Application of trichina succinic acid coenzyme A ligase beta-like protein SUCLA-beta

Abstract

The invention relates to an application of a trichina succinic acid coenzyme A ligase beta-like protein SUCLA-beta. The invention identifies a trichina recombinant protein SUCLA-beta which can be used for preventing or treating colitis. A mouse colitis model is successfully copied, it is proved that the protein can improve intestinal inflammation caused by TNBS, the disease activity indexes of mice treated by the trichina recombinant protein SUCLA-beta in a treatment group or a prevention group are reduced, intestinal injury and inflammation are relieved, the disease activity indexes are gradually reduced along with dosage increase, and the intestinal injury and inflammation are relieved. Intestinal injury and inflammatory symptoms are gradually relieved, and the trichina recombinant protein SUCLA-beta can relieve inflammatory response by adjusting the proportion of Th1 / Th2 / Th17 / Treg cells. The trichina recombinant protein SUCLA-beta has a relatively good application prospect in the aspect of preventing or treating colitis.

Description

Technical Field

[0001] The present invention belongs to the technical fields of veterinary immunology and molecular biology, and relates to the preparation and application of a parasite protein with immunosuppressive effects. Background Art

[0002] Trichinella spiralis (referred to as Trichinella for short) is a common zoonotic parasite that causes trichinellosis in humans and animals. Trichinellosis is widely distributed globally. Humans are infected by eating raw or undercooked meat products containing muscle larvae cysts of Trichinella. Patients show symptoms such as elevated body temperature, edema, abdominal pain and diarrhea, and may die from complications such as allergy, pneumonia, encephalitis or myocarditis in severe cases.

[0003] Trichinella can infect a variety of hosts such as humans, pigs, wild boars, rats, and mice. Its life cycle includes different developmental stages such as adult worms (AD), newborn larvae (NBL), and muscle larvae (ML). The host is infected by ingesting raw meat containing ML. The muscle larvae develop into adult worms in the small intestine. The male and female worms mate, and the female worm produces NBL. The NBL reaches the muscle tissue through the circulatory system and develops into ML. After Trichinella infects the host, it regulates the host immune system through various ways, and among them, inhibiting the host immune response is beneficial to the parasitism of the parasite.

[0004] Inflammatory bowel disease (IBD) is a common, chronic, inflammatory disease. There are many factors causing this disease. Generally, it is considered to be related to changes in intestinal flora, immune cell types, and the tight junction structure of vascular endothelial and colonic epithelial cells. In recent years, many scholars believe that parasite infection can protect the host from IBD. Some studies infected mice with Trichinella and induced colitis in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS), and analyzed indicators such as the body weight, DAI score, and macroscopic and microscopic damage of the colon in the mice. The results found that the inflammatory response of the colitis mice infected with Trichinella was significantly lower than that of the control group (colitis mice not infected with Trichinella), which indicates that Trichinella infection can effectively relieve colitis in mice. Although Trichinella infection can reduce the clinical symptoms of colitis, since the body cannot eliminate Trichinella after infection with Trichinella, it cannot be used clinically. Searching for the worm body protein that plays this role and using the worm body protein instead of the worm body infection has good application potential for the treatment and prevention of colitis. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of Trichinella succinyl-CoA ligase β-like protein SUCLA-β.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] Use of Trichinella succinyl-CoA ligase β-like protein SUCLA-β in the preparation of a drug for preventing and / or treating colitis, wherein the amino acid sequence of the Trichinella succinyl-CoA ligase β-like protein SUCLA-β is as shown in SEQ ID NO.4, and the nucleotide sequence is as shown in SEQ ID NO.3.

[0008] As a preference of the present invention, the Trichinella succinyl-CoA ligase β-like protein SUCLA-β is prepared by the following method: extracting RNA of Trichinella muscle larvae, reverse transcribing to synthesize the first strand of cDNA, using this cDNA as a template, performing RT-PCR amplification with the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and inserting the obtained product into the BamHⅠ and XhoI restriction enzyme cleavage sites of pET32a after verification to obtain a recombinant expression plasmid pET32a-SUCLA-β containing the SUCLA-β gene; transforming this plasmid into Escherichia coli for induced expression and separating and purifying to obtain the Trichinella succinyl-CoA ligase β-like protein SUCLA-β.

