Preparation and application of recombinant protein for treating inflammatory bowel disease

Abstract

The present application relates to a kind of preparation and application of recombinant protein for inflammatory bowel disease treatment, and the gene of the recombinant protein is derived from intestinal bacteria Akkermansia muciniphila, and the name is Amuc_1434.The present application research shows that the recombinant protein has good therapeutic effect on inflammatory bowel disease.We successfully cloned Amuc_1434 gene into e. coli expression vector, then after induction expression purification, the recombinant protein Amuc_1434 is obtained.Experiments show that the recombinant protein Amuc_1434 provided by the present application can significantly alleviate the symptoms of UC mice, slow down the shortening of colon, reduce the level of inflammatory cytokines, reduce the body oxidative stress, reduce the degree of intestinal tissue lesions.In addition, the recombinant protein Amuc_1434 also shows the protective effect on kidney, and the effect is better than the common colitis treatment drug mesalazine on the market.The present application provides scientific basis for the application of recombinant protein Amuc_1434 in inflammatory bowel disease treatment, and lays a foundation for further development of new recombinant protein drug for inflammatory bowel disease treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation and application of a recombinant protein for the treatment of inflammatory bowel disease, belonging to the field of biological medicine. BACKGROUND

[0002] Akkermansia muciniphila (AKK) is a potential probiotic bacteria, accounting for 1-5% of the fecal microbiota of healthy adults. It can adapt well to the mucus layer and plays a key role in maintaining the balance of the gastrointestinal tract and the integrity of the intestinal barrier. Although AKK has shown the potential to become a new generation of probiotics and has been proven to have a positive effect on the treatment of colorectal cancer, it still has many limitations in the process of actual clinical application. Compared with AKK, the protein encoded by AKK is single in composition, and its function and safety are more easily determined. Studies have shown that the abundance of Akkermansia muciniphila in humans and mice is related to a variety of diseases, and this regulation of disease may be related to the proteins encoded by itself. The genome of Akkermansia muciniphila ATCC BAA-835 has 2176 predicted protein-coding genes, and after sequence alignment and analysis, we targeted a hypothetical protein-coding gene Amuc_1434, which was then successfully cloned into an E. coli expression vector. After induction, expression and purification, the recombinant protein Amuc_1434 was obtained.

[0003] Inflammatory bowel disease (IBD) is a kind of recurrent non-specific intestinal inflammatory disease, mainly including Crohn's disease (CD) and ulcerative colitis (UC). Crohn's disease is a proliferative lesion that penetrates through all layers of the intestinal wall, can invade the mesentery and local lymph nodes, and the lesion is limited to the small intestine (mainly the terminal ileum) and the colon, and both can be involved. The lesion of this disease is segmental, separated from the normal intestinal segment, with clear boundaries, showing the characteristics of skip area. The pathological changes are divided into acute inflammation period, ulcer formation period, stenosis period and fistula formation period (perforation period). The acute period is mainly characterized by edema and inflammation of the intestinal wall; in the chronic period, the intestinal wall thickens and hardens, and the affected intestinal canal appears tubular, with the upper end of the intestinal canal dilated. The typical lesions of the mucosa are: ulcers, pebble-like nodules, granulomas, fistulas and abscess tubes. Ulcerative colitis is the most common type of IBD, and the lesion site is mainly in the rectum and colon, mainly in the colonic mucosa and submucosa, forming inflammatory lesions, and the clinical manifestations are intermittent diarrhea and hematochezia. This disease was commonly seen in developed western countries in the past, and in recent years the incidence rate in Asian regions has also shown an upward trend. However, the fundamental cause and pathogenesis are still unclear, and it is currently considered to be the result of the combined action of multiple factors such as genetics, immunity, intestinal flora and environment. In the development process of UC, excessive immune response can lead to tissue damage, intestinal flora imbalance and metabolic disorder. The commonly used treatment drugs at present include anti-inflammatory drugs, aminosalicylates, antidiarrheal drugs, corticosteroids, antibiotics, immunomodulators, etc., but these drugs will also produce serious side effects while exerting their effects, and long-term use will lead to decreased immunity of the body, making it more susceptible to diseases and having adverse effects on health. Therefore, it is necessary to find a new treatment strategy for colitis that is safe and effective and has no side effects. In view of this, the purpose of the present application is to provide a method for treating inflammatory bowel disease by using a recombinant protein Amuc_1434 derived from Akkermansia muciniphila. Experiments show that Amuc_1434 has good therapeutic effect on intestinal inflammation and inflammatory lesions, and has great application prospect in the treatment of inflammatory bowel disease. SUMMARY

[0004] The present application relates to the preparation and application of a recombinant protein for the treatment of inflammatory bowel disease.

