NZ619871B2 - Use of salmonella flagellin derivative in preparation of drug for preventing and treating inflammatory bowel diseases - Google Patents

Abstract

Disclosed use of a Salmonella flagellin protein derivative in the manufacture of a medicament for the treatment or prevention of an inflammatory bowel disease (IBD), wherein the protein derivative comprises the CLZC331 protein according to sequence ID NO:3 (as defined in the specification).

Description

USE OF SALMONELLA FLAGELLIN DERIVATIVE IN PREPARATION OF DRUG FOR PREVENTING AND TREATING INFLAMMATORY BOWEL DISEASES Technical Field This invention belongs to the new application of recombinant protein in medicine and drug, in particular to the new application of a flagellin derivative from Salmonella, named CZLC331, in the prevention and treatment of inflammatory bowel disease (IBD).

Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Ulcerative colitis (UC), also known as idiopathic ulcerative colitis or chronic nonspecific ulcerative colitis, is a disease of large intestine with infiltration of the mucosa and idiopathic chronic non-specific inflammation. The main clinical symptoms are abdominal pain, diarrhoea, mucus and bloody stools, and associated with optic neuritis, neuritis, and osteoporosis. It has been identified as the hardly disease by the World Health Organization (WHO) because of the difficulty to treatment, time-consuming and easy to relapse. Although currently there are a variety of drugs for curing IBD, such as amino salicylic acid, glucocorticoids and immunosuppressive agents, but they have many shortcomings, such as poor efficacy (especially to severe UC), slow onset, long course, and large side effects.

Therefore, the drug with an effective, rapid onset, short course, high safety, little side effects, convenient for IBD (especially chronic nonspecific ulcer colitis) is urgent needed.

It has been shown that CBLB502, one of the flagellin derivatives from Salmonella, has a protective effect on the hematopoietic system, and can extend the survival time of mice to the high-dose radiation injury and improve the survival rates of low-dose irradiated mice (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 study found that the flagellin protein derivatives from Salmonella and other derivatives containing the N-and C- terminal conserved domain have the radial protection, they can effectively improve the number of hematopoietic stem cells in the bone marrow of mice and effective preventing the death of mice caused by lethally irradiation. Its mechanism may be the anti-apoptotic role through NF- κB signal pathway. It means that this protein can be applied to development of anti-radiation drugs. There is no report on Salmonella flagellin derivatives except for the anti- radiation drug.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Summary of the Invention The present invention relates to a Salmonella flagellin protein derivative, CZLC331, which has been demonstrated to be effective in the prevention and treatment for IBD. The amino acid sequence of CZLC331 is shown in SEQ ID No:3, and the encoding nucleotide sequence is shown in SEQ ID No:2.

The CZLC331 has a good preventive and therapeutic effect for IBD such as ulcerative colitis (UC) and Crohn's disease (CD) in this invention. Therefore, this protein can be developed as a candidate drug for the prevention and treatment of IBD.

The CZLC331 protein includes an N-terminal Tat protein transduction peptide, and amino acid residues 28-203 and 220-323, which correspond to N- and C-terminal conserved domains of Salmonella flagellin proteins, wherein the two regions are connected by a flexible linker peptide.

Accordingly, in a first aspect of the present invention, there is provided use of a Salmonella flagellin protein derivative in the manufacture of a medicament for the treatment or prevention of an inflammatory bowel disease (IBD), wherein the protein derivative comprises the CLZC331 protein according to sequence ID NO:3.

In embodiments of the present, the medicament is a dosage form selected from the group consisting of a capsule, an enteric-coated tablet, a powder and a granule, and the protein derivative is present in the dosage form in an amount of 1-35 wt%.

In further embodiments of the present invention, the medicament is a dosage selected from the group consisting of an injectable liquid, an oral liquid and an enema liquid, and the protein derivative is present in the dosage form in a concentration of 2-64 g/L.

In other embodiments of the present invention, the dosage form is an injectable liquid comprising the protein derivative at a concentration of at least 5g/100mL.

In further embodiments of the present invention, the dosage form is an enteric-coated tablet comprising 100 parts by weight of the protein derivative, 60 parts by weight of lactose, parts by weight of microcrystalline cellulose, 20 parts by weight of sodium carboxymethyl starch, and 10 parts by weight of Povidone K .