[0009] As a preference of the present invention, use of the Trichinella succinyl-CoA ligase β-like protein SUCLA-β in the preparation of a veterinary drug for preventing and / or treating colitis in animals.

[0010] As a preference of the present invention, the Trichinella succinyl-CoA ligase β-like protein SUCLA-β can effectively relieve the clinical symptoms of patients or animals with colitis, reduce pathological changes, inhibit the production of inflammatory cytokines, and stimulate the production of regulatory immune cells.

[0011] Beneficial effects:

[0012] In the present invention, a colitis model was established in mice induced by TNBS, and preventive (used before onset) and therapeutic (used after onset) experiments were carried out on the diseased mice with 3 different doses of recombinant SUCLA-β protein at high, medium, and low levels. The results showed that this recombinant protein can effectively reduce the mortality rate of mice with colitis, relieve clinical symptoms, reduce pathological changes, inhibit the production of inflammatory cytokines, stimulate the production of regulatory immune cells, and the preventive and therapeutic effects of the medium-dose protein are the best.

[0013] The Trichinella recombinant protein SUCLA-β of the present invention for use in the preparation of a drug for treating colitis also has the following advantages:

[0014] (1) Low side effects and relatively safe. Glucocorticoid drugs are generally used to treat inflammatory bowel diseases, which have obvious side effects. The present invention uses Trichinella recombinant protein SUCLA-β for prevention and treatment, with good effects. Because parasites have "learned" to "coexist" with the host during long-term evolution, the immunomodulatory effect of their worm body proteins is relatively mild and there are no obvious adverse reactions.

[0015] (2) Low dosage and low cost. Whether in the treatment group or the prevention group, compared with the TNBS group, the disease activity index of mice treated with Trichinella recombinant protein SUCLA-β decreased, and intestinal damage and inflammation were alleviated. Moreover, as the dose increased, the disease activity index gradually decreased, and the symptoms of intestinal damage and inflammation also gradually alleviated. A good effect can be achieved with a dosage of 50 μg of the recombinant protein. The recombinant protein can be obtained by fermentation of genetically engineered bacteria, with relatively low costs and good application prospects. Description of the Drawings

[0016] Figure 1 PCR amplification product of the SUCLA-β gene

[0017] M: DNA Marker DL2000; 1: PCR amplification product of the SUCLA-β gene

[0018] Figure 2 Double digestion identification of the recombinant expression plasmid pET32a-SUCLA-β

[0019] M: DNA Marker DL2000; 1: Double digestion identification of pET32a-SUCLA-β

[0020] Figure 3 Purification of the recombinant protein SUCLA-β

[0021] M: Protein standard molecular weight; 1: Supernatant expression product 2: Inclusion body expression product

[0022] M: Protein standard molecular weight; 1: Recombinant protein SUCLA-β before purification 2: Recombinant protein SUCLA-β after purification

[0023] Figure 4 Western blot analysis of the recombinant protein SUCLA-β

[0024] M: Protein standard molecular weight; 1: Western blot analysis of recombinant protein SUCLA-β and serum of rats infected with Trichinella 2: Western blot analysis of recombinant protein SUCLA-β and negative serum of rats

[0025] Figure 5 TNBS-induced mouse colitis model and prevention and treatment protocol

[0026] Figure 6 For the body weight change of mice

[0027] Figure 7 For the disease activity index

[0028] Figure 8 For the macroscopic score of colon injury

[0029] Figure 9 For histopathological changes (A) and microscopic score (B)

[0030] Figure 10 For the change of cytokine levels in colon tissue

[0031] Figure 11 For the MPO analysis in colon tissue

[0032] Figure 12 For the detection of the content of Treg cells (A) and Th17 cells (B) in murine spleen lymphocytes Detailed implementation method

[0033] Basic materials:

[0034] 1. Trichinella spiralis is an isolate from pigs in Henan, China, with the international number ISS534, and has been passaged and stored in BALB / c mice in our laboratory.

[0035] 2. Experimental animals: 7 - 8-week-old Wistar rats, purchased from the Experimental Animal Center of Yangzhou University.

