在制备防治炎症性肠病药物中的应用 - Google Patents

沙门氏菌鞭毛蛋白衍生物在制备防治炎症性肠病药物中的应用 Download PDF

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WO2013004069A1
WO2013004069A1 PCT/CN2012/000097 CN2012000097W WO2013004069A1 WO 2013004069 A1 WO2013004069 A1 WO 2013004069A1 CN 2012000097 W CN2012000097 W CN 2012000097W WO 2013004069 A1 WO2013004069 A1 WO 2013004069A1
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czlc331
protein
salmonella flagellin
flagellin derivative
tat
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PCT/CN2012/000097
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English (en)
French (fr)
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张成岗
李军怀
徐阳
吴永红
李伟光
张艳春
高艳
李志慧
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苏州科景生物医药科技有限公司
中国人民解放军军事医学科学院放射与辐射医学研究所
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Priority to EP12807820.1A priority Critical patent/EP2730287B1/en
Priority to US14/131,050 priority patent/US9259453B2/en
Priority to NZ619871A priority patent/NZ619871B2/en
Priority to AU2012278744A priority patent/AU2012278744B2/en
Priority to JP2014517394A priority patent/JP5876930B2/ja
Priority to CA2840924A priority patent/CA2840924A1/en
Priority to KR1020147002932A priority patent/KR101814558B1/ko
Publication of WO2013004069A1 publication Critical patent/WO2013004069A1/zh
Priority to US14/940,648 priority patent/US9630999B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2806—Coating materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2893—Tablet coating processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • 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

Definitions

  • Salmonella flagellin derivative in preparing medicine for preventing and treating inflammatory bowel disease
  • the invention belongs to a new use of recombinant protein in the research and development of medicine in the field of medicine, and particularly relates to a new use of a Salmonella flagellin derivative (code CZLC331) in the development of a medicament for preventing and treating inflammatory bowel disease.
  • Ulcerative Colitis also known as idiopathic ulcerative colitis or chronic non-specific ulcerative colitis, is a chronic non-specific inflammatory disease characterized by mucosal and submucosal infiltration, which is characteristic of the large intestine. Intestinal diseases, the main clinical symptoms are: abdominal pain, diarrhea, mucus and bloody stools, accompanied by multiple sclerosis, optic neuritis and osteoporosis. Because the disease has the characteristics of high treatment difficulty, long course of treatment and easy recurrence, it has been identified as a modern refractory disease by the World Health Organization.
  • Salmonella flagellin derivatives studies have shown that the Salmonella flagellin derivative CBLB502 (standard nomenclature) has a protective effect on the hematopoietic system, prolongs the survival time of mice after high-dose radiation injury, and can improve low-dose irradiation of mice. Survival rate (Lyudmila G. Burdelya, et al, . An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity in Mouse and Primate Models. Science 2008; 320 (5873): 226-230).
  • the Salmonella flagellin derivative CZLC331 of the present invention has good preventive and therapeutic effects on inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, and therefore, the protein can be used as an active ingredient.
  • the Salmonella flagellin derivative CZLC331 of the present invention is a fusion protein comprising a Tat protein transduction peptide, a Salmonella flagellin from the N-terminal 1-176 amino acid residue and the 402-505 amino residue; the Salmonella flagellin The amino acid residues 1-176 at the N-terminus and the amino acid residues 402-505 can be linked by a flexible linker.
  • the Salmonella flagellin derivative CZLC331 can be prepared by a conventional prokaryotic expression method with a purity greater than 95%.
  • the method for specifically preparing the Salmonella flagellin derivative CZLC331 may include the following steps:
  • the Salmonella flagellin derivative CZLC296 is a flexible arm that positions the Salmonella flagellin from the N-terminal 1-176 An amino acid residue obtained by linking an amino acid residue at positions 402-505 of the N-terminus;
  • the synthetic Salmonella flagellin derivative CZLC296 gene was cloned into a prokaryotic expression vector carrying the Tat transduction peptide coding sequence to obtain prokaryotic expression of the Tat protein transduction peptide coding sequence and the Salmonella flagellin derivative CZLC296 coding gene.
  • a carrier the Salmonella flagellin derivative having a Tat protein transduction peptide linked to the N-terminus is designated as CZLC331, and the nucleotide sequence of the gene encoding the protein is as shown in SEQ ID NO: 2 in the Sequence Listing;
  • Inducible expression contains a Tat protein transduction peptide coding sequence and a Salmonella flagellin derivative
  • the prokaryotic expression vector carrying the Tat transduction peptide coding sequence in the step 2) is any prokaryotic expression vector which can be expressed in Escherichia coli, such as pET-22b series, The pET28 series or the pET-15 series, etc., preferably pET28b-Tat; the recombinant expression vector carrying the gene encoding the Salmonella flagellin derivative CZLC331 constructed with pET28b-Tat as the starting vector is pET28b-Tat-CZLC331 0
  • the host bacteria in 'Step 3' may be £ coli BL21 (DE3), E. coli ER2566 (DE3), E. coli BL2KDE3, plysS), E. JM109, £ £ ⁇ ' leg 01 or £ co ' ToplO, etc. It is preferably £ coli BL21 (DE3).
  • the drug containing the CZLC331 protein prepared by the present invention can be prepared into various forms such as an injection solution, an oral solution, an enema solution, a capsule, an enteric-coated tablet, a powder or a granule, and is preferably an injection solution, an enema solution or an enteric-coated tablet.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • the mass fraction of the active ingredient Salmonella flagellin derivative CZLC331 protein in the solid dosage form of the capsule, enteric tablet, powder and granule may be 1-35 %.
  • the concentration of the active ingredient Salmonella flagellin derivative CZLC331 protein in the liquid dosage form such as the injection solution, the oral solution and the enema solution may be 2-64 g/L.
  • one or more pharmaceutically acceptable excipients may be added to the above-mentioned drugs, including conventional absorption enhancers, surfactants, lubricants, stabilizers, diluents, binders, moisturizers in the pharmaceutical field.
  • a dissolving agent, a disintegrating agent, a diluent, an adsorption carrier, an excipient, etc., and if necessary, a coloring matter, a sweetener, a flavoring agent, and the like may be added.
  • the drug may be a protein injection consisting of CZLC331 protein at a weight ratio of 5% (5 g/100 mL), 0.85% (0.85 g/100 mL) of sodium chloride, and water for injection.
  • the drug is an enteric coated tablet, 100 parts by weight of CZLC331 protein, 60 parts by weight of lactose, 20 parts by weight of microcrystalline cellulose, 20 parts by weight of sodium carboxymethyl starch, and povidone K 3 . 10 parts by weight of the mixed granules, and the dry granules were added to the talc powder in a ratio of 2500:50 by weight to be uniformly compressed, and then sprayed with an enteric film coating layer on the surface of the tablet.
  • the above-mentioned drugs are generally used in an amount of 0.2-6.4 mg of CZLC331 protein/kg body weight, administered 1-2 times a day for 5 to 10 days.
  • the injection can be administered by intramuscular, intraperitoneal or intravenous injection.
  • the invention provides a new use of the Salmonella flagellin derivative CZLC331 protein in the development of inflammatory bowel disease drugs.
  • TNBS 2,4,6-trinitrobenzene sulfonic acid
  • Salmonella flagellin derivative CZLC331 protein has obvious prevention for inflammatory bowel disease.
  • therapeutic effects are as follows: 1) Modeling: The mice have significantly reduced food intake/drinking, reduced activity and blood in the stool, macroscopic congestion, edema, hemorrhage and ulceration in the colon, and cell structure disorder under the microscope.
  • the Salmonella flagellin derivative CZLC331 protein can be prepared as a pretreatment inflammatory bowel disease drug, and the drug has the following advantages: 1) The curative effect is obvious (the effective rate is 100%, the cure rate is 70%), and the clinical line The drug sulfasalazine has an efficiency of only about 90%, and the effective rate is only 50%. 2) It has a fast onset (it can take effect 24 hours after administration), and sulfasalazine is taken for about 2 weeks.
  • sulfasalazine Improvement 3) short course of treatment (usually 5-10 days), sulfasalazine treatment is generally about 6 weeks; 4) high safety (non-toxic), and patients have alanine after taking sulfasalazine Increased transaminase (ALT) and aspartate aminotransferase (AST) suggest hepatotoxicity; 5) small side effects (no obvious side effects), while sulfasalazine has nausea, rash, and neutropenia after administration Other adverse reactions; 6) Convenient medication (once per day intraperitoneal injection) and sulfasalazine 3-4g / d, divided into 3 - 4 times orally.
