Branchiostoma belcheri BjIM1 protein as well as gene and application thereof

Abstract

The invention provides a BjIM1 protein in Branchiostoma belcheri and a BjIM1 gene thereof, the amino acid sequence of the protein is as shown in SEQ ID NO. 1, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 2. The BjIM1 protein coded by the BjIM1 gene is applied to preparation of drugs for treating ulcerative enteritis.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to the BjIM1 protein and its gene, and its applications in the Qingdao amphioxus. Background Art

[0002] Amphioxus are transitional animals between invertebrates and vertebrates, and are among the most primitive chordates, specifically invertebrates. The amphioxus's immune system can be considered a "living fossil" of the primitive immune system of vertebrate ancestors; that is, the amphioxus's immune system largely represents the primitive immune system state of vertebrate ancestors. Amphioxus possess most of the molecular (genetic) material needed for adaptive immune evolution, such as members of the immunoglobulin superfamily, primitive MHC, and RAG.

[0003] Ulcerative colitis is an inflammation of the rectal and colonic mucosa and submucosa, the cause of which is not fully understood. The lesions may involve the rectum, a segment of the colon, or the entire colon. Clinical manifestations include gastrointestinal symptoms such as diarrhea, abdominal pain, and even hematochezia, or systemic symptoms such as fever and anemia. This disease is prone to recurrence, and treatment primarily involves medication and symptomatic relief. Currently, ulcerative colitis cannot be cured, but flare-ups can be managed. Mild or long-term remission generally results in a better prognosis. A small percentage of patients may develop complications such as toxic megacolon or gastrointestinal bleeding due to delayed treatment, or experience repeated relapses due to incomplete treatment. Based on the extent of the lesions, ulcerative colitis can be classified into: rectal type, left-sided colonic type, total colonic type, and ileocolonic type. Ulcerative colitis is a chronic, nonspecific inflammatory bowel disease with an unclear etiology. Clinical symptoms include diarrhea, abdominal pain, and bloody, mucous stools. It is not contagious, but severe cases can lead to complications such as intestinal perforation, massive bleeding, and cancer. The treatment of this disease is mainly based on medication. The disease is characterized by recurrent attacks and alternating remissions, which seriously affects the patient's quality of life. Summary of the Invention

[0004] The purpose of this invention is to provide the BjIM1 protein and its gene, as well as its applications, of the Qingdao amphioxus.

[0005] A BjIM1 protein of the BjIM1 gene screened from Qingdao amphioxus, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The BjIM1 gene encodes the aforementioned BjIM1 protein.

[0007] The BjIM1 gene mentioned above has a nucleotide sequence as shown in SEQ ID NO.2.

[0008] Vectors containing the aforementioned BjIM1 gene.

[0009] Host cells containing the BjIM1 gene or the vector described above.

[0010] The application of the above-mentioned BjIM1 protein, the above-mentioned BjIM1 gene, or the above-mentioned vector in the preparation of drugs for treating ulcerative colitis.

[0011] The application of the aforementioned BjIM1 gene or the aforementioned vector in the preparation of transgenic Qingdao amphioxus.

[0012] This application has achieved the following beneficial technical effects: Feeding *Yeruovia lipolyticis* expressing BjIM1 (Yl-Bj) significantly reduced weight loss, decreased the disease activity index (DAI), and reversed colonic shortening, while protecting colonic tissue structure and reducing inflammatory cell infiltration. Mechanistically, Yl-Bj enhances antioxidant defense by inhibiting the expression of pro-inflammatory factors, effectively maintaining intestinal barrier integrity. In summary, the BjIM1 protein and gene effectively alleviate DSS-induced colitis and ulcerative colitis through multiple mechanisms, including anti-inflammatory, antioxidant, and intestinal barrier protection, providing a new strategy for the treatment of ulcerative colitis. Attached Figure Description