In a second aspect of the present invention, there is provided a method for preparing a Salmonella flagellin protein derivative for the use of the invention, said method comprising: 1) providing a nucleic acid sequence according to SEQ ID NO:1; 2) preparing a recombinant expression vector by subcloning said nucleic acid sequence into a prokaryotic expression vector containing a coding sequence of a Tat transduction peptide; 3) transforming said recombinant expression vector into a host bacterium; 4) inducing expression of said protein derivative in the host bacterium; and ) isolating and purifying said protein derivative from the host bacterium.

The dosage of this drugs is generally 0. 2-6. 4mg CZLC331 protein / kg body weight, and is administered 1-2 times a day, the course is 5-10 days. The injection can be given by intramuscular injection, intraperitoneal injection or intravenous injection.

It is shown the new application of CZLC331 in IBD in this invention. CZLC331 has the obvious prevention and therapeutic effects for IBD through the simulate experiment of human ulcerative colitis by giving 2,4, 6-trinitrobenzene sulfonic acid (TNBS). The results are 1) model group: the feeding / drinking water of mice significantly reduced, the activities reduced and accompanied by blood in the stool. There are visible congestion, edema, bleeding and ulcers in the colon. And it is visible about the cell structural disorder, disappearance of goblet cells, lymphocytes, and neutrophils infiltration under the microscope. 2) intraperitoneal administration group: the feeding / water intake of mice return to normal, the activities increased significantly and reduce blood in the stool. The visible congestion, edema, bleeding and ulcers in the colon are significantly reduced. And the cell structural disorder, disappearance of goblet cells, lymphocytes, and neutrophils infiltration under the microscope are significantly reduced. The experimental results show that intraperitoneal injection CZLC331 may be effective against colon inflammation on intestinal damage, and has played a protective role in inflammation in mice. Thus, it is possible that CZLC331 protein was made as active drugs for IBD. The drug has the following advantages: 1) significant effect (effective rate is 100%, the cure rate is 70%), while the effective rate of clinical drug sulfasalazineis only about 90%, and the markedly effective rate was only 50%; 2) rapid onset (24 hours after administration to onset), sulfasalazine is about two weeks, and then the symptoms was improved; 3) short course (treatment is usually 5-10 days), sulfasalazine treatment is usually about 6 weeks; 4) safe (non-toxic), and patients who taking sulfasalazine alanine have elevated aminotransferase (ALT) and aspartate aminotransferase (AST), these suggesting that there is liver toxicity; 5) small side effects (no significant side effects), but there are are nausea, rash, neutropenia after taking sulfasalazine; 6) medication convenient (intraperitoneal injection once daily), and sulfasalazine is 3-4g / d, 3-4 times a day orally. In summary, the CZLC331 can solve the poor efficacy, slow onset, long course of treatment, side effects of existing treatment drugs for IBD, and it can significantly reduce the pain of patient, promote physical rehabilitation, and improve the patient's quality of life. It will be play an important role in the prevention and treatment of IBD in this invention, and it has broad application prospects.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

Brief Description of the Drawings Figure 1 The colon appearance of mice with UC after 0.5 h treating with 0.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 2 The colonic mucosa of mice with UC after 0.5 h treating with 0.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 3 The colonic mucosa HE staining of mice with UC after 0.5 h treating with 0.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 4 The colon appearance and colonic mucosa of mice with UC after 0.5 h treating with different concentrations CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 5 The colon appearance of mice with UC after different time treating with different concentrations CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modeling.

Figure 6 The colon appearance and colonic mucosa of mice with UC after different time treating with different concentrations CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 7 The colonic mucosa HE staining of mice with UC after different time treating with different concentrations CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 8 The expression of TLR 1-4 gene using RT-PCR with UC after 4 h treating with 3.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 9 The expression of TLR 6-9 gene using RT-PCR with UC after 4 h treating with 3.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 10 The colon appearance of mice with UC before modelling 0.5 h with 0.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 11 The colonic mucosa of mice with UC before modelling 0.5 h with 0.2mg/kg CZLC331 protein in trinitrobenzene sulfonic acid (TNBS) modelling.