[0036] 3. PCR primers:

[0037] F: 5'-CGCGGATCCATGGCAGCTGCATGGATACCACGAT-3';

[0038] R: 5'-CCGCTCGAGTTAGATAGGTAGTTCGTTCGAAACTGACATCAAGCG-3'

[0039] 4. Tool enzymes and reagents: DNA Marker, restriction endonuclease, prestained protein Marker, RPMI 1640 medium, DMEM medium, rat peripheral blood lymphocyte separation solution, FITC-dextran, Cy3-labeled goat anti-rat IgG(H+L), DAPI nuclear stain, anti-fluorescence quenching mounting medium, CellCounting Kit-8 (CCK-8) kit, semi-dry transfer system, Imject Alum, ELISA kit, Ms CD4 FITC, Ms CD25, PE Anti-MOUSE / RAT FOXP3, and Ms IL-17.

[0040] 5. Main instruments and equipment: Fluorescent quantitative PCR instrument, bench-top refrigerated centrifuge, stabilized voltage and current electrophoresis instrument, protein electrophoresis instrument, pressure steam sterilizer, ultrasonic cell disruptor, bench-top constant temperature oscillator, semi-dry transfer membrane instrument, water bath, water-jacketed constant temperature incubator.

[0041] Example 1. Preparation of Trichinella recombinant protein SUCLA-β

[0042] 1.1 Synthesis of primers

[0043] According to the SUCLA-β gene accession number (XM_003374581), SignalIP software was used to predict no signal peptide, and specific primers were designed using Primer 5.0 software respectively. The specific primers are designed as follows:

[0044] F: 5'CGC GGATCC ATGGCAGCTGCATGGATACCACGAT-3'

[0045] R: 5'CCG CTCGAG TTAGATAGGTAGTTCGTTCGAAACTGACATCAAGCG-3', where the underlines are the BamHI and XhoI restriction sites.

[0046] 1.2 Extraction of RNA from Trichinella muscle larvae

[0047] (1) Extract Trichinella muscle larvae RNA using the Trizol method: Add 1 mL of Trizol to Trichinella muscle larvae and homogenize in a glass homogenizer in ice water for 30 min;

[0048] (2) Transfer the homogenate to a 2 mL EP tube, add 200 μL of chloroform pre-cooled at 4℃, shake for 15 s, and let stand at room temperature for 2 min;

[0049] (3) At 4℃, transfer the supernatant to a new centrifuge tube again, add 200 μL of chloroform pre-cooled at 4℃, shake for 30 s and then let stand for 5 min;

[0050] (4) At 4℃, centrifuge at 12000g for 15 min, carefully transfer the upper aqueous phase to a new centrifuge tube; add cold isopropanol, invert and shake up and down for 1 min and then let stand for 10 min;

[0051] (5) At 4℃, centrifuge at 12000g for 10 min and discard the supernatant;

[0052] (6) Resuspend the precipitate with 75% ethanol prepared with 1 mL of DEPC water, centrifuge at 12000g in a 4℃ centrifuge for 10 min and discard the supernatant;

[0053] (7) Repeat step (6) once and then air-dry the centrifuge tube under the blower to promote the evaporation of residual ethanol. Dissolve the RNA in 30 μL of DEPC water.

[0054] (8) After measuring the purity and concentration of the RNA, store it at -80 °C for later use.

[0055] 1.3 Synthesis of cDNA

[0056] According to the reverse transcription kit instructions, use the RNA of Trichinella muscle larvae as a template to synthesize cDNA.

[0057] 1.4 Cloning of the SUCLA-β gene ( Figure 1 )

[0058] Using the above cDNA as a template, perform RT-PCR with the following reaction system: 2.0 μL of cDNA template, 12.5 μL of 2x Taq Master Mix, 1 μL of upstream primer F, 1 μL of downstream primer R, 8.5 μL of ddH2O. Mix well, pre-denature at 94 °C for 5 min on a PCR instrument, denature at 94 °C for 30 s, anneal at 55 °C for 30 s, extend at 72 °C for 1 min, for 35 cycles, and extend at 72 °C for 10 min.

[0059] Cut the gel with the target fragment after electrophoresis, and recover and purify the DNA according to the instructions. Double-digest the purified target fragment and the pET-32a vector with the corresponding enzymes, react in a water bath at 37 °C for 4 h. For the double-digestion system, take 25 μL of the RT-PCR product obtained above, electrophorese on a 1% agarose gel, cut the agarose gel at the position of the target band under ultraviolet light, and recover and purify the target fragment using the gel recovery kit from TaKaRa Biotechnology (Dalian) Co., Ltd. The method is referred to the instructions. Take the purified PCR product and ligate it with the vector overnight. Transform the ligation product into competent Escherichia coli DH5α, pick positive clone bacteria, extract the plasmid, and identify it by double digestion with BamHI and XhoI. 1.5 Expression of the SUCLA-β gene ( Figure 2 )

[0060] Double-digest the cloned plasmid vectors pMD19-T-SUCLA-β and pET32a with BamHI and XhoI respectively, recover the target gene and the large fragment of pET32a, ligate them in an appropriate ratio, transform the ligation product into competent Escherichia coli BL21, extract the plasmid, and identify it by double digestion with BamHI and XhoI and sequencing. The positive clone is named pET-32a-Ts-SUCLA-β.

