Preparation method of amphioxus bjim1 protein and gene and application thereof in lupus erythematosus disease

Abstract

This invention discloses a method for preparing the BjIM1 protein and gene from amphioxus and its application in lupus erythematosus, belonging to the field of biopharmaceutical technology. The process includes: S1. Material acquisition: collecting amphioxus from Qingdao; S2. Screening and preparation of BjIM1 protein: S2.1, screening for differentially expressed proteins under LPS stimulation; S2.2, screening for differentially expressed proteins with significantly upregulated LPS and low homology to known functional proteins, exhibiting anti-inflammatory activity by inhibiting pro-inflammatory factor expression and regulating immune tolerance; S3. Cloning and preparation of the BjIM1 gene: S3.1, extracting total RNA from amphioxus tissue; S3.2, designing specific primers based on the amino acid sequence of the BjIM1 protein; S3.3, amplifying the gene sequence using RACE technology, and assembling to obtain the full-length BjIM1 gene sequence containing a complete ORF; S3.4, verifying the result by PCR amplification and sequencing, obtaining the amphioxus BjIM1 gene encoding the BjIM1 protein. This invention possesses the technical advantages of definite anti-inflammatory effect, precise immune regulation, high biosafety, no risk of drug resistance, and stable and reproducible process.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to the preparation method of amphioxus BjIM1 protein and gene and its application in lupus erythematosus. Background Technology

[0002] Amphioxus is a transitional animal between invertebrates and vertebrates. It is the most primitive chordate and an invertebrate chordate. Amphioxus has most of the molecular (genetic) material needed for adaptive immune evolution, such as members of the immunoglobulin superfamily, primitive MHC and RAG.

[0003] Yarrowia lipolytica is widely considered a safe, non-pathogenic microorganism and has been widely used in biotechnology and industrial production. This yeast can degrade a variety of hydrophobic substrates, producing abundant metabolites, mainly monounsaturated fatty acids and high-value-added saturated lipids, which possess various potential anti-inflammatory mechanisms.

[0004] Systemic lupus erythematosus (SLE) is a complex and severe autoimmune disease characterized by immune system dysfunction, which mistakenly attacks the body's own tissues and organs, leading to multi-system damage. Clinical studies have shown that the pathogenesis of SLE involves the interaction of multiple factors, including genetic susceptibility, environmental triggers, and sex hormone imbalances, resulting in the destruction of immune tolerance, the production of large amounts of autoantibodies, the formation of immune complexes that deposit in tissues, and the subsequent induction of inflammatory responses. Patients exhibit highly heterogeneous clinical manifestations, with common symptoms including a characteristic butterfly rash, mucosal ulcers, joint pain, fatigue, and fever. It can also affect the kidneys, blood, nervous system, and cardiovascular system, leading to serious complications such as proteinuria, anemia, seizures, and pericarditis.

[0005] Currently, the diagnosis of SLE relies on a comprehensive assessment of clinical symptoms, serological marker detection, and histopathological examination. Treatment primarily involves glucocorticoids combined with immunosuppressants, supplemented by individualized interventions using novel biologics, aiming to control disease progression, prevent organ damage, and improve patients' quality of life. Despite ongoing medical research, a complete cure for SLE remains challenging, hampered by high costs, long testing cycles, and potential side effects and drug resistance associated with treatments.

[0006] There are currently no reports on the preparation and functional verification of amphioxus BjIM1 protein, nor on the use of *Yerlotia lipophila* surface display to treat systemic lupus erythematosus (SLE). This study will use amphioxus as the material and, based on previous work, utilize the *Yerlotia lipophila* surface display system to explore the anti-inflammatory activity of amphioxus BjIM1 protein. This will provide new insights into the prevention and treatment of SLE and offer new ideas for the rational utilization of marine biological resources. By exploring the potential applications of these natural active substances, we can effectively promote the development of the pharmaceutical field and facilitate the sustainable development and protection of marine resources. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a method for preparing the BjIM1 protein and gene from amphioxus and its application in lupus erythematosus. It has the advantages of definite anti-inflammatory effect, precise immune regulation, high biosafety, no risk of drug resistance, and stable and reproducible process. It solves the problems of large side effects, easy development of drug resistance, high treatment cost, and lack of natural targeted active therapeutic substances in the prior art for treating systemic lupus erythematosus.