Figure 12 Amino acid sequence of CZLC331 (SEQ ID No:3) and CZLC296 (SEQ ID No:4) with regions of interest mapped.

Detail Description of the Embodiments In order to study the new features of Salmonella flagellin and to develop as a new drug, it was found that CZLC331 may be cut TLRs expression in IBD, such as UC and Crohn's disease, and has a good preventive and therapeutic effect.

It was reported that intestinal epithelial cells can maintain intestinal immune tolerance by down-regulating the expression of TLR4 (Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Ardit i M. Decreased expression of Toll-like receptor - 4 and MD - 2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol, 2001 Aug 1;167(3):1609-16). It was unexpectedly found that Salmonella flagellin has a role in the prevention and treatment of UC, and the further evidence suggested that this effect is achieved by down-regulating the expression of TLR in colon.

Toll-like receptor (TLR) family is a receptor protein which has a homology with the Drosophila Toll protein, it belongs to the pattern recognition receptors. The main function is identifying the conservative structure of pathogenic microorganisms, improve immune system through a variety of signal transduction ways. The TLR family plays a key role in the anti- infective immunity and innate immunity, and it is also an important to acquired immune regulatory factors. The activation signal is delivered to the intracellular after TLR identifying pathogens, and activate NF- κB transcription factor. TL-1, IL-6, IL-8 are induced, upregulation of IL-12, TNF. IL-12 is a key regulator for inducting T and Β lymphocytes cells in the cell- mediated immune response. It can be used as biological adjuvants specific T cell responses against pathogens. Therefore, it can be said TLR family is the early signal for IL-12 and Thl cells involved in the immune response. When the balance between TLRs and the normal flora is broken, it will cause pathological intestinal inflammation. In particular, Salmonella flagellin has the effect for down-regulating the expression of TLR family in colon tissue, and it may be the important mechanism for its role in the treatment of UC.

The coding sequence of 1-176 and 402-505 amino acids for Salmonella flagellin were synthesis, and a Salmonella flagellin amino acids what the total length is 331 is prepared by the prokaryotic expression, and named as CZLC331. It has been proved that it has a good prevention and treatment for IBD, such as UC and Crohn's disease. It will provide important reference for the prevention and treatment of IBD.

The detailed embodiments and specific procedure are given as follows, but the scope of protection of this invention is not limited to the following embodiments. The methods used in this invention are conventional methods.

Example 1 The preparation of Salmonella flagellin protein derivative, CZLC331 1. The construction of prokaryotic expression vector pET28b-Tat-CZLC331 for expressing CZLC331 a) The coding gene for the N- and C- terminal conserved domains of Salmonella flagellin, separated by a linker, was synthesised and the nucleotide sequence is shown in SEQ ID No:1. The length of the nucleotide sequence is 891bp, and it was synthesized by Beijing Bo Mai De Science and Technology Development Co., Ltd.

The gene was tested using 2% agarose gel electrophoresis, and the result showed that the target gene, 891bp, was confirmed to match the expected results.

The amino acid for the protein encoded by SEQ ID No:1 is shown in SEQ ID No:4, and called CZLC296. The mapped regions of interest of this protein can be seen in Figure 12. The 891bp coding gene for the CZL296 protein was cloned into a cloning vector. b) The coding gene of the CZLC296 protein, SEQ ID No:1, was amplified using conventional PCR for insertion into a cloning vector, pGH.

The reaction system is 50 μl amplified coding gene: plasmid template 0.5 μl (the cloning vector pGH). The CZLC296 coding gene was inserted into the SmaI restriction sites of the cloning vector pGH. The resulting vector was named pGH-CZLC296.

The PCR conditions used to amplify the CZLC296 coding gene were: 10 × dNTP 5 μl, 10 × Ex Taq buffer 5 μl, the upstream and downstream primers 0.5 μl, and the upstream primer sequence is 5'-CGCGGGATCCATGGCTCAAGTTATCA-3', reverse primer sequence is 5'- CCGCTCGAGTCTCAACAAAGACAAGTT-3'. Ex Taq enzyme 0.25 μl, ddH O 38.25 μl. The reaction condition of PCR is 95 C 4 min, 95 °C 45 sec, 56 °C 30 sec, 72 °C 45 sec, total 30 cycles, and then 72 °C 7 min. The PCR product was subjected to 1% agarose gel electrophoresis after completing reaction, and the results show that the amplified DNA fragments what is 891bp is consistent with the expected results, and then the target fragment was recovered and purified. c) The digestion of the target gene encoding CZLC296 and pET28b-TAT vector using restriction endonuclease.