[0005] The technical scheme of the present application is as follows:

[0006] A recombinant protein Amuc_1434 for treating inflammatory bowel disease, the amino acid sequence and gene sequence of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0007] The preparation method of the recombinant protein Amuc_1434 for treating inflammatory bowel disease is as follows: the whole gene synthesized recombinant plasmid pET25b(+)-Amuc_1434 contains a His tag, and the enzyme cutting sites are BamHI and XhoI. After being identified by sequencing, the recombinant plasmid is transformed into an Escherichia coli expression strain. After the bacteria are cultured, the recombinant protein is induced by IPTG. After the induction is completed, the bacterial solution is centrifuged by using a large-capacity refrigerated centrifuge, the supernatant is discarded, and then the bacterial body precipitate is resuspended by using PBS, and is broken by ultrasonic at low temperature. The supernatant protein solution is collected after centrifugation again. The protein is purified by Ni 2+ -NTA affinity chromatography. The His tag in the protein is combined with the Ni 2+ -NTA affinity chromatography. The His tag in the protein is combined with the Ni

[0008] The recombinant protein Amuc_1434 obtained above is injected into a mouse with inflammatory bowel disease to observe the therapeutic effect of Amuc_1434 on inflammatory bowel disease.

[0009] The mouse with an inflammatory bowel disease model is injected with a sterile PBS solution containing Amuc_1434 every day.

[0010] The recombinant protein Amuc_1434 provided by the application is derived from a human intestinal bacterium Akkermansia muciniphila. Akkermansia muciniphila is a potential probiotic bacterium that can well adapt to the mucus layer and plays a key role in maintaining the balance of the gastrointestinal tract and the integrity of the intestinal barrier. Many studies have shown that the bacterium plays a beneficial role in the treatment of various diseases, which may be related to the proteins encoded by itself. The recombinant protein Amuc_1434, like mesalazine, can alleviate the weight loss, colon shortening and oxidative stress level of a DSS-induced inflammatory bowel disease mouse, reduce the disease activity index (DAI) and the content of pro-inflammatory cytokines, regulate the composition of intestinal flora, increase the content of beneficial bacteria, and thus effectively treat DSS-induced inflammatory bowel disease in mice.

[0011] The recombinant protein Amuc_1434 used in the application is found to have no toxic side effects on the organs of mice through organ toxicity detection, and can reduce the damage to the colon tissue, and is a safe and efficient product for treating inflammatory bowel disease.

[0012] The recombinant protein Amuc_1434 provided by the application can be used for treating inflammatory bowel disease, and has great application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1Effect of Amuc_1434 on body weight of mice with inflammatory bowel disease.

[0014] Figure 2 Effect of Amuc_1434 on DAI index of mice with inflammatory bowel disease.

[0015] Figure 3 Effect of Amuc_1434 on colon length of mice with inflammatory bowel disease.

[0016] Figure 4 Effect of Amuc_1434 on inflammatory factors in serum and colon of mice with inflammatory bowel disease.

[0017] Figure 5 Effect of Amuc_1434 on colon histopathology of mice.

[0018] Figure 6 Effect of Amuc_1434 on kidney histopathology of mice.

[0019] Figures 1-5 In the following Examples, data are expressed as mean ± SD, # P<0.05, ## P<0.01 and ### P<0.001,versus CON group.*P<0.05,**P<0.01 and***P<0.001,versus DSS group. DETAILED DESCRIPTION

[0020] The mice involved in the following Examples are 6-8 week old male SPF C57BL / 6J mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The ELISA kit involved in the following Examples is purchased from Quanzhou Ruixin Biological Technology Co., Ltd. The fecal occult blood detection kit involved in the following Examples is purchased from Ebio Biopharm (Hangzhou) Co., Ltd. The mesalazine enteric-coated tablets (Salofalk) involved in the following Examples is purchased from Dr. Falk Pharma GmbH. The dextran sulfate sodium (DSS) involved in the following Examples is purchased from Shanghai Yisen Biotechnology Co., Ltd. The H&E staining kit involved in the following Examples is purchased from Quanzhou Ruixin Biological Technology Co., Ltd.

[0021] The application will be further described below in conjunction with the accompanying drawings.