  • ALT transaminase
  • AST aspartate aminotransferase
  • the present invention can solve the existing treatment of inflammatory bowel disease
  • the poor efficacy of the drug, slow onset of action, long course of treatment, and large side effects can significantly reduce the pain of the patient's treatment process, promote the patient's physical recovery and improve the quality of life.
  • the invention plays an important role in the prevention and treatment of inflammatory bowel disease and other parts of inflammation, and has broad application prospects.
  • Figure 1 is a colonic appearance of 0. 2mg / kg CZLC331 protein after treatment with trinitrobenzene sulfonic acid (TNBS) model 0. 5h treatment of ulcerative colitis mice
  • Figure 2 is 0. 2mg / kg CZLC331 protein in the treatment of trinitrobenzene sulfonic acid (TNBS) model 0. 5h treatment of colonic mucosa in mice with ulcerative colitis
  • TNBS trinitrobenzene sulfonic acid
  • Figure 3 is 0. 2mg / kg CZLC331 protein after the application of trinitrobenzene sulfonic acid (TNBS) model 0. 5h treatment of ulcerative colitis mice colonic mucosal tissue section HE staining results
  • FIG 4 shows the colon appearance and colonic mucosa of mice treated with ulcerative colitis at different concentrations of CZLC331 protein after administration of trinitrobenzenesulfonic acid (TNBS).
  • TNBS trinitrobenzenesulfonic acid
  • Figure 5 shows the colon appearance of CZLC331 protein in mice with ulcerative colitis at different concentrations of different administration time.
  • Figure 6 shows the colonic mucosa of mice with ulcerative colitis treated with CZLC331 protein at different concentrations.
  • Figure 7 shows different doses of different doses of CZLC331.
  • HE staining results after treatment of colonic mucosal tissue sections of mice with ulcerative colitis
  • FIG 8 shows the expression of 2.5%/kg CZLC331 protein in mice with ulcerative colitis after intraperitoneal injection of trinitrobenzenesulfonic acid (TNBS) for 4 hours.
  • TNBS trinitrobenzenesulfonic acid
  • Fig. 9 shows that 2. 2mg/kg CZLC331 protein was intraperitoneally injected into mice with ulcerative colitis 4 hours after modeling with trinitrobenzenesulfonic acid (TNBS), and the expression of TLR6-9 family gene in colon was detected by RT-PCR. A), and its columnar analysis (B)
  • Figure 10 is a colonic appearance of 0. 2mg/kg CZLC331 protein in mice with ulcerative colitis after prolonged administration of trinitrobenzenesulfonic acid (TNBS).
  • TNBS trinitrobenzenesulfonic acid
  • Figure 11 is a colonic mucosa of a mouse with ulcerative colitis after 0. 2mg/kg CZLC331 protein in the form of trinitrobenzene sulfonic acid (TNBS).
  • TNBS trinitrobenzene sulfonic acid
  • CZLC331 may down-regulate the expression of TLRs against inflammatory bowel diseases such as ulcerative colitis and gram. Ron disease and the like have good preventive and therapeutic effects.
  • Study on inflammatory bowel disease (Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Ardit i M. Decreased expression of Tol ll ike receptor - 4 and MD - 2 correlates with intest inal epithel ial cel l protect ion / dysregulated proinflammatory gene expression in response to bacterial l ipopolysaccharide.
  • intestinal epithelial cells can maintain intestinal immune tolerance by down-regulating TLR4 expression and signaling.
  • Salmonella flagellin has an effect of preventing and treating ulcerative colitis, and further proves that this effect is achieved by down-regulating the expression of the Tol l-like receptor family of colon tissues.
  • TLR Tol l ike receptor
  • IL-12 is a key regulator of the induction of T and sputum lymphocytes producing Th1 cells, which can be used as a bioadjuvant to amplify the response of specific T cells to pathogens.
  • TLR family is an early signal that IL-12 and Th1 cells are involved in the production of immune responses.
  • the balance between TLRs and the normal flora is broken, causing pathological intestinal inflammation.
  • Salmonella flagellin has the effect of down-regulating the expression of TLR family in colon tissue, which may be an important mechanism for its therapeutic effect on ulcerative colitis.
  • the present invention is directed to the nucleotide sequence of amino acid No. 1-176 and amino acid 402-505 of Salmonella flagellin, and the nucleotide coding sequence of the nucleotide sequence is synthesized by prokaryotic expression, and a 331 amino acid full-length Salmonella flagellin derivative is prepared, which is named CZLC331. And through experiments to prove that it has good preventive and therapeutic effects on inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. The practice of the present invention will provide an important reference for the prevention and treatment of inflammatory bowel disease.
  • the 891 bp Salmonella flagellin derivative CZLC296 protein was amplified by conventional PCR using a synthetic nucleotide sequence as a template.
  • the 50 ⁇ l PCR reaction system was: Plasmid template (carrying Salmonella flagellin derivative CZLC296 protein
  • the cloning vector P GH-CZLC296 encoding the gene was constructed by inserting the synthetic CZLC296 gene into the Smal restriction site of the cloning vector pGH) 0.
  • upstream primer sequence 5'-CGCG6 ⁇ mTGGCTCAAGnATCA-3', and the downstream primer sequence: 5'HXGCmCTCTCMCAAAGACMGTT-3'
  • upstream primer sequence 5'-CGCG6 ⁇ mTGGCTCAAGnATCA-3'
  • downstream primer sequence 5'HXGCmCTCTCMCAAAGACMGTT-3'
  • PCR reaction conditions are: 95V for 4 minutes; then 95 ° C for 45 seconds, 56 ° C for 30 seconds, 72 ⁇ 45 seconds, a total of 30 cycles; finally 72 ° C for 7 minutes.
  • the PCR product was subjected to 1% agarose gel electrophoresis, and the result showed that a 891 bp DNA fragment was obtained by amplification, which was consistent with the expected result, and the target fragment was recovered and purified.
  • the Salmonella flagellin derivative CZLC296 gene and the vector pET28b-Tat vector were constructed by first synthesizing a double-stranded TAT core sequence containing Ncol (upstream) and Ndel (downstream) by chemical synthesis. Then the restriction endonuclease TVcoI and ⁇ 3 ⁇ 43 ⁇ 4 double-digested the TAT core fragment and the vector pET28b, and recovered the target fragment, which was ligated and identified by T4 DNA ligase, and finally sent to Yingjun for direct sequencing to confirm the prokaryotic expression of the core fragment containing TAT. The vector pET28b-TAT was constructed correctly.
  • the clones grown after the culture were confirmed by restriction endonuclease digestion and sequencing, respectively.
  • the sequencing results showed that the recombinant expression vector carrying the Tat protein transduction peptide coding sequence and the Salmonella flagellin derivative CZLC296 gene was obtained, which was named pET28b - Tat-CZLC331.
  • the Salmonella flagellin derivative CZLC296 having a Tat protein transduction peptide linked to the N-terminus is designated as CZLC331, and the nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO: 2 in the Sequence Listing.
  • the correct prokaryotic expression vector pET28b-Tat-CZLC331 was constructed in step 1 and transformed into E. coli BL21 (DE3) and plated onto LB plates containing Kana + (final concentration 100 g/mL), and clones were grown. Take one The clones were inoculated into 5 mL of LB medium containing Kana+ (final concentration 100 pg/mL), and shaken at 37 ° C, 220 rpm for about 16 hours, until the cells were completely resuscitated.
  • the above-mentioned induced expression broth was centrifuged at 12000 rpra for 10 minutes at 4 ° C to collect the cells, and the sample was diluted with 20 mM sodium phosphate buffer, and then subjected to sonication to prepare a CZLC331 protein sample, and the supernatant was left to be separated and purified.
  • the supernatant prepared in step 4 was equilibrated with initial buffer (20 mM Na 3 P0 4 +0.5 M NaCl, pH 7.4) and directly loaded onto a well-balanced HisTrap HP 5 mL column (purchased from GE), and then used. 4-5 column volumes of the above buffer were used to rinse the column to the baseline of the elution peak, and finally the protein sample was eluted with an elution buffer (20 mM Na 3 P0 4 + 0.5 M NaCl + 0.5 M imidazole, pH 7.4). After desalting, the Salmonella flagellin derivative CZLC331 protein was obtained, and the purity was over 95%.