[0013] Figure 1 Mice were randomly divided into four groups: control group, DSS group, yeast group (Yl group) and yeast surface display of BjIM1 protein (IM1-Yl group); Figure 2 IM1-Yl is an indicator of weight change in mice with DSS-induced colitis; Figure 3 The Disease Activity Index (DAI) score was used to assess the remission of DSS-induced colitis in mice by IM1-Yl. Figure 4 The colon length was used as an indicator of IM1-Yl in alleviating DSS-induced colitis in mice. Figure 5 HE staining of mouse colon tissue showed that the IM1-Yl group protected colon tissue structure and reduced inflammatory cell infiltration. Figure 6 MPO staining of mouse colon tissue showed that the IM1-Yl group significantly reduced the level of inflammation in the colon tissue. Figure 7a , Figure 7b as well as Figure 7c The expression levels of pro-inflammatory factors in mouse colon tissue; Figure 8a and Figure 8b The expression levels of pro-inflammatory and anti-inflammatory factors in mouse serum; Figure 9a , Figure 9b as well as Figure 9cThe level of oxidative damage in mouse colon tissue; Figure 10 This represents the mRNA expression level of tight junction protein (ZO-1) in colon tissue. Detailed Implementation

[0014] The following implementations will help those skilled in the art to further understand the present invention, but do not limit the invention in any way.

[0015] This application provides a BjIM1 protein of the BjIM1 gene screened from Qingdao amphioxus, the amino acid sequence of which is shown in SEQ ID NO.1: MKLLLLLAILGAAAAAEQTPWSPWRWWPFSSSDSAAAEPHWNTLKVGWGINPLTSFQSLPRTRSEAVKQGWTMYGRTTCAGTYWQGLRMIYQDDPAVILLFDRNGFIAGMQMGVKQSDLPADRSIPARDVIPPWVQDRNNDMWLITTYFIRP DTICTVGRTATEFQAKGTGADLYLQTGASPRTDFVIIPKYEKDLEGTDWTPGKCFWTMGKHYFKNLRENMRCEELYPLFPLYNEGKLNAFGLLIGASVPSPTSRYESPIASKTFYQLFMTPVPKCLQSHTEEKGMTTLHVFLENDPRLNNFC.

[0016] This application provides the BjIM1 gene encoding the aforementioned BjIM1 protein.

[0017] In the embodiments of this application, the nucleotide sequence of the BjIM1 gene is shown in SEQ ID NO.2: ATGAAGCTTCTCCTTCTTCTGGCGATTCTCGGTGCGGCCGCGGCGGCCGAGCAGACGCCATGGTCGCCATGGCGGTGGTGGCCCTTCTCTAGCTCTGACTCTGCAGCCGCTGAACCACACTGGAATACCCTGAAAGTTGGATGGGGCATCAACCCACTGACCTCGTTCCAGTCCCTTCCGCGCACGCGCAGTGAGGCTGTCAAACAGGGATGGACCATGTACGGACGGACCACATGTGCAGGAACCTACTGGCAGGGTCTGCGGATGATCTACCAGGACGATCCCGCAGTGATTCTGCTGTTCGACAGGAACGGCTTCATCGCCGGAATGCAGATGGGAGTGAAACAGAGCGACCTTCCCGCGGACAGGTCCATCCCAGCCCGTGACGTCATCCCACCATGGGTGCAGGACAGGAATAATGACATGTGGCTCATCACTACCTACTTCATCAGACCAGATACCATCTGTACTGTGGGGCGCACCGCGACAGAGTTCCAGGCAAAGGGAACAGGCGCAGACCTGTACCTCCAGACCGGGGCGAGCCCACGTACGGACTTTGTCATCATCCCCAAGTACGAGAAAGACCTGGAGGGAACGGACTGGACCCCGGGGAAGTGCTTCTGGACCATGGGCAAGCACTACTTTAAGAATCTGCGGGAGAACATGCGCTGTGAGGAGCTGTACCCGCTATTCCCGTTGTACAACGAGGGGAAGCTGAACGCATTTGGGCTGCTGATCGGCGCAAGTGTCCCCAGCCCCACCTCTCGTTATGAGTCACCAATCGCCTCCAAGACGTTCTATCAGTTGTTCATGACTCCGGTACCCAAGTGCCTCCAGAGCCACACCGAGGAGAAGGGCATGACCACCCTGCACGTCTTCCTGGAGAATGACCCTCGCCTGAACAACTTCTGTTAA。

[0018] This application provides a vector (PINA1317 plasmid) containing the above-mentioned BjIM1 gene.