The CZLC296 coding gene and pET28b-TAT vector were digested using restrictionendonuclease BamH I and Xho I. The construction of pET28b-TAT vector is synthesizing the TAT sequence for having the upstream of NcoI and the downstream of NdeI, then digested the TAT sequence and pET28b using restriction endonuclease. The recovered fragment was recycled by T DNA ligase and restriction digestion. The sequence was confirmed by Invitrogen Co., Ltd. And then the digested production was transformed into competent cells E. coli DH5 α using T DNA ligase at 16 °C. d) Identification The transformed E. coli DH5 α cultures were grown and the sequences of clones were confirmed by digestion of restriction enzyme and sequencing. The sequencing results showed that the obtained sequence and the insertion position of the CZLC296 coding gene and TAT sequence were correct. The recombinant expression vector comprising the coding sequence of Tat protein transduction peptide and the coding gene for the CZLC296 protein was named pET28b-Tat-CZLC331. The protein corresponding to the CZLC296 protein with an N-terminal Tat protein transduction peptide was named CZLC331. The nucleotide sequence encoding CZLC331 is shown in SEQ ID No:2 and the amino acid sequence of CZLC331 is SEQ ID No:3. The amino acid sequence of CZLC331 with the regions of interest mapped is shown in Figure 12. 2. Transformation and bacterial recovery The prokaryotic expression vector pET28b-Tat-CZLC331 what was constructed correctly was transformed into E. coli BL21 (DE3), and then coating to the LB plate what contain the Kana 100 μg/ml. Then the clone was inoculated into 5mL LB containing Kana 100 μg/mL, the bacteria was shake at 220 rpm for 16 h to be fully recovered bacteria. 3. The inducible expression of prokaryotic expression vector pET28b-Tat-CZLC331.

The bacteria was recovered, and it was diluted to OD =0.8. Then the bacteria (5 ml) were inoculated into 150 ml LB containing Kana (final concentration is 100 μg / ml). The bacteria were shaking at 37 °C approximately 4-5 hours at 220 rpm. The inducing agent IPTG (final concentration 1mM) was added rapidly to the bacteria when the OD is 0.6-1.0, and it was induced and expressed at 30 °C, 220rpm by 8 hours. 4. The preparation of CZLC331 protein The induced and expressed bacteria were collected at 12000 rpm for 10 minutes at 4 °C.

The bacteria were broke by ultrasound using 20 mM sodium phosphate buffer, and was prepared a CZLC331 protein, and then CZLC331 protein was to be separated and purified. 5. The separation and purification of CZLC331 protein.

The unpurified CZLC331 was directly loaded onto the HisTrap HP 5ml column (purchased from GE Corporation), and then the peak baseline was washed using 4-5 column volumes of the above buffer. Finally, the protein was washed using elution buffer (20mM Na PO + 0.5M NaCl + 0.5M imidazole, pH7.4), and then CZLC331 was obtained, which purity is more than 95%.

Example 2 The effect of CZLC331 for treatment of IBD Experimental Animal: Male BALB/c mice (7–8 weeks old, 25±5 g) were obtained from Experimental Animal Center of Academy of Military Medical Sciences (Beijing, China). Mice were provided sterile food and water, and 12 h light/12 h dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee at Academy of Military Medical Sciences.

Reagents: 2,4,6-trinitrobenzene sulfonic acid (TNBS, were purchased from Sigma Chemical Co.), CZLC331 (prepared in Example 1), RT PCR kit (ID isFSK100, purchased from Toyobo Biotechnology Co., Ltd.).