  • Example 2 Therapeutic effect of Salmonella flagellin derivative CZLC331 protein on inflammatory bowel disease Experimental animals: Clean grade 7-8 weeks Balb/c male mice (25 ⁇ 5 g), purchased from the Experimental Animal Center of the Academy of Military Medical Sciences Single cage isolation for 24 hours and free diet.
  • Trinitrobenzenesulfonic acid (TNBS, purchased from Sigma); CZLC331 (prepared in Example 1); RT PCR kit (Cat. No. FSK100, purchased from Toyobo Biotech Co., Ltd.)
  • mice were first anesthetized with an anesthesia machine, then gently inserted into the anus by a silicone tube with a diameter of 2.0 mm and a length of about 10 ca (approximately 4 cm deep) and the enema reagent (containing 150 g/L TNBS + 380 mL/L ethanol) Solution, 150 mg/kg) was slowly pushed into the colon to prepare an inflammatory bowel disease model.
  • the enema reagent containing 150 g/L TNBS + 380 mL/L ethanol
  • Experimental grouping Experimental animals were grouped into: model group, treatment group and control group, with 8 mice in each group.
  • control group 380 mL/L ethanol solution (without TNBS) was enema for normal mice for 2 days, and the normal group was fed for 2 days.
  • the treatment group was for mice with ulcerative colitis model, and the ulcerative colon was to be treated. 5-6.
  • mice in the model group had significantly reduced food intake/water consumption, reduced activity, and blood in the stool.
  • the mice were visually observed with hyperemia, edema, hemorrhage and ulceration (0. 2 mg/kg CZLC331 protein in trinitrobenzenesulfonate).
  • the colon appearance and colonic mucosa of mice treated with acid (TNBS) were 0.5 h for the treatment of ulcerative colitis, as shown in Figures 1 and 2, respectively: A: control group; B: model group; C: treatment group) Under the microscope, the cell structure disorder, goblet cell disappearance, lymphocyte and neutrophil infiltration were observed in the model group (0.
  • mice in the control group had normal feeding and drinking water, no blood in the stool and decreased activity.
  • the cells were neatly arranged under the microscope, and there were no goblet cell reduction and lymphocyte and neutrophil infiltration.
  • detection of intestinal TLR family expression level extraction of CZLC331 protein before the model group, treatment group, The total RNA of the colonic mucosa of the mouse group was detected, and the expression level of the TLR family gene was detected by PCR after reverse transcription.
  • the specific TLR family and the reference gene ⁇ -actin primer information are shown in Table 1 below.
  • the PCR reaction system is: Ex Taq 0. 25 ⁇ 1 , 10 X loading buffer 5 ⁇ 1 , dNTP 5 ⁇ 1 , template 2 ⁇ 1 , 1 ⁇ 1 of the upstream and downstream primers, 1 double distilled water to 50 ⁇ 1.
  • the PCR reaction conditions were: 95 °C 5rain ⁇ (95°C 45s ⁇ 62°C 45s ⁇ 72°C lrain) X 35 cycles ⁇ 72°C 5min ⁇ 4°C stop.
  • the PCR amplification product was subjected to 2% agarose gel electrophoresis detection and image analysis was performed. The results are shown in Fig. 8 and Fig. 9.
  • the expression of TLR family PCR bands after CZLC331 treatment showed significant difference from the model group, and the expression levels of TLR2, TLR3, TLR4, TLR8 and TLR9 models were significantly up-regulated.
  • CZLC331 can reverse its up-regulation after treatment; TLR6, TLR7 model group expression is significantly up-regulated, and CZLC331 treatment after reversal of its up-regulation, although there is no statistical difference, but its reverse trend is obvious.
  • the results indicate that the treatment process and mechanism may be related to the down-regulation of TLR family expression. It is speculated that the Salmonella flagellin derivative CZLC331 protein can exert therapeutic effects on inflammatory bowel disease through TLR family and other pathways, and it can be used as active ingredient to prepare inflammatory bowel disease. medicine.
  • mice were divided into three groups: model group, prevention group and control group, with 8 mice in each group.
  • the prevention group was given intraperitoneal injection of CZLC331 protein 3. 2 mg / kg body weight (0. 2-6. 4m g CZLC331 protein / kg body weight), and then according to the model to prepare the model, normal feeding after modeling 2
  • the mice were intraperitoneally injected with the same volume of normal saline for normal mice, and then modeled according to the model preparation method. The mice were fed normally for 2 days after modeling; the control group used 380 mL/L ethanol for normal mice. The solution was enema once and then fed normally.
  • Salmonella flagellin derivative CZLC331 protein has a preventive effect on inflammatory bowel disease, and it can be used as a prophylactic agent for the preparation of inflammatory bowel disease.
  • Example 4 Preparation of Salmonella flagellin derivative CZLC331 protein injection
  • Composition CZLC331 protein injection consisting of CZLC331 protein with a weight-to-volume ratio (mg/mL) of 5%, 0.85% sodium chloride and water for injection.
  • the protein sample was eluted with an elution buffer (20 mM Na 3 P0 4 +0.5 M NaCl +0.5 M imidazole, pH 7.4), and desalted to obtain the Salmonella flagellin derivative CZLC331 protein.
  • a CZLC331 protein sheet with a diameter of 1.2 cm was used as a base.
  • the surface of the sheet was spray coated with a layer of an insoluble film coating having a thickness of 1.0 mm.
  • the coating material is Yixinke E0BS68.
  • the film coating layer is airtight and moisture-tight. It has strong film strength and anti-gastric properties. It controls the solution by changing the pH value to ensure effective and safe release of the drug.
  • the Salmonella flagellin derivative CZLC331 protein of the present invention is prepared by using the active ingredient as a pre-treatment inflammatory bowel disease drug, and has the following characteristics:
  • Example 2 High efficiency: The experimental data of Example 2 and Example 3 showed that 8 mice in each of the treatment group and the prevention group improved the symptoms of less food, less movement and blood in the stool after the experiment, indicating that the effective rate was 100%. In the treatment group, 8 mice were found to have complete colonic mucosal tissue recovery, and the cure rate was 75%. The clinical first-line drug sulfasalazine was only about 90% effective, and the effective rate was only 50%.
  • Example 2 Fast onset of action: The experimental data of Example 2 and Example 3 showed that the mice could take effect 24 hours after administration; while the sulfasalazine was administered for about 2 weeks, the symptoms were improved. 3) The course of treatment is short (the course of treatment is generally 5-10 days): The course of sulfasalazine is generally about 6 weeks; the effect of this experiment on mice is 1-2 days, and the treatment of sulfasalazine in mice is In a week or so, the clinical onset was 6 weeks, and the course of the drug of the present invention was estimated to be 5-10 days.
  • the side effects are small (no obvious side effects): sulfasalazine has adverse reactions such as nausea, rash, and neutropenia after administration, and the CZLC331 of the present invention belongs to protein drugs and has been used for anti-radiation damage drug development. There are no obvious side effects on the body.
  • the invention provides the use of the Salmonella flagellin derivative CZLC331 in the research and development and preparation of the medicament for preventing and treating inflammatory bowel diseases, the medicament has high efficiency, quick onset, short treatment period, no toxicity, small side effects and convenient administration, and can solve the existing inflammatory
  • the problem of poor efficacy, slow onset, long course of treatment and side effects of enteropathic treatment drugs can significantly reduce the pain of the patient's treatment process, promote the physical recovery of patients and improve the quality of life.
  • the present invention will play an important role in the prevention and treatment of inflammatory bowel disease and other parts of inflammation, and has broad application prospects.