[0019] This application provides a host cell (Yerovia lipophila) containing the BjIM1 gene described above or containing the vector described above.

[0020] This application provides the use of the above-mentioned BjIM1 protein, the above-mentioned BjIM1 gene, or the above-mentioned vector in the preparation of a medicament for treating ulcerative colitis.

[0021] This application provides the application of the above-mentioned BjIM1 gene or the above-mentioned vector in the preparation of transgenic Qingdao amphioxus.

[0022] Example 1 1. Origin and Aquaculture of Amphioxus in Qingdao The Qingdao amphioxus (Branchiostoma japonicum) selected in this invention was collected from the Shazikou waters of Shandong Province and was cultured in the laboratory.

[0023] 2. The process of extracting BjIM1 protein from amphioxus Discovery of BjIM1 protein: 2.1. Proteomics analysis: First, proteomics methods were used to analyze the body fluid proteins of saury stimulated by lipopolysaccharide (LPS). By two-dimensional electrophoresis and mass spectrometry, 26 differentially expressed proteins were identified between the LPS-treated group and the control group.

[0024] 2.2. Screening for special proteins: Among these differentially expressed proteins, one uncharacterized protein (called BjIM1) was significantly upregulated in the LPS-treated group and had very low similarity to other proteins with known functions, so it was selected for further study.

[0025] 2.3. Functional inflammation: BjIM1 inhibits LPS-induced upregulation of TLR pathway genes. The TLR signaling pathway plays an important role in the immune response, and the expression level of TLR pathway genes is related to the inflammatory response.

[0026] 3. The process of extracting the BjIM1 gene from amphioxus Obtain the BjIM1 gene sequence: 3.1. cDNA Cloning: Total RNA was extracted from amphioxus and cDNA was synthesized by reverse transcription. Primers were designed based on the predicted amino acid sequence of the BjIM1 protein, and partial cDNA fragments of BjIM1 were obtained by PCR amplification.

[0027] 3.2. RACE technology: Using the BD SMART™ RACE cDNA amplification kit, 3′ and 5′ RACE amplification were performed to obtain the complete sequence of BjIM1 cDNA. The obtained fragments were cloned and sequenced. The overlapping regions were assembled to finally obtain the full-length ORF BjIM1 sequence.

[0028] 3.3. Validation: Primer pairs were designed for PCR amplification, and the obtained full-length BjIM1ORF sequence was verified by sequencing.

[0029] 4. The process and results of animal experiments To investigate the anti-inflammatory effect of yeast surface-displayed BjIM1 protein on colitis, mice were randomly divided into four groups: control group, DSS group, yeast group (Yl group), and yeast surface-displayed BjIM1 protein (IM1-Yl group) (e.g., control group, DSS group, yeast group (Yl group), and yeast surface-displayed BjIM1 protein (IM1-Yl group). Figure 1 (As shown).

[0030] 4.1 IM1-Yl alleviates DSS-induced colitis in mice: Weight change, disease activity index (DAI) score, and colon length are key indicators for assessing the severity of colitis (e.g., Figure 2-4 shown); The results showed that mice in the DSS group experienced a significant decrease in body weight, while feeding IM1-Yl significantly reduced this weight loss. Mice in the Yl group showed a slight increase in body weight compared to the DSS group, but the difference was not statistically significant. The DAI score of the IM1-Yl group was significantly lower than that of other model groups, indicating that IM1-Yl treatment effectively alleviated the severity of colitis. Furthermore, IM1-Yl treatment effectively reversed DSS-induced colonic shortening, suggesting a protective effect against colitis. Histopathological analysis further confirmed that both the Yl group and the IM1-Yl group alleviated DSS-induced tissue damage (e.g., Figure 5 As shown in the figure, the colonic tissue structure of mice in the IM1-Yl group was well preserved, with minimal inflammatory cell infiltration. The results indicate that Yl, as a carrier, can significantly alleviate DSS-induced colitis in mice, maintain tissue integrity, and effectively reduce clinical symptoms by displaying BjIM1 protein on its surface.