[0008] This invention is achieved through a method for preparing the BjIM1 protein and gene from amphioxus, comprising the following steps: S1. Material Acquisition: Amphioxus were collected from Qingdao and artificially domesticated in the laboratory. The amphioxus were stimulated with lipopolysaccharide (LPS), and body fluid protein samples were collected. Preparation of S2 and BjIM1 proteins for screening: S2.1. By combining two-dimensional electrophoresis with mass spectrometry proteomics analysis, the LPS-treated group and the control group were compared to screen for differentially expressed proteins under LPS stimulation. S2.2. Differential proteins with significantly upregulated LPS and low homology with known functional proteins were screened and identified and named BjIM1 protein. The BjIM1 protein has anti-inflammatory activity that inhibits the expression of pro-inflammatory factors and regulates the body's immune tolerance. S3 and BjIM1 gene cloning preparation: S3.1 Extract total RNA from amphioxus tissue and reverse transcribe it to synthesize cDNA; S3.2. Based on the amino acid sequence of BjIM1 protein, specific primers were designed, and PCR amplification was performed to obtain a partial cDNA fragment of the BjIM1 gene. S3.3. The 3′ and 5′ gene sequences were amplified separately using RACE technology and assembled to obtain the full-length BjIM1 gene sequence containing the complete ORF. S3.4. The BjIM1 gene encoding the BjIM1 protein in amphioxus was obtained by PCR amplification and sequencing verification.

[0009] Application of BjIM1 protein from amphioxus in lupus erythematosus.

[0010] As a preferred embodiment of the present invention, Yersinia lipophila is used as an expression vector to express BjIM1 protein on its surface, and an oral formulation is prepared for the prevention or treatment of systemic lupus erythematosus.

[0011] As a preferred embodiment of the present invention, the BjIM1 protein can alleviate weight loss, lymphadenopathy, splenomegaly, and skin lupus lesions in systemic lupus erythematosus model mice, and reduce kidney pathological damage.

[0012] As a preferred embodiment of the present invention, the BjIM1 protein can reduce the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β in the kidney tissue of systemic lupus erythematosus model mice, inhibit the inflammatory response, and rebuild the body's immune tolerance.

[0013] As a preferred embodiment of the present invention, the BjIM1 protein can reduce serum creatinine and serum urea concentrations, reduce urinary protein content, and improve lupus-induced renal function damage in systemic lupus erythematosus model mice.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention establishes a standardized preparation process for the isolation, screening, and cloning of the amphioxus BjIM1 protein and its full-length coding gene. The method is stable, reproducible, and suitable for large-scale bioprocessing. BjIM1 protein can significantly improve typical lupus signs in lupus mice, such as weight loss, skin lesions, lymph node enlargement, and splenomegaly. It can significantly reduce glomerular sclerosis, immune complex deposition, and interstitial inflammatory cell infiltration in lupus model mice, protecting kidney tissue structure and reducing renal pathological damage. It also significantly downregulates IL-6 and TNF levels in kidney tissue. The expression of pro-inflammatory factors such as -α and IL-1β can rebuild the body's immune tolerance, reduce the production of autoantibodies and the deposition of immune complexes in tissues; it can effectively reduce the levels of serum creatinine, serum urea and urinary protein in model mice, and significantly repair lupus-induced renal function damage; BjIM1 is a natural marine bioactive protein, which has low toxicity and no obvious risk of drug resistance compared with traditional hormones and immunosuppressants. It can be developed into an oral formulation with good compliance, providing a new candidate molecule and research and development idea for the prevention and treatment of systemic lupus erythematosus, while expanding the high-value utilization of amphioxus marine biological resources. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of random grouping of mice provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the changes in indicators that alleviate weight loss in MRL / lpr mice provided by the embodiments of the present invention using BjIM1; Figure 3This is a schematic diagram of the indicators of BjIM1 alleviating skin lesions in MRL / lpr mice provided in the embodiments of the present invention; Figure 4 This is a schematic diagram illustrating the indicators of BjIM1 in alleviating splenomegaly in MRL / lpr mice provided in this embodiment of the invention. Figure 5 This is a schematic diagram of PAS staining of mouse kidney tissue provided in an embodiment of the present invention; Figure 6 This is a schematic diagram illustrating the expression levels of inflammatory factors in four groups of mouse kidney tissues provided in this embodiment of the invention; Figure 7 This is a schematic diagram of the concentrations of serum creatinine and serum urea in four groups of mice provided in the embodiments of the present invention; Figure 8 This is a schematic diagram of urinary protein concentration in four groups of mice from 10 to 21 weeks, provided in an embodiment of the present invention. Detailed Implementation