Model preparation: Mice (hunger for 36 h) were anesthetized with isoflurane using anesthesia machine. To induce IBD, 150 mg/kg of TNBS (Sigma-Aldrich) in 38% ethanol (to break the intestinal epithelial barrier) was administered by the mouse gavage needle equipped with a 1 ml syringe, what the diameter of silicone tube is 2. 0 mm and the length is about 10 cm, the gavage needle was advanced into the rectum until the tip was 4 cm proximal to the anal verge. The total injection volume was 100 μl.

Experimental animal groups: model group, treatment group and control group (n = 8).

The control mice received 38% ethanol alone by using the same method described above, and normal feeding two days. The treatment group: BALB/c mice were treated therapeutically with CBLB502 (3.2 mg/kg) at 0.5 h after TNBS administration. At the same time, BALB/c mice were treated therapeutically with CBLB502 (0.2-6.4 mg/kg) from 0.5 h to 8 h after TNBS administration, and normal feeding two days. The mice of model group were normal feeding two days after TNBS administration.

Mice were monitored for the appearance of diarrhoea, changes of body weight, and overall mortality. At the end of the experiment after TNBS administration two days, surviving mice were killed, blood samples were collected by cardiac puncture, and a 7-cm segment of the colon was excised for macroscopic (Figure 1, Figure 4 left, and Figure 5) and microscopic damage evaluation (Figure 2, Figure 4 right, and Figure 6). And the end of the colon after washing using physiological saline was kept at -80 °C frozen for follow-up testing. 1. The results shown that feeding / water and activity were significantly reduced, and there were blood in the stool in model mice. There were significant congestion, edema, haemorrhage, and ulceration in modelling. The colon appearance and colonic mucosa of mice with ulcerative colitis after 0.5 h treating with 0.2mg/kg CZLC331 protein in TNBS modelling is shown in Figure 1 and Figure 2, A is the control group, B is the model group, C is treatment group. The results were structural disorder of cells, disappearance of goblet cells, lymphocytes and neutrophils infiltration under the microscope in model group. The colonic mucosa HE staining of mice with ulcerative colitis after 0.5 h treating with 0.2mg/kg CZLC331 protein in TNBS modelling is shown in Figure 3, A is the control group, B is the model group, C is treatment group. The phenomenon of blood in the stool, eat less and move less were reduced. The phenomenon of congestion, edema, haemorrhage began reduce, such as Figure 1, Figure 2, Figure 4 (The colon appearance and colonic mucosa of mice with ulcerative colitis after 0.5 h treating with different concentrations CZLC331 protein in TNBS modelling. A. 0.2mg/kg CZLC331, B. 0.4mg/kg CZLC331, C. 0.8mg/kg CZLC331, D. 1.6mg/kg CZLC331), Figure 5 (The colon appearance of mice with ulcerative colitis after different time treating with different concentrations CZLC331 protein in TNBS modelling. 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), Figure 6 (The colon appearance and colonic mucosa of mice with ulcerative colitis after different time treating with different concentrations CZLC331 protein in TNBS modelling. 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).

The structures of cells were arranged in neat, a small number of goblet cells and lymphocyte and neutrophil infiltration were disappeared (Figure 3 and Figure 7).

The food intake and water intake is normal, and there were no blood in the stool and reduction activities in control mice. The cells arranged in neat rows, no decrease in goblet cells and infiltration of lymphocytes and neutrophils. 2. Histological examination: The colonic samples were fixed in 4% buffered formalin overnight and then transferred to 70% ethanol, embedded in paraffin for sectioning, and then stained with haematoxylin and eosin (H&E) to examine the histological differences.

The colonic mucosa HE staining of mice with ulcerative colitis after 0.5 h treating with 0.2mg/kg CZLC331 protein in TNBS modelling is shown in Figure 3. The colonic mucosa HE staining of mice with ulcerative colitis after different time treating with different concentrations CZLC331 protein in TNBS modelling is shown in Figure 7 (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). The cells arranged in neat rows, no decrease in goblet cells and infiltration of lymphocytes and neutrophils means that there were well results for treating. 3. The expression of TLR family The colon samples were removed at the indicated times, washed with phosphate- buffered saline (pH 7.2), and cut in small pieces. Total RNA of colon samples what were control group, modelling group and treatment group were extracted by Total RNA kits II (Omega, Japan) according to the manufacturers’ instructions, and then reverse-transcribed by the Reverse Transcription System (Sigma, USA). Subsequently, the TLRs and β-actin were amplified by PCR using the following primers (Table 1). The reaction system of PCR was 50 μl, Ex Taq 0.25 μl, 10 × buffer 5 μl, dNTP 5 μl, template 2 μl, the upstream and downstream primers 1 μl, ddH O 35.75 μl. The reaction condition of PCR is 95 C 5 min, 95 °C 45 sec, 62 °C 45 sec, 72 °C 1 min, total 25 cycles, and then 72 °C 5 min. PCR products were identified on 2% agarose-gel electrophoresis followed by image analysis software.