Abstract

本发明公开了沙门氏菌鞭毛蛋白衍生物CZLC331蛋白在制备预防和治疗炎症性肠病药物中的应用。沙门氏菌鞭毛蛋白衍生物CZLC331蛋白是包括TAT蛋白转导肽、沙门氏菌鞭毛蛋白自N端第1-176位氨基酸残基和第402-405位氨基酸残基的融合蛋白。

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沙门氏菌鞭毛蛋白衍生物在制备防治炎症性肠病药物中的应用 技术领域
本发明属于医药领域中重组蛋白在药物研发中的新用途， 特别是涉及一种沙门氏 菌鞭毛蛋白衍生物 （代号 CZLC331 ) 在研制用于防治炎症性肠病药物中的新用途。 背景技术
溃疡性结肠炎 (Ulcerative Colitis , UC)又称特发性溃疡性结肠炎或慢性非特异 性溃疡性结肠炎， 是一种以粘膜及粘膜下层浸润为主、 特发于大肠的慢性非特异性炎 症性肠病， 主要临床症状表现为： 腹痛、 腹泻、 粘液便和血便， 并伴有多发性硬化症、 视神经炎和骨质疏松等病症。 由于该病具有治疗难度大、 疗程长及易复发等特点， 已 被世界卫生组织确定为现代难治病。 虽然现己有多种炎症性肠病治疗药物， 如氨基水 杨酸类药物、 糖皮质激素类药物和免疫抑制剂等， 但是它们却具有疗效差 （尤其是中 重度 UC )、起效慢、疗程长、 副作用大等缺点， 因此， 市场上迫切需要一种疗效显著、 起效快、 疗程短、 安全性高、 副作用小、 用药方便的预防和治疗炎症性肠病 （特别是 慢性非特异性溃疡性结肠炎） 的药物。
关于沙门氏菌鞭毛蛋白衍生物，已有研究表明，沙门氏菌鞭毛蛋白衍生物 CBLB502 (标准命名） 对造血系统有保护作用， 可延长高剂量放射损伤后小鼠的生存时间， 并 可提高低剂量照射小鼠的生存率（Lyudmila G. Burdelya, et al， . An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity in Mouse and Primate Models. Science 2008 ; 320 (5873) : 226-230 ) 。 深入研究发现沙门氏菌鞭毛蛋白及包含其 N 和 C-端保守结构域的衍生物均具有辐射防护作用，能够有效提高小鼠骨髓中造血干细 胞数量有效地预防致死剂量照射引起的小鼠死亡， 其机制为通过 NF- i B 信号通路发 挥抗凋亡作用。 提示该沙门氏菌鞭毛蛋白衍生物 CBLB502可向抗辐射药物方向发展。 目前并没有任何关于沙门氏菌鞭毛蛋白衍生物向抗辐射药物以外其它药用方向的研 究报道。 发明内容
本发明的目的是提供一种沙门氏菌鞭毛蛋白衍生物 CZLC331在防治炎症性肠病药 物研发与制备中的用途。
本发明所述沙门氏菌鞭毛蛋白衍生物 CZLC331对炎症性肠道疾病如溃疡性结肠炎 和克罗恩病等具有良好的预防和治疗效果， 因此， 以该蛋白为活性成分， 可以制 备成预防和治疗炎症性肠病的药物。
本发明所述沙门氏菌鞭毛蛋白衍生物 CZLC331是包括 Tat蛋白转导肽、 沙门氏菌 鞭毛蛋白自 N端第 1-176位氨基酸残基和第 402-505位氨基 残基的融合蛋白； 所述 沙门氏菌鞭毛蛋白自 N端第 1-176位氨基酸残基和第 402-505位氨基酸残基可通过柔 性连接肽连接。
可采用常规原核表达方法制备沙门氏菌鞭毛蛋白衍生物 CZLC331 ,纯度大于 95 %。 具体制备沙门氏菌鞭毛蛋白衍生物 CZLC331的方法可包括以下步骤：
1 ) 合成沙门氏菌鞭毛蛋白衍生物 CZLC296编码基因， 其核苷酸序列如序列表中序 列 1所示； 所述沙门氏菌鞭毛蛋白衍生物 CZLC296是通过柔性臂将沙门氏菌鞭毛蛋白自 N端第 1-176位氨基酸残基和自 N端第 402-505位氨基酸残基连接得到的；
2 ) 将人工合成的沙门氏菌鞭毛蛋白衍生物 CZLC296编码基因克隆到携带 Tat转导 肽编码序列的原核表达载体中， 得到携带 Tat蛋白转导肽编码序列和沙门氏菌鞭毛蛋 白衍生物 CZLC296编码基因的原核表达载体； 所述 N端连接有 Tat蛋白转导肽的沙门氏 菌鞭毛蛋白衍生物命名为 CZLC331 , 编码该蛋白的基因的核苷酸序列如序列表中序列 2 所示；
3 ) 将重组表达载体 pET28b- Tat-CZLC331转化入宿主菌；
4) 诱导表达含有携带 Tat蛋白转导肽编码序列和沙门氏菌鞭毛蛋白衍生物
CZLC296基因的原核表达载体的宿主菌；
5 ) 分离及纯化表达的重组蛋白， 得到沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白。 在上述沙门氏菌鞭毛蛋白衍生物 CZLC331制备方法中， 所述步骤 2 ) 中携带 Tat转 导肽编码序列的原核表达载体为任意一种可在大肠杆菌中表达的原核表达载体， 如 pET-22b系列、 pET-28系列或 pET- 15系列等， 优选为 pET28b- Tat ; 以 pET28b- Tat为出 发载体构建的携带沙门氏菌鞭毛蛋白衍生物 CZLC331编码基因的重组表达载体为 pET28b-Tat-CZLC3310
'步骤 3 ) 中的宿主菌可为£ coli BL21 (DE3) , E. coli ER2566 (DE3 ) 、 E. coli BL2KDE3 , plysS) 、 E. JM109、 £ £^'腿01或£ co ' ToplO等， 优选为 £ coli BL21 (DE3) 。
本发明所制备的含有 CZLC331蛋白的药物可以制成注射液、 口服液、 灌肠液、 胶 囊、 肠溶片、 粉剂或粒剂等多种形式， 优选为注射液、 灌肠液或肠溶片。
上述各种剂型的药物均可以按照药学领域的常规方法制备。
所述胶囊、 肠溶片、 粉剂和粒剂等固态剂型的药物中活性成分沙门氏菌鞭毛蛋白 衍生物 CZLC331蛋白的质量百分含量可为 1-35 %。 所述注射液、 口服液和灌肠液等液态剂型的药物中活性成分沙门氏菌鞭毛蛋白衍 生物 CZLC331蛋白的浓度可为 2- 64g/L。
需要的时候， 在上述药物中还可以加入一种或多种药学上可接受的辅料， 包括药 学领域常规的吸收促进剂、 表面活性剂、 润滑剂、 稳定剂、 稀释剂、 粘合剂、 湿润剂、 崩解剂、 稀释剂、 吸附载体、 赋形剂等， 必要时还可加入色素、 甜味剂及香味剂等。
具体的，所述药物可以为蛋白注射液，由重量体积比为 5% ( 5g/100mL)的 CZLC331 蛋白、 0. 85% ( 0. 85g/100mL) 的氯化钠和注射用水组成。
或者， 所述药物为肠溶片， 由 CZLC331蛋白 100重量份， 乳糖 60重量份， 微晶 纤维素 20重量份、 羧甲基淀粉钠 20重量份、 聚维酮 K3。 10重量份混合制粒， 再将干 颗粒按重量份 2500: 50 的比例加入滑石粉混合均匀压片， 再在压片表边喷雾涂覆肠 溶薄膜衣层制成。
上述药物的用量一般为 0. 2- 6. 4mg CZLC331蛋白 /kg体重， 每天给药 1-2次， 疗 程为 5至 10天。 注射液可经肌肉注射、 腹腔注射或静脉注射给药。
本发明提供了沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白在炎症性肠病药物研发中的 新用途。 通过给予小鼠三硝基苯磺酸（2，4, 6- trinitrobenzene sulfonic acid, TNBS) 灌肠以模拟人溃疡性结肠炎实验发现， 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白对炎症 性肠病具有明显的预防和治疗作用， 具体表现为： 1)造模组： 小鼠摄食 /饮水量明显 减少， 活动减少并伴有便血， 结肠出现肉眼可见的的充血、 水肿、 出血及溃疡、 显微 镜下可见细胞结构紊乱、 杯状细胞消失、 淋巴细胞和中性粒细胞浸润等； （2)腹腔给 药后： 小鼠摄食 /饮水量恢复正常、 活动明显增加且便血减少， 结肠肉眼可见的充血、 水肿、 出血及溃疡等现象明显减轻， 显微镜下可见的细胞结构紊乱和杯状细胞消失、 淋巴细胞和中性粒细胞浸润现象明显减轻等。 上述实验结果表明腹腔注射 CZLC331蛋 白可以有效对抗结肠炎症对肠粘膜的损伤， 并对炎症条件下的小鼠起到保护作用。 因 而， 可以沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白为活性成分制备成预治炎症性肠病药 物， 该药具有以下优点： 1 ) 疗效明显 （有效率达 100%， 治愈率达 70% ) ， 而临床 一线药物柳氮磺胺吡啶有效率仅达 90%左右， 显效率仅 50% ; 2 )起效快 （用药后 24 小时即可起效） ， 柳氮磺胺吡啶则在服用 2周左右， 症状方有所改善； 3 )疗程短（疗 程一般为 5-10天） ， 柳氮磺吡啶的疗程一般为 6周左右； 4) 安全性高 （无毒性） ， 而服用柳氮磺胺吡啶后患者有丙氨酸转氨酶（ALT)和天冬氨酸转氨酶（AST)的升高， 提示其有肝脏毒性； 5 ) 副作用小 （无明显副作用） ， 而柳氮磺胺吡啶在服用后则有 恶心、 皮疹、 粒细胞减少等不良反应； 6) 用药方便 （每日腹腔注射一次即可） 而柳 氮磺胺吡啶则 3-4g/d， 分 3- 4次口服。 综上所述， 本发明可解决现有炎症性肠病治疗 药物疗效差、 起效慢、 疗程长、 副作用大的问题， 可显著减少患者治疗过程的痛苦， 促进患者的身体康复和提高生存质量。 本发明将在炎症性肠病及其它部位炎症的预防 和治疗中发挥重要作用， 具有广阔的应用前景。 附图说明