[0061] 1.6 Purification of the expression product

[0062] 1.6.1 Expression and purification of the recombinant protein

[0063] (1) Inoculate 1:100 of the fresh bacterial solution of the correctly sequenced positive clone into 1 L of LB liquid medium containing ampicillin.

[0064] (2) Incubate in a shaker at 37 °C at 180 rpm until the logarithmic growth phase.

[0065] (3) Add IPTG with a working concentration of 1 mL.

[0066] (4) Continue the incubation, and take 1 mL of the bacterial solution at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively for phase expression analysis.

[0067] (5) After 5 h of induced expression, centrifuge at 8000 rpm at 4 °C for 15 min, discard the supernatant, and resuscitate the recombinant strains BL21(DE3)pET-32a and BL21(DE3)pET-32a-Ts-SUCLA-β. Transfer them to a new liquid LB medium containing ampicillin antibiotic, add IPTG to a final concentration of 1 mM when it reaches the logarithmic growth phase at 37 °C, and continue the incubation for 5 h. Collect the bacteria, wash them twice with PBS, and resuspend the bacteria with the supernatant Binding Buffer; after three cycles of freeze-thawing, ultrasonically disrupt them, centrifuge at 8000 r / min for 10 min, dissolve the precipitate overnight with the inclusion body binding buffer, filter, and purify the recombinant protein with a His protein purification column. The operation steps are shown in the instruction manual of the His protein purification column. After identification by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), place the recombinant protein in a dialysis bag and renature it in renaturation buffers containing different gradient concentrations of urea (6 mol·L⁻¹, 4 mol·L⁻¹, 2 mol·L⁻¹, 0 mol·L⁻¹) respectively, and finally dialyze it in potassium-free PBS. After concentration by polyethylene glycol, filter and sterilize, and aliquot and store at -80 °C. SDS-PAGE shows that the molecular weight of the recombinant protein is about 65 kDa. After subtracting the tag protein pET-32a of 18 - 20 kDa, the protein molecular weight is about 47 kDa, which is consistent with the expectation. The phase expression of the protein shows that the expression level of the recombinant protein reaches the maximum at 5 h of induction and is mainly expressed in inclusion bodies. A relatively pure fusion protein rSUCLA-β ( Figure 3 ) was obtained.

[0068] 1.6.2 Western blot analysis of the recombinant protein

[0069] (1) Perform 12% SDS-PAGE electrophoresis on the recombinant protein rSUCLA-β.

[0070] (2) Use a semi-dry transfer apparatus to transfer the protein on the gel after electrophoresis to a methanol-activated PVDF membrane.

[0071] (3) Transfer the transferred PVDF membrane to TBST and wash for 1 min, then transfer it to a blocking solution prepared with 5% BSA in TBST and incubate at 37 °C for 2 h.

[0072] (4) Add the serum of rats infected with Trichinella spiralis as the primary antibody (diluted 1:100 with the blocking solution), and the serum of healthy rats as the negative control, and incubate overnight at 4 °C.

[0073] (5) Wash the membrane three times with TBST, 5 - 10 min each time.

[0074] (6) Place the membrane into HRP goat anti-rat IgG (diluted 1:5000 with the blocking solution) respectively, and incubate at 37 °C in the dark for 2 h.

[0075] (7) Wash the membrane 3 times with TBST, 5 - 10 min each time.

[0076] (8) Use DAB for color development, observe the results and take pictures.

[0077] The results are as Figure 4 shown. There is a target band at 65 kDa, while there is no band in the negative serum. It indicates that the host immune system can recognize the recombinant protein SUCLA-β.