[0022] Example 1: Preparation of Amuc_1434

[0023] The recombinant plasmid pET25b(+)-Amuc_1434 synthesized by full gene synthesis contains a His tag and enzyme digestion sites of BamHI and Xhol. After sequencing and identification, the recombinant plasmid is transformed into an Escherichia coli expression strain. After the bacteria are cultured, the recombinant protein is induced by IPTG. After induction, the bacterial solution is centrifuged by a large-capacity refrigerated centrifuge, and the supernatant is discarded. The bacterial body precipitate is resuspended in PBS, and is broken by ultrasonic crushing at low temperature. The supernatant protein solution is collected after centrifugation again. The protein is purified by Ni 2+ -NTA affinity chromatography. The His tag in the protein is combined with the protein to hang on the chromatography column, and then the target protein is eluted by using an elution buffer. The whole purification process needs to be carried out on ice, and the solutions involved need to be precooled. Then the buffer is replaced to PBS by ultrafiltration, and the protein is concentrated. The elution buffer is imidazole. 2+ The His tag in the protein is combined with the protein to hang on the chromatography column, and then the target protein is eluted by using an elution buffer. The whole purification process needs to be carried out on ice, and the solutions involved need to be precooled. Then the buffer is replaced to PBS by ultrafiltration, and the protein is concentrated. The elution buffer is imidazole.

[0024] Example 2: Animal experiment design

[0025] C57BL / 6J mice, male, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 18-20 g. After the mice are adaptively fed for one week, they are randomly divided into four groups, five in each group, namely a blank group (Con), a model group (Dss), a positive drug group (AC) and an Amuc_1434 treatment group (Amuc_1434). Except for the blank group, the mice freely drink sterile water containing 3% DSS (36-50 kDa) for 8 days to establish an inflammatory bowel disease mouse model, and the treatment group is given enema administration every day during the treatment.

[0026] Blank group: free diet and water for 1-7 days; injection of sterile normal saline every day for 8-15 days.

[0027] Model group: free diet and water for 1-7 days; 3% DSS free water for 8-15 days, while sterile normal saline is injected every day.

[0028] Positive drug group: free diet and water for 1-7 days; 3% DSS free water for 8-15 days, while mesalazine is given by gavage every day.

[0029] Amuc_1434 treatment group: free diet and water for 1-7 days; 3% DSS free water for 8-15 days, while sterile PBS solution containing Amuc_1434 is injected every day.

[0030] Table 1 Animal experiment design

[0031]

[0032] On the 16th day of the experiment, the blood of the mice was collected under anesthesia with a sterile tube, and the blood was allowed to clot naturally at room temperature for 30 minutes. The serum of the mice was collected by centrifugation at 3000 rpm for 20 minutes at 2-8°C and stored at -80°C for later use. After the mice were euthanized with CO2, the heart, liver, kidney, pancreas, spleen, and part of the colon tissue were quickly removed, weighed, and fixed with 4% paraformaldehyde. Part of the colon tissue was washed with PBS solution and stored frozen. The length of the mouse cecum from the front end to the end of the rectum in a naturally stretched state was measured with a vernier caliper and recorded.

[0033] Example 3: Effect of Amuc_1434 on DSS-induced inflammatory bowel disease in mice

[0034] In the experiment, the body weight, stool shape, and blood in the stool of the mice were recorded, and the hair, mental state, food intake, and activity of the mice were observed. The disease activity index (DAI) score standard of the inflammatory bowel disease model mice is shown in Table 1. DAI = (weight loss + stool shape + occult blood) / 3.

[0035] Table 2 Disease activity index (DAI) score table

[0036]

[0037] As shown in Table 3, the DAI index of the mice with intestinal inflammation was significantly increased. On the eighth day, the DAI index of the negative control DSS group reached 3.79. Amuc_1434 and the positive drug had a significant effect on reducing the DAI index, which were 3.13 and 3.07, respectively. Figure 1 As shown in Table 3, the DAI index of the mice with intestinal inflammation was significantly increased. On the eighth day, the DAI index of the negative control DSS group reached 3.79. Amuc_1434 and the positive drug had a significant effect on reducing the DAI index, which were 3.13 and 3.07, respectively.

[0038] Figure 2 As shown in Table 3, the DAI index of the mice with intestinal inflammation was significantly increased. On the eighth day, the DAI index of the negative control DSS group reached 3.79. Amuc_1434 and the positive drug had a significant effect on reducing the DAI index, which were 3.13 and 3.07, respectively.