图 1为 0. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡 性结肠炎小鼠的结肠外观
图 2为 0. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡 性结肠炎小鼠的结肠粘膜
图 3为 0. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡 性结肠炎小鼠结肠粘膜组织切片后 HE染色结果
图 4为不同浓度 CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡 性结肠炎小鼠的结肠外观和结肠粘膜
图 5为不同浓度不同给药时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠外观 图 6为不同浓度不同给药时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠粘膜 图 7为不同浓度不同给药时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠粘膜组织 切片后 HE染色结果
图 8为 3. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后 4h腹腔注射治疗溃 疡性结肠炎小鼠， 使用 RT-PCR技术检测结肠 TLR1-4家族基因表达的情况 （A幅） ， 及其柱状分析图 （B幅）
图 9为 3. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模后 4h腹腔注射治疗溃 疡性结肠炎小鼠， 使用 RT- PCR技术检测结肠 TLR6-9家族基因表达的情况 （A幅） ， 及其柱状分析图 （B幅）
图 10为 0. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模前 0. 5h预防给药后 溃疡性结肠炎小鼠的结肠外观
图 11为 0. 2mg/kg CZLC331蛋白在三硝基苯磺酸 (TNBS)造模前 0. 5h预防给药后 溃疡性结肠炎小鼠的结肠粘膜 具体实施方式
为了深入研究沙门氏菌鞭毛蛋白的新功能并将其开发为新领域的保护药物， 本发 明的发明人通过系统研究， 发现 CZLC331可能通过下调 TLRs的表达对炎症性肠道疾 病如溃疡性结肠炎和克罗恩病等具有良好的预防和治疗效果。 关于炎症性肠病的研究 （Abreu MT, Vora P， Faure E， Thomas LS， Arnold ET, Ardit i M. Decreased expression of Tol l-l ike receptor - 4 and MD - 2 correlates with intest inal epithel ial cel l protect ion against dysregulated proinflammatory gene expression in response to bacterial l ipopoly saccharide. / Immunol, 2001 Aug 1 ; 167 (3) : 1609-16 ) 表明， 肠上皮细胞可以通过下调 TLR4 表 达及其信号传导来维持肠道免疫耐受。 本发明的发明人意外研究发现沙门氏菌鞭毛蛋 白具有防治溃疡性结肠炎的作用， 而且还进一步证明这种作用是通过下调结肠组织 Tol l样受体家族的表达实现的。
Tol l样受体（Tol l l ike receptor, TLR)是与果蝇 Tol l蛋白具有同源性的受体蛋 白家族， 属于模式识别受体， 主要识别病原微生物的保守结构， 通过多种信号转导途 径改善免疫系统功能。 TLR家族既在抗感染免疫及先天性免疫中起关键性作用， 也是 重要的获得性免疫调节因子。 TLR识别病原体后， 向胞内传递活化信号， 激活 NF- K B 等转录因子， 诱导促炎症因子 TL- 1、 IL-6、 IL-8等的产生， 上调 IL-12、 TNF等重要 炎症因子的表达水平及对病原体的杀伤水平。 在细胞介导的免疫应答中， IL-12 对于 诱导 T、 Β淋巴细胞产生 Thl细胞来说， 是一个关键性的调节物， 可作为生物佐剂放 大特异性 T细胞对病原体的应答， 因此， 可以说 TLR家族是 IL- 12以及 Thl细胞参与 免疫应答产生的早期信号。 TLRs和正常菌群之间的平衡被打破， 便会造成病理性肠道 炎症。 特别的， 沙门氏菌鞭毛蛋白具有下调结肠组织 TLR家族表达的效果， 可能是其 对溃疡性结肠炎治疗作用的重要机制。
本发明针对沙门氏菌鞭毛蛋白第 1-176位氨基酸和第 402-505位氨基酸， 从头合 成其核苷酸编码序列， 通过原核表达制备了全长为 331个氨基酸的沙门氏菌鞭毛蛋白 衍生物， 命名为 CZLC331 , 并通过实验证明其对炎症性肠道疾病如溃疡性结肠炎和克 罗恩病等具有良好的预防和治疗效果。 本发明的实施将为炎症性肠病的预防和治疗 提供重要参考。
以下给出了详细的实施方式和具体的操作过程， 但本发明的保护范围不限于下述 实施例。
本发明所用方法如无特别说明均为常规方法。 实施例 1、 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白的制备
1. 构建沙门氏菌鞭毛蛋白衍生物 CZLC331的原核表达载体 pET28b-Tat- CZLC331 1 )人工合成沙门氏菌鞭毛蛋白衍生物 CZLC296(沙门氏菌鞭毛蛋白衍生物 CZLC296 是通过柔性臂将沙门氏菌鞭毛蛋白自 N端第 1-176位氨基酸残基和自 N端第 402-505 位氨基酸残基连接得到的） 的编码基因， 其核苷酸序列如序列表中序列 1所示， 长度 为 891bp， 由北京博迈德科技发展有限公司合成。对合成的基因进行 2 %琼脂糖凝胶 电泳进行检测， 检测结果表明获得了 891bp的目的基因， 与预期结果相符。
2 ) 重组表达载体 pET28b- Tat- CZLC331的构建
a) PCR扩增沙门氏菌鞭毛蛋白衍生物 CZLC296蛋白编码基因
采用常规 PCR的方法以人工合成的核苷酸序列为模板扩增出 891bp的沙门氏菌鞭 毛蛋白衍生物 CZLC296蛋白的编码基因， 50 μ 1 PCR反应体系为： 质粒模板 （携带沙 门氏菌鞭毛蛋白衍生物 CZLC296蛋白编码基因的克隆载体 PGH-CZLC296， 构建方法为 将人工合成的 CZLC296编码基因插入克隆载体 pGH的 Smal酶切位点获得） 0. 5 μ 1， 10 X dNTP 5 μ 1 , 10 X Ex Taq 缓冲液 5 μ 1， 上、 下游引物 （上游引物序列为： 5' -CGCG6^mTGGCTCAAGnATCA-3'， 下游引物序列为： 5' HXGCmCTCTCMCAAAGACMGTT- 3' )各 0. 5 μ 1， Ex Taq酶 0. 25 μ 1， dd¾0 38. 25 μ 1； PCR反应条件为： 先 95V 4分钟； 然 后 95°C 45秒， 56°C 30秒， 72Ό 45秒， 共 30个循环； 最后 72°C 7分钟。 反应结 束后， 对 PCR产物进行 1 %琼脂糖凝胶电泳检测， 检测结果表明经扩增获得了 891bp 的 DNA片段， 与预期结果相符， 回收并纯化该目的片段。