[0031] 4.2 IM1-Yl reduces intestinal inflammation in DSS-induced colitis: Myeloperoxidase (MPO) is highly expressed primarily in neutrophils, and its activity is often used as a marker of inflammation, such as... Figure 6 As shown, MPO expression was highest in the colon of mice in the DSS group, followed by the Yl group, and lowest in the IM1-Yl group. This indicates that IM1-Yl can reduce DSS-induced MPO expression, thereby reducing the inflammatory response. Next, the expression levels of pro-inflammatory factors in each group of mice were examined (e.g., ... Figure 7a , Figure 7b as well as Figure 7c As shown in the figure, compared with the DSS group, the mRNA expression levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) in the Yl group and the IM1-Yl group were decreased, with the most significant decrease in the IM1-Yl group. Furthermore, serum IL-10 and TNF-α levels were significantly increased in both the DSS group and the Yl group, while the expression levels in the IM1-Yl group were close to those in the control group (as shown in the figure). Figure 8a and Figure 8b (As shown). These results indicate that the DSS and Yl groups generated a systemic immune response, and that IM1-Yl treatment effectively alleviated the level of inflammation.

[0032] 4.3 IM1-Yl alleviates DSS-induced colonic oxidative stress: The level of oxidative damage in mouse colon tissue was assessed (e.g. Figure 9a , Figure 9b as well as Figure 9c As shown in the figure, the antioxidant capacity of mice in the DSS group and Yl group was significantly reduced, while IM1-Yl treatment could effectively restore antioxidant capacity, including SOD, CAT and GSH activities. These results indicate that Yl itself did not enhance antioxidant capacity, but IM1-Yl could significantly improve antioxidant defense capacity in DSS-induced colitis.

[0033] 4.4 IM1-Yl protects against DSS-induced intestinal barrier: Excessive oxidative stress disrupts the redox balance, ultimately leading to damage to the intestinal barrier. Therefore, the mRNA expression level of tight junction protein (ZO-1) in colonic tissue was further examined (e.g., Figure 10 As shown in the figure, the DSS group had the lowest expression level of tight junction protein, while IM1-Yl treatment could significantly restore its expression.

[0034] The working principle of the BjIM1 protein in this application for treating ulcerative colitis is as follows: BjIM1 is upregulated in amphioxus upon stimulation with lipopolysaccharide (LPS) and exerts immunomodulatory effects by inhibiting key genes in the TLR signaling pathway. Previous studies have shown that BjIM1 can significantly inhibit the IL-17 (interleukin-17) signaling pathway, and excessive activation of IL-17 and various immune diseases, including IL-17 dysregulation, are key factors leading to ulcerative colitis.

[0035] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A BjIM1 protein of the BjIM1 gene screened from Qingdao lancelet, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The BjIM1 gene encoding the BjIM1 protein according to claim 1.

3. The BjIM1 gene according to claim 2, characterized in that The nucleotide sequence of the BjIM1 gene is shown in SEQ ID NO.

2.

4. A vector containing the BjIM1 gene according to claim 2 or 3. A host cell comprising the BjIM1 gene according to claim 2 or 3 or the vector according to claim 4.

6. Use of the BjIM1 protein according to claim 1, the BjIM1 gene according to claim 2 or 3, or the vector according to claim 4 in the preparation of a medicament for treating ulcerative enteritis.

7. Use of the BjIM1 gene according to claim 2 or 3 or the vector according to claim 4 in preparing transgenic Qingdao amphioxus.

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