The results were that the expression of TLR family has significant differences with the model group after CZLC331 treatment in Figure 8 and Figure 9. The expression of TLR2, TLR3, TLR4, TLR8 and TLR9 were significantly raised,in model group, and the expression would change after CZLC331 therapy. The expression of TLR6 and TLR7 were significantly raised, in model group too, but there were no significant differences after CZLC331 therapy.

The treatment and mechanism may also be related to reduce expression of TLR family.

And it suggested that the therapy of CZLC331 for IBD was effective through the pathway of TLR family, it can be become the drug for treating IBD.

Table 1 RT-PCR amplification with a primer sequence information Primer Primer sequence pU ：5 ′-TTGTGCCACCCAACAGTCAGCC-3 ′ TLR1 pD ：5 ′-ACCGCTCAACCCCAGGAACTGT-3 ′ pU ：5 ′-TTCCCTGCTCGTTCTCCCAGCA-3 ′ TLR2 pD ：5 ′-TAGAGCTCTTGCAGCCGAGGCA-3 ′ pU ：5 ′-AGCGTCTGTCCCCTCGCTCTTT-3 ′ TLR3 pD ：5 ′-GGCGGCCCGAAAACATCCTTCT-3 ′ pU ：5 ′-GCTTCCACAAGAGCCGGAAGGT-3 ′ TLR4 pD ：5 ′-TGGCCAGGCTATCTGTGAGCGT-3 ′ pU ：5 ′-TTCATCCACATGGTGTGCCCGC-3 ′ TLR6 pD ：5 ′-ATATGCTCTCAGCCCAGGCGCA-3 ′ pU ：5 ′-TCAGCATGTGCCCCCAACATGG-3 ′ TLR7 pD ：5 ′-CAACGGCCAGAGTTCACTGCCA-3 ′ pU ：5 ′-TGGCTGCTCTGGTTCACCACCT-3 ′ TLR8 pD ：5 ′-TGTTGGGCCACTGGAGGATGGA-3 ′ pU ：5 ′-GCCTGGTGGACTGCAATTGGCT-3 ′ TLR9 pD ：5 ′-TCACAGCGACGGCAATTCCCAC-3 ′ pU ：5 ′-GCGAGCACAGCTTCTTTGCAGC-3 ′ β-actin pD ：5 ′-AATACAGCCCGGGGAGCATCGT-3 ′ Example 3. The preventive effect of CZLC331 protein for IBD Experimental Animals: the same as in Example 2.

Reagents: the same as in Example 2.

Model preparation: the same as in Example 2.

Experimental animal groups: model group, prevention group and control group (n = 8).

The prevention group: BALB/c mice were treated therapeutically with CBLB502 (3.2 mg/kg) before TNBS administration ， and then the mice were given TNBS and normal feeding two days. The model group was normal feeding two days after TNBS administration. The control mice received 38% ethanol alone by using the same method described above, and normal feeding.

Test contents and test methods: the same as in Example 2.

The results shown that feeding / water and activity were significantly reduced, and there were blood in the stool in model mice. There were significant congestion, edema, haemorrhage, and ulceration in modelling (Figure 10, A. control group, B. modeling group, C. prevention group). The blood in the stool, eat less and move less were reduced, and the congestion, edema, hemorrhage began reduce (Figure 11, A. control group, B. modeling group, C. prevention group). These results showed that CZLC331 has protective effects for the prevention of IBD, and it can be become the drug for preventing IBD.