b) 限制性内切酶分别酶切目的基因 CZLC296和 pET28b-Tat载体
分别对沙门氏菌鞭毛蛋白衍生物 CZLC296编码基因和载体 pET28b- Tat载体 （构 建方法为： 首先通过化学合成的方法合成含有酶切位点为 Ncol (上游） 和 Ndel (下 游） 的双链 TAT核心序列， 然后采用限制性内切酶 TVcoI和 Λ¾¾双酶切 TAT核心片段 和载体 pET28b， 回收目的片段后经 T4 DNA连接酶连接及酶切鉴定， 最后送英骏公司 直接测序确证含有 TAT核心片段的原核表达载体 pET28b-TAT构建正确） 用限制性内 切酶 BamHl和 Xho I双酶切， 然后将酶切产物在 16°C下用 T4 DNA连接酶连接过夜并 转化到大肠杆菌 DH5 a 感受态细胞中。
c)鉴定
将培养后生长出的克隆分别通过限制性内切酶酶切及测序确证。
测序结果表明获得了序列及插入位置均正确的携带 Tat蛋白转导肽编码序列和沙 门 氏菌鞭毛蛋 白衍生物 CZLC296编码基因 的重组表达载体， 命名为 pET28b - Tat-CZLC331。 所述在 N端连接有 Tat蛋白转导肽的沙门氏菌鞭毛蛋白衍生物 CZLC296命名为 CZLC331 , 编码该蛋白的基因的核苷酸序列如序列表中序列 2所示。
2. 转化及菌体复苏
将步骤 1构建正确的原核表达载体 pET28b- Tat- CZLC331转化大肠杆菌 BL21 (DE3) 并涂布至含有 Kana+ (终浓度 lOO g/mL)的 LB平皿上， 待长出克隆， 各组分别挑取一 个克隆接种到 5mL含有 Kana+ (终浓度 100 p g/mL)的 LB培养基中， 37°C、 220rpm摇菌 约 16小时， 待菌体完全复苏。
3. 原核表达载体 pET28b- Tat- CZLC331的诱导表达
将复苏的菌液稀释至 0D6。。=0. 8， 然后吸取 5mL菌液接种到 150mL含有 Kana+ (终浓 度 100 g/mL)的 LB 培养基中， 37°C、 220rpra 摇菌约 4-5 小时后测定 0D6。。， 待 0DMO=0. 6-1. 0时迅速加入诱导剂 IPTG (终浓度 ImM) , 并于 30°C、 220rpm诱导表达 8 小时。
4. 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白样品的制备
上述诱导表达菌液于 4°C、 12000rpra离心 10分钟以收集菌体， 采用 20mM磷酸钠 缓冲液稀释样品后超声破碎， 制备 CZLC331蛋白样品， 留取上清液待分离纯化。
5. 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白的分离、 纯化
将步骤 4制备的上清液采用初始缓冲液（20mM Na3P04+0. 5M NaCl, pH7. 4)平衡后直 接上样至平衡好的 HisTrap HP 5mL柱 （购自 GE公司） ， 然后采用 4-5个柱体积的上 述缓冲液冲洗柱子至洗脱峰基线， 最后采用洗脱缓冲液（20mM Na3P04+0. 5M NaCl+0. 5M 咪唑， pH7. 4)洗脱蛋白样品， 脱盐后得到沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白， 纯 度达 95 %以上。
实施例 2、沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白对炎症性肠病的治疗效果验证 实验动物： 清洁级 7-8周 Balb/c雄性小鼠 （25 ± 5克） ， 购自军事医学科学院实 验动物中心； 单笼隔离饲养 24小时并允许自由饮食。
实验试剂： 三硝基苯磺酸 (TNBS, 购自 Sigma公司）； CZLC331 (实施例 1制备）； RT PCR试剂盒 （货号 FSK100, 购自东洋纺生物科技有限公司）
模型制备： 首先将小鼠采用麻醉机麻醉， 然后采用直径 2. 0mm、 长约 lOcra的硅胶 管由肛门轻缓插入 （深约 4cm) 并将灌肠试剂 （含有 150g/L TNBS+380mL/L乙醇溶液， 150mg/kg) 缓慢推入结肠以制备炎症性肠病模型。
实验分组： 实验动物分组为： 模型组、 治疗组和对照组， 每组 8只小鼠。 对照组 是针对正常小鼠用以上方法将 380mL/L乙醇溶液 （不含 TNBS ) 灌肠 1次， 正常喂养 2 天; 治疗组是针对已制备溃疡性结肠炎模型的小鼠， 待其溃疡性结肠炎形成后 0. 5h， 腹腔注射 CZLC331蛋白（3. 2mg/kg体重)进行治疗， 同时， 可根据损伤程度在模型制造 成功后 0. 5 8h时间范围内，在 0. 2-6. 4mg/kg体重的浓度范围内用药，正常喂养 2天; 模型组是针对已制备溃疡性结肠炎模型的小鼠进行正常喂养 2天。
1、 药效检测： 首先对注射 CZLC331蛋白前、 后小鼠摄食 /水量、 活动度、 便血等 一般指标进行观察； 然后于 2d后颈椎脱臼处死小鼠并分离全结肠， 拍照观察肠壁形 态（如图 1、 图 4左幅、 图 5所示） ； 最后沿肠系膜剪开肠腔， 观察结肠黏膜组织（如 图 2、 图 4右幅、 图 6所示） ， 生理盐水冲洗后留取远端结肠， - 80°C冰冻保存以备后 续检测。
结果可见模型组小鼠摄食 /水量明显减少， 活动减少， 有便血； 小鼠结肠肉眼观 察， 会出现明显的充血、 水肿、 出血及溃疡形成 （0. 2mg/kg CZLC331蛋白在三硝基苯 磺酸 (TNBS)造模后给药 0. 5h治疗溃疡性结肠炎小鼠的结肠外观和结肠粘膜分别如图 1 和图 2所示， A: 对照组； B: 模型组； C: 治疗组） ， 显微镜下可见模型组细胞结构 紊乱、 杯状细胞消失、 淋巴细胞和中性粒细胞浸润等现象 （0. 2mg/kg CZLC331蛋白在 三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡性结肠炎小鼠结肠粘膜组织切片后 HE 染色结果如图 3所示， A: 对照组； B: 模型组； C: 治疗组） ； CZLC331经腹腔注射后， 小鼠便血、 少食少动现象有所减轻 （摄食 /水量、 活动性明显增加） ； 解剖发现， 肠 粘膜充血、 水肿、 溃疡等现象亦有所减轻， 如图 1、 图 2、 图 4 (不同浓度 CZLC331蛋 白在三硝基苯磺酸 (TNBS)造模后给药 0. 5h治疗溃疡性结肠炎小鼠的结肠外观和结肠 粘膜。 A: 0. 2mg/kg CZLC331 ; B: 0. 4rag/kg CZLC331 ; C: 0. 8mg/kg CZLC331 ; D: 1. 6mg/kg CZLC331 ) 、 图 5 (不同浓度不同给药时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠外 观。 A: 0. 2mg/kg CZLC331 ; B: 0. ½g/kg CZLC331 ; C: 0. 8mg/kg CZLC331 ; D: 1. 6mg/kg CZLC331 ; E: 3. 2mg/kg CZLC331 ; F: 6. 4mg/kg CZLC331 ) 和图 6 (不同浓度不同给药 时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠粘膜。 A: 0. 2mg/kg CZLC331 ; B: 0. 4mg/kg CZLC331 ; C: 0. 8mg/kg CZLC331 ; D: 1. 6mg/kg CZLC331 ; E: 3. 2mg/kg CZLC331 ; F: 6. 4mg/kg CZLC331 ) 所示， 显微镜下可见治疗组细胞排列结构比较整齐、 少量杯状细 胞消失及淋巴细胞和中性粒细胞浸润等现象 （如图 3和图 7所示） 。
对照组小鼠摄食、 饮水量正常， 无便血及活动减少等现象， 显微镜下可见细胞排 列整齐， 无杯状细胞减少及淋巴细胞和中性粒细胞浸润等现象。