[0078] Example 2. Animal model establishment and prevention and treatment of colitis

[0079] Randomly divide 100 mice into 10 groups, namely PBS control group, TNBS group, treatment groups (TNBS + 250 SUCLA-β group, TNBS + 50 SUCLA-β group, TNBS + 10 SUCLA-β group, and TNBS + pET-32a group), and prevention groups (250 SUCLA-β + TNBS group, 50 SUCLA-β + TNBS group, 10 SUCLA-β + TNBS group, and pET-32a + TNBS group).

[0080] On the -1st day, the mice in the TNBS group, TNBS + 250 μg rSUCLA-β group, TNBS + 50 μg rSUCLA-β group, TNBS + 10 μg rSUCLA-β group, TNBS + pET-32a group, 250 μg rSUCLA-β + TNBS group, 50 μg rSUCLA-β + TNBS group, 10 μg rSUCLA-β + TNBS group, and pET-32a + TNBS group were fasted for 24 h. On the 0th day, the mice were anesthetized with anhydrous ether. A thin catheter with a diameter of 1 mm was inserted into the colon, and then 100 μL of TNBS solution (50 μL of 50% ethanol + 50 μL of 5% TNBS) was rapidly injected. After that, the anus of the mice was pinched, and the mice were inverted for 3 - 4 minutes. The mice were transferred to a new cage and allowed to freely access food and water. The mice in the PBS group were normally raised without any treatment.

[0081] The mice in the TNBS + 250 μg rSUCLA-β group were intraperitoneally injected with 250 μg of recombinant SUCLA-β protein on the 1st, 2nd, and 3rd days. The mice in the TNBS + 50 μg rSUCLA-β group were intraperitoneally injected with 50 μg of recombinant SUCLA-β protein on the 1st, 2nd, and 3rd days. The mice in the TNBS + 10 μg rSUCLA-β group were intraperitoneally injected with 10 μg of recombinant SUCLA-β protein on the 1st, 2nd, and 3rd days. The mice in the TNBS + pET-32a group were intraperitoneally injected with 50 μg of tag protein (pET-32a) on the 1st, 2nd, and 3rd days.

[0082] The mice in the 250 μg rSUCLA-β + TNBS group were intraperitoneally injected with 250 μg of recombinant protein on the -21st, -14th, and -7th days. The mice in the 50 μg rSUCLA-β + TNBS group were intraperitoneally injected with 50 μg of recombinant protein on the -21st, -14th, and -7th days. The mice in the 10 μg rSUCLA-β + TNBS group were intraperitoneally injected with 10 μg of recombinant protein on the -21st, -14th, and -7th days. The mice in the pET-32a + TNBS group were intraperitoneally injected with 50 μg of recombinant protein (pET-32a) on the -21st, -14th, and -7th days (the protocol is as Figure 5 )

[0083] Example 3. Detection of mouse survival rate and body weight change

[0084] Starting from the 0th day, the death situation and body weight change of the mice were recorded every day.

[0085] The mice in the PBS control group showed normal behavior and no deaths. After 24 hours of TNBS induction, the mortality rate of the mice in the TNBS group was 80%. In the treatment groups of TNBS+250SUCLA-β, TNBS+50SUCLA-β, and TNBS+10SUCLA-β, and in the prevention groups of 250SUCLA-β+TNBS, 50SUCLA-β+TNBS, and 10SUCLA-β+TNBS, the symptoms were milder and there were no deaths. The mortality rates of the mice in the TNBS+pET-32a group and the pET-32a+TNBS group were similar to those in the TNBS group, both being 80%.

[0086] In the treatment groups of TNBS+250SUCLA-β, TNBS+50SUCLA-β, and TNBS+10SUCLA-β, and in the prevention groups of 250SUCLA-β+TNBS, 50SUCLA-β+TNBS, and 10SUCLA-β+TNBS, the body weights were higher than those in the TNBS, TNBS+pET-32a, and pET-32a+TNBS groups. The results were as Figure 6 shown. The body weight began to decrease on the 1st day, reached the lowest value on the 2nd day, and then gradually increased.

[0087] Example 4. Analysis of disease activity index

[0088] After TNBS-induced inflammation, the mental state, activity, hair luster, appetite, and defecation (such as whether the feces contained blood, the type of feces, and the defecation frequency) of the mice were observed daily. The mice in each group were scored for the disease activity index (DAI) according to the international standard (Table 1), where DAI = (weight loss score + stool performance score + bloody stool score) / 3. The fecal occult blood was detected by the benzidine method.