Example 4 The preparation of CZLC331 injection 1. Composition: the CZLC331 injection (5% by weight / volume (mg / mL) CZLC331 protein), 0.85% sodium chloride and water for injection. 2. The preparation method of CZLC331 protein injection: 1) The prokaryotic expression vector pET28b-Tat-CZLC331 what was constructed correctly was transformed into E. coli BL21 (DE3), and then coating to the LB plate what contain the Kana+ 100 μg/ml. Then the clone was inoculated into 5ml LB containing Kana+ 100 μg/ml, the bacteria was shake at 220 rpm for 16 h. 2) The bacteria was recovered, and it was diluted to OD =0.8. Then the bacteria (5 ml) were inoculated into 150 ml LB containing Kana+ (final concentration is 100 μg / ml). The bacteria were shaking at 37 °C approximately 4-5 hours at 220 rpm. The inducing agent IPTG (final concentration 1mM) was added rapidly to the bacteria when the OD is 0.6-1.0, and it was induced and expressed at 30 °C, 220rpm by 8 hours. 3) The induced and expressed bacteria were collected at 12000 rpm for 10 minutes at 4 °C. The bacteria were broke by ultrasound using 20 mM sodium phosphate buffer, and was prepared a CZLC331 protein. The unpurified CZLC331 was directly loaded onto the HisTrap HP 5ml column (purchased from GE Corporation), and then the peak baseline was washed using 4-5 column volumes of the above buffer. Finally, the protein was washed using elution buffer (20mM Na3PO4 + 0.5M NaCl + 0.5M imidazole, pH7.4), and then CZLC331 was obtained. 4) The CZLC331 protein was dissolved in injection water, and then sodium chloride was added, and uniformly stirred. The activated carbon what was treated by dry heat at 0.3% (weight / volume) was added to the solution for adsorbing 15 min, the solution was filtered to clarity and safekeeping. The filter includes three filters: a titanium filter for decarburizations, a 0.45 μm cartridge filter filtering and a 0.22 μm drum filter cleaning strainer.

Example 5. The preparation of CZLC331 protein enteric-coated tablets 1. The CZLC331 protein 100g is weight, and it is through the 100-mesh sieve. The lactose is 60 g, microcrystalline cellulose is 20g, carboxymethyl starch sodium is 20 g, povidone K 10g through a 80-mesh sieve. The main drugs and accessories were mixed in accordance to the principle of equal increments, and then other materials is added for a 30% volume ratio. Finally, the drug is granulated through the 20 mesh and gets the wet granules. 2. The CZLC331 protein was dried for 3 hours at 55 - 65 °C, and got dry pellets after 20- mesh sieve. The 50 g talcs were added to 2500 g pellets of CZLC331, and was mixed. The content of mixed protein was determined. The determined pellets were sending to a tablet machine for fitting speed and compress, and were suppressed into platode CZLC331 protein tablet what is a diameter of 1.2 cm. 3. The enteric coating was painting on platode CZLC331 tablet for about 1.0 mm. The coating material is E0BS68, the enteric coating is airtight and moisture, it has strongly coat strength and resistance to the gastric juice. The effective and safe release of drug is done through changing the pH of solution.

Efficacy Analysis: The CZLC331 protein as the active ingredient was prepared to the drug for preventing and curing IBD. It has the following characteristics: 1) High efficiency: the experimental data show that all animals less fooding and activity, and blood in the stool have improved significantly in the treatment group and the prevention group in example 2 and example 3. These results indicated that the effective rate is 100%.

The effective rate of recovered colon mucosa is 75% in treatment group. The clinical drugs sulfasalazine has the effective rate of only about 90%, and the markedly effective rate was only 50%. 2) Rapid onset: the experimental data show that all animals have effective after treating at 24 h, while the sulfasalazine take is about two weeks in example 2 and example 3. 3) The course of treatment is short (usually 5-10 days): The sulfasalazine treatment is generally about 6 weeks. The course of treatment using this drug is 1-2 days, the effect of sulfasalazine is a week or so, and the clinical onset is six weeks. Therefore, the course of treatment is 5-10 days in this invention. 4) High safe (non-toxic): The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of patients who take sulfasalazine increased, it suggested that there is liver toxicity. The CZLC331 belongs to the protein drugs, it has been used as anti-radiation drug, and it has non-toxic to the body.