2、 肠粘膜组织病理学评分： 所取肠道组织置于 4 %甲醛溶液中固定后石蜡包埋、 切片和 HE染色， 并进行光学显微镜观察检测。 0. 2mg/kg CZLC331蛋白在三硝基苯磺 酸 (TNBS)造模后给药 0. 5h治疗溃疡性结肠炎小鼠结肠粘膜组织切片后 HE染色结果如 图 3所示， 不同浓度不同给药时间 CZLC331蛋白治疗溃疡性结肠炎小鼠结肠粘膜组织 切片后 HE染色结果如图 7所示 (A: 0. 2mg/kg CZLC331 ; B: 0. 4mg/kg CZLC331 ; C: 0. 8mg/kg CZLC331 ; D: 1. 6mg/kg CZLC331 ; E: 3. 2mg/kg CZLC331 ; F: 6. 4rag/kg CZLC331 ) , 经 CZLC331蛋白治疗后细胞排列结构趋于整齐、 少量杯状细胞消失并出现淋巴细胞和 中性粒细胞浸润等现象， 说明达到了良好的治疗效果。
3、 肠道 TLR家族表达水平检测： 提取注射 CZLC331蛋白前模型组、 治疗组、 对 照组小鼠结肠粘膜组织的总 RNA， 反转录后通过 PCR检测 TLR家族基因表达水平， 具 体 TLR家族及内参基因 β -actin引物信息如下表 1所示。 PCR反应体系为： Ex Taq 0. 25 μ 1、 10 X上样缓冲液 5 μ 1、 dNTP 5 μ 1 , 模板 2 μ 1、 上、 下游引物各 1 μ 1， 双蒸水 补齐至 50 μ 1。 PCR 反应条件为： 95 °C 5rain→ ( 95°C 45s→62°C 45s→72°C lrain ) X 35个循环→72°C 5min→4°C停止。 反应结束后， 对 PCR扩增产物进行 2 %琼脂糖凝 胶电泳检测并采集图像分析。结果如图 8和图 9所示，经 CZLC331治疗后 TLR家族 PCR 条带的表达量显示与模型组明显的差异， 其中 TLR2、 TLR3、 TLR4、 TLR8、 TLR9 模型 组的表达量都明显上调， 给予 CZLC331治疗后可逆转其上调； TLR6、 TLR7模型组表达 量明显上调， 给予 CZLC331治疗后逆转其上调虽然没有统计学差异， 但其逆转下调趋 势明显。 检测结果表明治疗过程和机理还可能与 TLR家族表达下调有关， 推测沙门氏 菌鞭毛蛋白衍生物 CZLC331蛋白可通过 TLR家族等通路对炎症性肠病发挥治疗作用， 可以其为活性成分制备成炎症性肠病治疗药物。
表 1 RT-PCR扩增用引物序列信息表
Figure imgf000010_0001
引物序列
pU: 5 '-TTGTGCCACCCA ACAGTC AGCC-3 '
TLR1
pD: 5'-ACCGCTCAACCCCAGGAACTGT-3'
pU： 5 '-TTCCCTGCTCGTTCTCCCAGCA-3 '
TLR2
pD: 5'-TAGAGCTCTTGCAGCCGAGGCA-3'
pU: 5 '-AGCGTCTGTCCCCTCG CTCTTT-3 '
TLR3
pD: 5 '-GGCGGCCCGAAAACATCCTTCT-3 '
pU: 5 '-GCTTCCAC A AGAGCCGGAAGGT-3 '
TLR4
pD: 5 '-TGGCCAGGCTATCTGTG AGCGT-3 '
pU: 5'-TTCATCCACATGGTGTGCCCGC-3'
TLR6
pD: 5'-ATATGCTCTCAGCCCAGGCGCA-3'
pU: 5 '-TCAGCATGTGCCCCCA AC ATGG-3 '
TLR7
pD: 5'-CAACGGCCAGAGTTCACTGCCA-3'
pU: 5'-TGGCTGCTCTGGTTCACCACCT-3'
TLR8
pD: 5 '-TGTTGGGCCACTGGAGGATGGA-3 '
pU: 5 '-GCCTGGTGG ACTGC A ATTGGCT-3 '
TLR9
pD: 5'-TCACAGCGACGGCAATTCCCAC-3'
pU: 5'-GCGAGCACAGCTTCTTTGCAGC-3'
β-actin
pD: 5 '-A ATACAGCCCGGGG AGCATCGT-3 ' 实施例 3、 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白对炎症性肠病的预防效果验 证
实验动物： 与实施例 2相同。
实验试剂： 与实施例 2相同。
模型制备： 与实施例 2相同。
实验分组： 实验动物分三组： 模型组、 预防组和对照组， 每组 8只小鼠。 预防组 针对正常小鼠先给予腹腔注射 CZLC331蛋白 3. 2mg/kg体重 （0. 2-6. 4mg CZLC331蛋白 /kg体重均可） ， 然后再按模型制备造模， 造模后正常喂养 2天； 模型组针对正常小 鼠先使用相同体积的生理盐水对小鼠进行腹腔注射， 然后再按模型制备方式造模， 造 模后正常喂养 2天； 对照组针对正常小鼠用 380mL/L乙醇溶液灌肠 1次后正常喂养。
检测项目及检测方法： 与实施例 2相同
实验结果：
模型组小鼠摄食 /水量明显减少， 活动减少， 有便血， 小鼠结肠肉眼观察， 出现 明显的充血、 水肿、 出血及溃疡形成 （如图 10所示， A: 对照组； B : 模型组； C : 预 防组） CZLC331预防组小鼠便血、 少食少动现象有所减轻， 摄食 /水量、 活动性明显 增加， 解剖发现， 肠粘膜充血、 水肿、 溃疡等现象亦有所减轻 （如图 1 1所示， A : 对 照组； B : 模型组； C : 预防组） ， 整体病理表现与对照组非常接近， 无摄食 /水量减 少及活动减少， 无便血， 小鼠结肠肉眼观察无明显血便、 水肿、 出血及溃疡形成。 结 果表明沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白对炎症性肠病具有预防作用， 可以其为 活性成分制备成炎症性肠病的预防药物。 实施例 4、 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白注射液的制备
1. 组成成分： CZLC331蛋白注射液， 由重量体积比 （mg/mL ) 为 5 %的 CZLC331蛋 白， 0. 85 %的氯化钠和注射用水组成。
2. CZLC331蛋白注射液制备方法：
1 )将构建正确的原核表达载体 pET28b- Tat-CZLC331转化大肠杆菌 BL21 (DE3)并 涂布至 LB平皿， 待长出克隆， 各组分别挑取一个克隆接种到 5mL含有 Kana+ (终浓度 100 μ g/wL)的 LB培养基中， 37 °C、 220rpm摇菌约 16小时。
2)待菌体完全复苏， 将复苏的菌液稀释至 OD6。。=0. 8， 然后吸取 5raL 菌液接种到 150mL含有 Kana+ (终浓度 100 μ g/mL)的 LB培养基中， 37 °C、 220rpm摇菌约 4— 5小时 后测定 0D冊，待 (¾。。=0. 6— 1. 0时迅速加入诱导剂 IPTG (终浓度 IraM)，并于 30 °C、220rpm 诱导表达 8小时。
3)诱导表达菌液于 4°C、 12000rpm离心 10分钟以收集菌体， 采用 20raM磷酸钠缓 冲液稀释样品后超声破碎制备蛋白样品， 留取上清液；将上清液采用初始缓冲液 (20mM Ν¾Ρ04+0. 5Μ NaCl, pH7. 4)平衡后直接上样至平衡好的 HisTrap HP 5mL柱 （购自 GE 公司） ， 然后采用 4- 5个柱体积的上述缓冲液冲洗柱子至洗脱峰基线， 最后采用洗脱 缓冲液（20mM Na3P04+0. 5M NaCl+0. 5M 咪唑， pH7. 4)洗脱蛋白样品， 脱盐后得到沙门 氏菌鞭毛蛋白衍生物 CZLC331蛋白。
4) 首先将 CZLC331蛋白采用注射用水溶解， 然后加入氯化钠， 搅拌均匀； 最后加 入重量体积比 0. 3 %的经干热处理的活性炭搅拌吸附 15分钟，过滤至澄明度合格后供 灌封。 过滤包括钛棒过滤器粗滤脱碳、 0. 45 μ m筒式过滤器过滤和 0. 22 μ m筒式过滤 器精滤三重过滤。 实施例 5、 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白肠溶片的制备
1. 称取 CZLC331蛋白 100g， 过 100目筛， 乳糖 60g， 微晶纤维素 20g、 羧甲基淀 粉钠 20g、 聚维酮 K3。 10g过 80 目筛， 将主药与辅料按照等量递增的原则混合， 加入 体积比 30 %的水制软材， 20目制粒， 得湿颗粒。
2. 55- 65°C通风干燥 3小时， 20目整粒， 得干颗粒； 将 2500g千颗粒加入滑石粉 50g, 混合均匀， 测定含量； 调整好压片机的转速和压力， 将定量后的颗粒送入压片 机， 压制成直径 1. 2cm的扁形 CZLC331蛋白片。