[0016] To further understand the invention's content, features, and effects, the following embodiments are provided, and detailed descriptions are given in conjunction with the accompanying drawings.

[0017] The present invention will now be described in detail with reference to the accompanying drawings.

[0018] 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 cultured in a laboratory.

[0019] 2. The process of extracting BjIM1 protein from amphioxus Discovery of BjIM1 protein: 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. 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; Functional inflammation: BjIM1 inhibits the expression of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β, improves immune tolerance disruption, reduces the production of a large number of autoantibodies, and thus reduces the induction of inflammatory responses.

[0020] 3. The process of extracting the BjIM1 gene from amphioxus Obtain the BjIM1 gene sequence: 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 BjIM1 protein, and partial cDNA fragments of BjIM1 were obtained by PCR amplification. RACE technology: Using the BD SMARTRACE 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. Validation: Primer pairs were designed for PCR amplification, and the obtained full-length BjIM1ORF sequence was verified by sequencing.

[0021] 4. The process and results of animal experiments To investigate the anti-inflammatory effect of yeast surface-displayed BjIM1 protein on systemic lupus erythematosus (SLE), mice were randomly divided into four groups: control group, MRL / lpr group, YL group, and BjIM1 protein group (e.g., control group, MRL / lpr group, YL group, and BjIM1 protein group). Figure 1 (As shown).

[0022] 4.1. BjIM1 alleviates lupus-like signs and reduces lupus-related kidney pathological damage in MRL / lpr mice: Weight changes, lymphadenopathy, and splenomegaly are key indicators for assessing the severity of systemic lupus erythematosus (SLE). Figure 2-4 (As shown); The results showed that the body weight of mice in the MRL / lpr group and the MRL / lpr+YL group decreased significantly, while feeding BjIM1 could significantly reduce the body weight loss. The degree of splenomegaly in the BjIM1 group was significantly lower than that in the MRL / lpr group and the YL group, indicating that BjIM1 treatment can effectively alleviate the severe condition of splenomegaly. In addition, the YL treatment group showed a slight improvement in the splenomegaly, indicating that BjIM1 has a protective effect against organ damage. Histopathological analysis further confirmed that feeding BjIM1 alleviated renal tissue damage in MRL / lpr (e.g. Figure 5 As shown in the figure, the overall glomerular structure of mice in the BjIM1 group was better preserved, glomerular sclerosis symptoms were reduced, and inflammatory cell infiltration was minimal. In contrast, the MRL / lpr and YL groups showed more severe glomerular immune complex deposition, accompanied by interstitial inflammatory cell infiltration and glomerular crescent formation. The results indicate that feeding BjIM1 can significantly alleviate glomerular lesions in MRL / lpr mice, improve renal tissue damage, maintain tissue integrity, and effectively reduce clinical symptoms.

[0023] 4.2. BjIM1 reduces kidney inflammation in MRL / lpr mice: The expression levels of inflammatory factors in each group of mice were detected (e.g., Figure 6As shown in the figure, compared with the MRL / lpr group, the mRNA expression levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) in the YL group were slightly reduced, while the mRNA expression levels of inflammatory cytokines in the BjIM1 group were significantly reduced. These results indicate that the MRL / lpr group generated a systemic immune response, while BjIM1 treatment can effectively alleviate the level of inflammation.