[0089] Table 1 Disease activity index scoring criteria

[0090]

[0091] The mice in each group showed varying degrees of disheveled hair, arched backs, listlessness, soft stools or bloody stools. The symptoms were most severe on the second day after TNBS induction, with the highest DAI score, and gradually improved starting from the third day. The DAI results of each group were as Figure 7 shown. The DAI scores of the treatment groups of TNBS+250SUCLA-β, TNBS+50SUCLA-β, and TNBS+10SUCLA-β, and of the prevention groups of 250SUCLA-β+TNBS, 50SUCLA-β+TNBS, and 10SUCLA-β+TNBS were lower than those of the control groups such as TNBS, TNBS+pET-32a, and pET-32a+TNBS.

[0092] Example 5. Evaluation of Colonic Injury

[0093] Macroscopic evaluation: Mice were sacrificed on the 7th day. The abdominal cavity was opened to find the cecum, and the colon was located 7 - 8 cm posterior to the ileocecal orifice. The colon was removed, the intestinal wall was cut open, and the colon was observed and scored macroscopically with the scoring criteria shown in Table 2.

[0094] Table 2 Macroscopic Lesion Evaluation of Colon

[0095]

[0096]

[0097] The results were as Figure 8 shown. In the TNBS group, there were slight bleeding points in the colon of mice. Compared with the PBS group, the intestinal wall was thickened with ulcers or congestion. In the groups treated and prevented with SUCLA-β, the intestinal wall was basically normal with only very few bleeding points. Comparing the colon lengths of each group, it was found that the colon in the TNBS group was the shortest, the PBS group was the longest, and the colon lengths in the SUCLA-β treatment group or prevention group were longer than that in the TNBS group.

[0098] Microscopic evaluation: Mouse colon was selected, the contents in the intestinal lumen were rinsed with PBS, the colon was fixed with 4% tissue fixative, and HE sections of the colon tissue were prepared. Observation and scoring were performed under a microscope with the scoring criteria shown in Table 3.

[0099] Table 3 Microscopic Evaluation of Colonic Injury

[0100]

[0101] Observation of HE sections of colon tissue under a microscope, the results were as Figure 9 shown. In the PBS group, TNBS + 50SUCLA-β group, and 50SUCLA-β + TNBS group, the colon tissue structure was intact, the mucosal epithelium showed no necrosis, exfoliation, or loss, the glandular structure was normal, and there were no pathological changes such as hyperemia, edema, or inflammatory cell infiltration in the stroma; in the TNBS group and the two pET-32a control groups (TNBS + pET-32a and pET-32a + TNBS), there was slight or mild focal exfoliation of the mucosal layer epithelium accompanied by inflammatory cell infiltration and disappearance of the glandular structure in the mouse colon; in the TNBS + 10SUCLA-β group, 10SUCLA-β + TNBS group, and TNBS + 250SUCLA-β group, there was slight focal exfoliation of the mucosal epithelium and inflammatory cell infiltration in the mouse colon; in the 250SUCLA-β + TNBS group, there was slight inflammatory cell infiltration on the surface of the colon mucosa.

[0102] Example 6. Detection of Changes in Cytokines in Mouse Colon Tissue

[0103] Take a small section of colon, weigh it, add PBS at a weight ratio of 1:1, grind it thoroughly, centrifuge at 13,000 rpm for 10 min, take the supernatant, and according to the instructions of the mouse ELISA detection kit, detect the contents of IFN-γ, IL-4, IL10, IL-17 and IL-9 in the colon tissue, and count the changes in the contents of different cytokines.

[0104] Determine the cytokines IFN-γ, IL-4, IL-10, IL-6, IL-9 and IL-17 in the mouse colon tissue. The ELISA results are as Figure 10 shown. The IFN-γ, IL-4 and IL-10 in the colon tissue of the TNBS group mice were significantly lower than those of the PBS control group, while the IL-6, IL-9 and IL-17 were significantly higher than those of the PBS control group.