[0039] As shown in Table 4, except for the blank group, drinking 3% DSS caused the colon of the mice to be significantly shortened. The average colon length of the mice in the blank group was 8.53 cm, and that in the DSS group was 4.95 cm. Each treatment group significantly reduced the shortening of the colon of the mice with intestinal inflammation. Except for the positive drug, the best treatment effect was Amuc_1434 (6.43 cm). Figure 3 Example 5: Determination of organ index

[0040]

[0041] ​​The heart, spleen, liver, and kidneys were separated, rinsed with physiological saline, and dried with filter paper before being weighed quickly. Organ index (%) = the ratio of an organ's weight to the body weight. An increased organ index indicates congestion, edema, or hyperplasia / hypertrophy in the organ; a decreased organ index indicates organ atrophy and other degenerative changes.

[0042] As shown in Table 3, except for the control group, drinking 3% DSS induced significant liver enlargement in mice, while Amuc_1434 could alleviate the liver enlargement. There were no significant changes in the heart, spleen, and kidney indices in any group, indicating that the treatment had no toxic side effects on the mouse organs.

[0043] Table 3 Organ Index

[0044]

[0045] Example 4: Effects of Amuc_1434 on inflammatory factors in mice with DSS-induced inflammatory bowel disease

[0046] The levels of pro-inflammatory factors IL-8 and TNF-α in serum and colon homogenate were detected using an ELISA kit. The specific operating procedures were performed according to the instructions.

[0047] like Figure 4 As shown, the levels of inflammatory factors IL-8 and TNF-α in the colon homogenate and serum of mice in the DSS group were significantly higher than those in normal mice (P < 0.05). Compared with the model group, administration of Amuc_1434 significantly reduced the levels of inflammatory factors (P < 0.05).

[0048] Example 4: Effects of Amuc_1434 on the histopathology of colon and kidney tissues in mice with DSS-induced inflammatory bowel disease

[0049] The effects of paraffin-embedded tissue sections and H&E staining on the structure of mouse kidney and colon tissues were observed, and the results are as follows: Figure 5 and Figure 6 As shown.

[0050] Under a 100x microscope, the colonic tissue structure in the Con group was normal, with normal gland morphology, normal goblet cells between glands, and normal crypts. No obvious neutrophil infiltration or ulceration was observed. In the DSS group, the colonic mucosa was extensively damaged, with significant neutrophil infiltration, a significantly reduced number of goblet cells, obvious mucosal congestion and edema, gland disappearance, and destroyed and distorted crypt structure. Ulcers and abscesses were observed in some areas. Amuc_1434, like mesalazine, can improve colonic lesions, with relatively intact colonic tissue structure, less damage to goblet cells, and a small amount of neutrophil infiltration.

[0051] The most common renal damage in patients with ulcerative colitis is kidney stones, glomerulonephritis. As shown in Figure 6 As shown in Figure 8, under 200 times microscope observation, the glomerulus size of the Con group was normal, the shape was normal, the boundary was clear, and there was no inflammatory cell infiltration. In the DSS group, the proximal tubule was severely damaged, there was severe congestion and edema, the renal interstitium was obviously increased, there were a large number of neutrophil infiltrations in the diffuse or patchy distributed renal interstitium, and there were ulcers. The degree of renal tubular congestion and edema of the Amuc_1434 group was reduced compared with the DSS group, and there was a small amount of neutrophil infiltration.

[0052] The above is the preferred embodiment of the present application, and other related personnel in the field of research can make some improvements and modifications without departing from the theory and scope of the present application, and the protection scope of the present application is subject to the scope of the claims.

Claims

1. Use of a recombinant protein for the treatment of inflammatory bowel disease, characterized in that, A recombinant protein drug for preparing a treatment or an adjuvant treatment of inflammatory bowel disease; the recombinant protein gene is from a bacterium Akkermansia muciniphila, named Amuc_1434; the amino acid sequence and the gene sequence of the recombinant protein Amuc_1434 are respectively shown as SEQ ID NO: 1 and SEQ ID NO:

2.

2. Use according to claim 1, characterized in that, The drug is used for relieving the symptoms of abdominal pain, diarrhea, hematochezia, colon shortening and colon tissue damage caused by inflammatory bowel disease.

3. Use according to claim 1, characterized in that, The recombinant protein Amuc_1434 is applied in the form of injection or oral administration.

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