3. 以直径 1. 2cm的 CZLC331蛋白片为片基，片表边喷雾涂覆有一层厚 1. 0mm的肠 溶薄膜衣层。 包衣材料为易信克 E0BS68, 该薄膜衣层不透气、 不透潮， 具有较强薄膜 强度及抗胃液的性质， 通过对 pH值变化控制溶液， 保证药物有效、 安全释放。 药效分析：
本发明沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白为活性成分制备成预治炎症性肠病 药物， 具有以下特点：
1 ) 有效率高： 实施例 2和实施例 3实验数据显示， 治疗组和预防组各 8只小鼠， 实验后所有动物的少食少动及便血等症状改善明显， 表明有效率达 100 %， 而治疗组 8只小鼠结肠解剖发现， 结肠黏膜组织完全恢复 6只， 治愈率达 75 % ; 而临床一线药 物柳氮磺胺吡啶有效率仅达 90 %左右， 显效率仅 50%。
2 ) 起效快： 实施例 2和实施例 3实验数据显示小鼠用药后 24小时即可起效； 而 柳氮磺胺吡啶则在服用 2周左右， 症状方有所改善。 3 ) 疗程短 （疗程一般为 5-10天） ： 柳氮磺吡啶的疗程一般为 6周左右； 本实验 作用于小鼠起效为 1-2天， 柳氮磺吡啶作用于小鼠疗程为一周左右， 其临床起效为 6 周， 以此估算本发明药物疗程为 5-10天。
4 ) 安全性高 （无毒性） ： 服用柳氮磺胺吡啶后患者有丙氨酸转氨酶 （ALT) 和天 冬氨酸转氨酶 （AST ) 的升高， 提示其有肝脏毒性； 而本发明的 CZLC331 属于蛋白类 药物， 己被用于抗放射损伤药物研发， 对机体无毒性。
5 ) 副作用小 （无明显副作用） ： 而柳氮磺胺吡啶在服用后则有恶心、 皮疹、 粒 细胞减少等不良反应， 而本发明的 CZLC331属于蛋白类药物， 已被用于抗放射损伤药 物研发， 对机体无明显副作用。
6 ) 用药方便 （每日腹腔注射一次即可） ： 而柳氮磺胺吡啶则 3- 4g/d， 分 3- 4次 口服。 工业应用性
本发明提供沙门氏菌鞭毛蛋白衍生物 CZLC331在防治炎症性肠病药物研发与制备 中的用途， 该药物效率高、 起效快、 疗程短、 无毒性、 副作用小且用药方便， 可解决 现有炎症性肠病治疗药物疗效差、 起效慢、 疗程长、 副作用大的问题， 可显著减少患 者治疗过程的痛苦， 促进患者的身体康复和提高生存质量。 本发明将在炎症性肠病及 其它部位炎症的预防和治疗中发挥重要作用， 具有广阔的应用前景。

Claims

权利要求
1、 沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白在防治炎症性肠病药物研发中的应用。
2、 根据权利要求 1 所述的应用， 其特征在于： 所述炎症性肠病包括溃疡性结肠 炎和克罗恩病。
3、 根据权利要求 1或 2所述的应用， 其特征在于： 所述沙门氏菌鞭毛蛋白衍生 物 CZLC331是包括 Tat蛋白转导肽、 沙门氏菌鞭毛蛋白自 N端第 1-176位氨基酸残基 和第 402-505位氨基酸残基的融合蛋白； 所述沙门氏菌鞭毛蛋白自 N端第 1-176位氨 基酸残基和第 402-505位氨基酸残基可通过柔性连接肽连接。
4、 根据权利要求 1至 3任一所述的应用， 其特征在于： 所述沙门氏菌鞭毛蛋白 衍生物 CZLC331蛋白可采用常规的原核表达的方法制备得到， 纯度大于 95 %。
5、 根据权利要求 1至 4任一所述的应用， 其特征在于： 所述沙门氏菌鞭毛蛋白 衍生物 CZLC331蛋白按包括以下步骤的方法制备得到：
1 ) 合成沙门氏菌鞭毛蛋白衍生物 CZLC296编码基因， 其核苷酸序列如序列表中序 列 1所示； 所述沙门氏菌鞭毛蛋白衍生物 CZLC296是通过柔性臂将沙门氏菌鞭毛蛋白自 N端第 1-176位氨基酸残基和自 N端第 402-505位氨基酸残基连接得到的；
2 )将人工合成的沙门氏菌鞭毛蛋白衍生物 CZLC296编码基因克隆到携带 Tat转导肽 编码序列的原核表达载体中， 得到携带 Tat蛋白转导肽编码序列和沙门氏菌鞭毛蛋白 衍生物 CZLC296编码基因的原核表达载体； 所述 N端连接有 Tat蛋白转导肽的沙门氏菌 鞭毛蛋白衍生物命名为 CZLC331 , 编码该蛋白的基因的核苷酸序列如序列表中序列 2所 示；
3 ) 将重组表达载体 pET28b- Tat-CZLC331转化入宿主菌；
4 ) 诱导表达含有携带 Tat蛋白转导肽编码序列和沙门氏菌鞭毛蛋白衍生物
CZLC296基因的原核表达载体的宿主菌；
5 ) 分离及纯化表达的重组蛋白， 得到沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白。
6、 根据权利要求 5所述的应用， 其特征在于： 所述步骤 2 ) 中携带 Tat转导肽编码 序列的原核表达载体为任意一种可在大肠杆菌中表达的原核表达载体， 如 pET-22b系 列、 pET-28系列或 pET-15系列， 优选为 pET28b- Tat ; 以 pET28b-Tat为出发载体构建的 携带沙门 氏菌鞭毛蛋 白衍生物 CZLC331编码基因的重组表达载体为 pET28b-Tat-CZLC331 ; 所述步骤 3 ) 中的宿主菌为 £ coii BL21 (DE3) , E. coli ER2566
(DE3 ) 、 E. coii BL2KDE3, plysS)、 E. coii JM109, E. 。 1¾101或£ co7i ToplO 等， 优选为^: coli BL21 (DE3) 0
7、 根据权利要求 1-6 任一项所述的应用， 其特征在于： 所述药物可以制成注射 液、 口服液、 灌肠液、 胶囊、 肠溶片、 粉剂或粒剂， 优选为注射液、 灌肠液或肠溶片。
8、 根据权利要求 7 所述的应用， 其特征在于： 所述胶 ¾、 肠溶片、 粉剂和粒剂 等固态剂型的药物中活性成分沙门氏菌鞭毛蛋白衍生物 CZLC331蛋白的质量百分含量 为 1一 35 % ; 所述注射液、 口服液和灌肠液等液态剂型的药物中活性成分沙门氏菌鞭 毛蛋白衍生物 CZLC331蛋白的浓度可为 2- 64g/L; 在所述药物中可以加入一种或多种 药学上可接受的辅料， 包括药学领域常规的吸收促进剂、 表面活性剂、 润滑剂、 稳定 剂、 稀释剂、 粘合剂、 湿润剂、 崩解剂、 稀释剂、 吸附载体、 赋形剂， 必要时还可加 入色素、 甜味剂及香味剂。
9、 根据权利要求 7 所述的应用， 其特征在于： 所述药物为蛋白注射液， 由重量 体积比为 5 % ( 5g/100mL) 的 CZLC331蛋白、 0. 85 % ( 0. 85g/100mL) 的氯化钠和注射 用水组成。
10、根据权利要求 7或 8所述的应用，其特征在于:所述药物为肠溶片，由 CZLC331 蛋白 100重量份， 乳糖 60重量份， 微晶纤维素 20重量份、 羧甲基淀粉钠 20重量份、 聚维酮 K3。 10重量份混合制粒， 再将干颗粒按重量份 2500: 50的比例加入滑石粉混 合均匀压片， 再在压片表边喷雾涂覆肠溶薄膜衣层制成。
PCT/CN2012/000097 2011-07-05 2012-01-19 沙门氏菌鞭毛蛋白衍生物在制备防治炎症性肠病药物中的应用 WO2013004069A1 (zh)

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NZ619871A NZ619871B2 (en) 2011-07-05 2012-01-19 Use of salmonella flagellin derivative in preparation of drug for preventing and treating inflammatory bowel diseases
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JP2014517394A JP5876930B2 (ja) 2011-07-05 2012-01-19 炎症性腸疾患の予防および治療のための薬物の調製におけるサルモネラフラゲリン誘導体の使用
CA2840924A CA2840924A1 (en) 2011-07-05 2012-01-19 Use of salmonella flagellin derivative in preparation of drug for preventing and treating inflammatory bowel diseases
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