[0105] In the treatment group, the IFN-γ in the colon tissue of the TNBS + 250SUCLA-β group mice was significantly higher than that of the TNBS group and the TNBS + pET-32a group; the IL-4 in the colon tissue of the TNBS + 250SUCLA-β group and the TNBS + 10SUCLA-β group mice was significantly higher than that of the TNBS group and the TNBS + pET-32a group; the IL-10 concentration in the colon tissue of the TNBS + 50SUCLA-β group mice was higher than that of the TNBS group and significantly higher than that of the TNBS + pET-32a group; the IL-6 levels in the colon tissue of the TNBS + 50SUCLA-β group and the TNBS + 10SUCLA-β group mice were significantly lower than those of the TNBS group and the TNBS + pET-32a group; the IL-9 in the colon tissue of the TNBS + 250SUCLA-β group and the TNBS + 50SUCLA-β group mice was significantly lower than that of the TNBS group and the TNBS + pET-32a group, and the TNBS + 10SUCLA-β group was significantly higher than that of the TNBS group and the TNBS + pET-32a group; the IL-17 levels in the colon tissue of the TNBS + 50SUCLA-β group / TNBS + 50SUCLA-β group and the TNBS + 10SUCLA-β group mice were lower than those of the TNBS group and the TNBS + pET-32a group.

[0106] In the prevention group, intraperitoneal injection of different doses of SUCLA-β before TNBS induction did not cause changes in the level of IFN-γ in the colon tissue of mice; the IL-4 levels in the colon tissue of mice in the 250SUCLA-β+TNBS group and the 10SUCLA-β+TNBS group were significantly higher than those in the TNBS group and the pET-32a+TNBS group, and there was no significant difference between the 50SUCLA-β+TNBS group and the TNBS group; the IL-10 levels in the colon tissue of mice in the 50SUCLA-β+TNBS group were significantly higher than those in the TNBS group and the pET-32a+TNBS group, while the IL-10 levels in the colon tissue of mice in the 250SUCLA-β+TNBS group and the 10SUCLA-β+TNBS group were significantly lower than those in the TNBS group and the pET-32a+TNBS group. BS group; the IL-6 in the colon tissue of mice in the 250SUCLA-β+TNBS group and the 10SUCLA-β+TNBS group was significantly lower than that in the TNBS group and the pET-32a+TNBS group, and there was no significant difference between the 50SUCLA-β+TNBS group and the TNBS group; the IL-9 in the colon tissue of mice in the 250SUCLA-β+TNBS group, the 50SUCLA-β+TNBS group and the 10SUCLA-β+TNBS group was significantly lower than that in the TNBS group and the pET-32a+TNBS group; the IL-17 in the colon tissue of mice in the 250SUCLA-β+TNBS group, the 50SUCLA-β+TNBS group and the 10SUCLA-β+TNBS group was significantly lower than that in the TNBS group and the pET-32a+TNBS group.

[0107] Example 7. Detection of myeloperoxidase (MPO) levels in mouse colon tissue

[0108] A small section of colon was taken out, weighed and added with PBS at a ratio of 1:1. After grinding thoroughly, it was centrifuged at 13000 rpm for 10 min, and the supernatant was taken out. The MPO content in the colon tissue was detected according to the instructions of the mouse MPO detection kit.

[0109] The MPO level in colon tissue was measured. Figure 11 As shown, compared with the PBS control group, the content of MPO in the colon of the TNBS group was significantly increased and significantly higher than that of the other groups.

[0110] In the treatment model, the MPO levels in the colon tissues of mice in the TNBS+250SUCLA-β, TNBS+50SUCLA-β and TNBS+10SUCLA-β groups were significantly lower than those in the TNBS group, and the MPO levels in the colon tissues of mice in the TNBS+250SUCLA-β and TNBS+10SUCLA-β groups were significantly different from those in the TNBS+pET-32a groups.

[0111] In the prevention model, the MPO levels in the colon tissues of mice in each group treated with SUCLA-β were significantly lower than those in the TNBS group, and there were significant differences between each group and the pET-32a + TNBS group.

[0112] Example 8. Detection of the Contents of Treg Cells and Th17 Cells in Mouse Spleen Lymphocytes

[0113] Collection of mouse spleen lymphocytes:

[0114] (1) After collecting blood from the eyes of 5 mice until they died, place them in 75% ethanol;

[0115] (2) Take the mice soaked in 75% ethanol into the laminar flow hood, open the abdomen and take out the spleen;

[0116] (3) Isolate spleen lymphocytes according to the instructions of the spleen lymphocyte separation solution, and dilute them with cell culture medium to a cell suspension of 1×10 6 -mL 1 ;

[0117] Detection of the Contents of Treg Cells and Th17 Cells in Mouse Spleen Lymphocytes:

[0118] Stain the spleen lymphocytes separated above in the following manner:

[0119] (1) Activate lymphocytes: Quickly thaw Leukocyte Activation Cocktail in a 37°C water bath, add 2 μL of the cocktail to each 1 mL of cell suspension and mix well. Place the mixture in a 37°C CO2 incubator and culture for 4 - 6 hours. After activation, wash the cells with FACS staining buffer. Finally, resuspend the cells with 100 μL of FACS staining buffer to obtain a single-cell suspension;

[0120] (2) Surface staining: Add 1 μL of Ms CD4 FITC and 1 μL of Ms CD25 PE to the single-cell suspension in (1), incubate at 2 - 4°C for 30 - 60 min, and then add 2 mL of FACS staining buffer to wash the cells.

[0121] (3) Intracellular cytokine staining: Aspirate the excess FACS staining buffer, add 1 mL of freshly prepared 1×Fix / Ferm Buffer to permeabilize and fix the cells, mix well, and incubate at 2 - 8°C in the dark for 1 h; add 1 mL of 1×Ferm / Wash Buffer, centrifuge at 2 - 8°C and 350 g for 6 min; wash the cells with 2 mL of 1×Ferm / Wash Buffer again, and resuspend the cells with 100 μL of 1×Ferm / Wash Buffer; add 1 μL of Anti-MOUSE / RAT FOXP3 and 1 μL of Ms II-17 to the cells, mix well, and incubate at 2 - 4°C in the dark for 1 h; wash twice with 2 mL of 1×Ferm / Wash Buffer; finally, resuspend the cells with 350 μL of stainbuffer;

[0122] (4) Detect and analyze the data by flow cytometry.

[0123] Detect the contents of Treg cells and Th17 cells in mouse spleen lymphocytes, and the results are as Figure 12 shown: Compared with the PBS control group, the Treg cells in the TNBS group decreased significantly, while the Th17 cells increased significantly. In the treatment group, the Treg cells in the mouse spleen lymphocytes treated with different doses of SUCLA-β were significantly higher than those in the TNBS group. As the injection dose increased, the Treg cells also gradually increased, showing a significant difference from the TNBS + pET-32a group; the Th17 cells in the mouse spleen lymphocytes treated with different doses of SUCLA-β were significantly lower than those in the TNBS group. As the injection dose of SUCLA-β increased, the Th17 cells also gradually decreased, showing a significant difference from the TNBS + pET-32a group. In the prevention group, there was no significant difference compared with the PBS group; in the prevention model, the Treg cells in the 250SUCLA-β + TNBS group, 50SUCLA-β + TNBS group, and 10SUCLA-β + TNBS group were significantly higher than those in the TNBS group, and the Th17 cells in each group were significantly lower than those in the TNBS group, showing a dose-dependent relationship and a significant difference from the pET-32a group.

Claims

1. Use of Trichinella succinyl-CoA ligase β-like protein SUCLA-β in the preparation of a drug for preventing and / or treating colitis, wherein the amino acid sequence of the Trichinella succinyl-CoA ligase β-like protein SUCLA-β is as shown in SEQ ID NO.

4.

2. The application according to claim 1, wherein The Trichinella succinyl-CoA ligase β-like protein SUCLA-β is prepared by the following method: extracting RNA from Trichinella muscle larvae, reverse-transcribing to synthesize the first strand of cDNA, using this cDNA as a template, performing RT-PCR amplification with the primers shown in SEQ ID NO.1 and SEQ ID NO.2, inserting the obtained product into the BamHⅠ and XhoI restriction enzyme cleavage sites of pET32a after verification to obtain a recombinant expression plasmid pET32a-SUCLA-β containing the SUCLA-β gene; transforming this plasmid into Escherichia coli for induced expression and separating and purifying to obtain the Trichinella succinyl-CoA ligase β-like protein SUCLA-β.

3. The application according to claim 1 or 2, characterized in that, Use of the Trichinella succinyl-CoA ligase β-like protein SUCLA-β in the preparation of a veterinary drug for preventing and / or treating colitis in animals.

4. The application according to claim 1 or 2, characterized in that, The Trichinella succinyl-CoA ligase β-like protein SUCLA-β can effectively relieve the clinical symptoms of colitis patients or animals, reduce pathological changes, inhibit the production of inflammatory cytokines, and stimulate the production of regulatory immune cells.

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