) Side effects (no significant side effects): There have nausea, rash, neutropenia after taking the sulfasalazine, this is a protein drug, it has been used as anti-radiation drug, and it has no significant side effects to the body. 6) Convenient (only once daily intraperitoneal injection): The usage of sulfasalazine is 3 - 4 g / d orally, and needed divided into 3 - 4 times a day.

Industrial Applicability The invention provides a flagellin derivative of Salmonella CZLC331 in the prevention and treatment for IBD. It has high efficacy, fast onset, short course, non-toxic, small side effects, and medication convenient. The CZLC331 can solve the poor efficacy, slow onset, long course of treatment, side effects of existing treatment drugs for IBD, and it can significantly reduce the pain of patient, promote physical rehabilitation, and improve the patient's quality of life. It will be play an important role in the prevention and treatment of IBD in this invention, and it has broad application prospects.

Claims (17)

Claims

1. Use of a Salmonella flagellin protein derivative in the manufacture of a medicament for the treatment or prevention of an inflammatory bowel disease (IBD), wherein the protein derivative comprises the CLZC331 protein according to sequence ID 5 NO:3.

2. The use according to claim 1, wherein the IBD is selected from ulcerative colitis (UC) and Crohn's disease (CD).

3. The use according to claim 1 or claim 2, wherein the protein derivative is prepared using a recombinant prokaryotic expression system. 10

4. The use according to any one of claims 1 to 3, wherein the protein derivative has a purity of at least 95%.

5. The use according to any one of claims 1 to 4, wherein the medicament is in a dosage form selected from the group consisting of an injectable liquid, an oral liquid, an enema liquid, a capsule, an enteric-coated tablet, a powder and a granule. 15

6. The use according to claim 5, wherein the dosage form is selected from the group consisting of a capsule, an enteric-coated tablet, a powder and a granule, and wherein the protein derivative is present in the dosage form in an amount of 1-35 wt%.

7. The use according to claim 5, wherein the dosage form is selected from the group consisting of an injectable liquid, an oral liquid and an enema liquid, and wherein the protein 20 derivative is present in the dosage form in a concentration of 2-64 g/L.

8. The use according to any one of claims 1 to 7, wherein the medicament further comprises one or more pharmaceutically acceptable auxiliary materials selected from the group consisting of absorption accelerators, surfactants, lubricants, stabilizers, diluents, bonding agents, wetting agents, disintegrants, adsorption carriers, excipients, sweeteners, flavoring agents and 25 pigments.

9. The use according to claim 7, wherein the dosage form is an injectable liquid comprising the protein derivative at a concentration of at least 5g/100mL.

10. The use according to claim 9, wherein the injectable liquid further comprises sodium chloride at a concentration of at least 0.85g/100mL.

11. The use according to claim 6, wherein the dosage form is an enteric-coated tablet comprising the protein derivative, lactose, microcrystalline cellulose, sodium carboxymethyl starch, 5 and Povidone K in a weight ratio of 10:6:2:2:1.

12. A method for preparing a Salmonella flagellin protein derivative for the use of claim 1, said method comprising: 1) providing a nucleic acid sequence according to SEQ ID NO:1; 2) preparing a recombinant expression vector by subcloning said nucleic acid sequence into a 10 prokaryotic expression vector containing a coding sequence of a Tat transduction peptide; 3) transforming said recombinant expression vector into a host bacterium; 4) inducing expression of said protein derivative in the host bacterium; and 5) isolating and purifying said protein derivative from the host bacterium.

13. The method of claim 12, wherein the host bacterium is E. Coli. 15

14. The method of claim 12 or claim 13, wherein the prokaryotic expression vector is selected from the group consisting of pET-22b, pET-28, and pET-15 vectors.

15. The method of any one of claims 12 to 14, wherein the prokaryotic expression vector is pET28b-Tat.

16. The method of claim 15, wherein the recombinant expression vector is pET28b-Tat- 20 CZLC331.

17. The method of any one of claims 12 to 16, wherein the host bacterium is selected from the group consisting of E. coli BL2l(DE3), E. coli ER2566(DE3), E. coli BL2l(DE3) plysS, E. coli JM109, E. coli HB101 and E. coli Topl0.