[0024] 4.3. BjIM1 alleviated renal function in MRL / lpr mice: The serum creatinine and serum urea concentrations of mice in each group were measured and found to be (e.g.) Figure 7 As shown in the figure, compared with the control group mice, the serum creatinine and urea concentrations of the other three groups of mice were increased, indicating that the renal function of the lupus model mice was impaired. However, compared with the MRL / lpr group and the YL group mice, the serum creatinine and serum urea concentrations of the BjIM1 protein group mice were decreased. Meanwhile, the urinary protein concentration of four groups of mice from week 10 to week 21 was measured. The urinary protein concentration of all three groups of MRL / lpr mice was higher than that of the normal group. However, with the accumulation of food, the urinary protein concentration of the BjIM1 protein group was significantly lower than that of the MRL / lpr model group and the YL group, and showed a decreasing trend. The results indicate that BjIM1 alleviated lupus kidney injury in MRL / lpr mice. Figure 8 This alleviated the kidney function condition.

[0025] 4.4 The working principle of the BjIM1 protein in this application for treating systemic lupus erythematosus: BjIM1 is upregulated in amphioxus by lipopolysaccharide (LPS) stimulation and plays an immunomodulatory role by inhibiting the expression of various inflammatory factors. Previous studies have shown that BjIM1 can significantly inhibit the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α. The overactivation of these inflammatory factors and various immune diseases are key factors leading to systemic lupus erythematosus.

[0026] 4.5. Experimental Results and Effects Analysis of the Invention: Feeding patients with Yerba Lipolytica expressing BjIM1 (Yl-Bj) significantly reduced skin lesions, lymphadenopathy, and weight loss, decreased the disease activity index (DAI), improved splenomegaly, and reduced inflammatory cell infiltration. Mechanistically, Yl-Bj effectively improved immune tolerance disruption by inhibiting the expression of inflammatory factors, reduced the production of large amounts of autoantibodies, and reduced the deposition of immune complexes in tissues, thereby reducing the induced inflammatory response. In summary, the BjIM1 protein and BjIM1 gene effectively alleviate immune damage and improve kidney function through multiple mechanisms, including anti-inflammatory effects, reduction of immune system dysfunction, and protection against organ damage, thereby improving the symptoms of systemic lupus erythematosus.

[0027] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0028] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing BjIM1 protein and gene from amphioxus, characterized in that, Includes the following steps: S1. Material Acquisition: Amphioxus were collected from Qingdao and artificially domesticated in the laboratory. The amphioxus were stimulated with lipopolysaccharide (LPS), and body fluid protein samples were collected. Preparation of S2 and BjIM1 proteins for screening: S2.

1. By combining two-dimensional electrophoresis with mass spectrometry proteomics analysis, the LPS-treated group and the control group were compared to screen for differentially expressed proteins under LPS stimulation. S2.

2. Differential proteins with significantly upregulated LPS and low homology with known functional proteins were screened and identified and named BjIM1 protein. The BjIM1 protein has anti-inflammatory activity that inhibits the expression of pro-inflammatory factors and regulates the body's immune tolerance. S3 and BjIM1 gene cloning preparation: S3.1 Extract total RNA from amphioxus tissue and reverse transcribe it to synthesize cDNA; S3.

2. Based on the amino acid sequence of BjIM1 protein, specific primers were designed, and PCR amplification was performed to obtain a partial cDNA fragment of the BjIM1 gene. S3.

3. The 3′ and 5′ gene sequences were amplified separately using RACE technology and assembled to obtain the full-length BjIM1 gene sequence containing the complete ORF. S3.

4. The BjIM1 gene encoding the BjIM1 protein in amphioxus was obtained by PCR amplification and sequencing verification.

2. The application of the amphioxus BjIM1 protein as described in claim 1 in lupus erythematosus.

3. The application of amphioxus BjIM1 protein as described in claim 2 in lupus erythematosus, characterized in that: Using *Yeroia lipophila* as an expression vector, the BjIM1 protein was expressed on its surface and formulated into an oral preparation for the prevention or treatment of systemic lupus erythematosus.

4. The application of amphioxus BjIM1 protein as described in claim 2 in lupus erythematosus, characterized in that: The BjIM1 protein can alleviate weight loss, lymphadenopathy, splenomegaly, and skin lupus lesions in systemic lupus erythematosus model mice, and reduce kidney pathological damage.

5. The application of amphioxus BjIM1 protein as described in claim 2 in lupus erythematosus, characterized in that: The BjIM1 protein can reduce the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β in the kidney tissue of mice with systemic lupus erythematosus, inhibit the inflammatory response, and rebuild the body's immune tolerance.

6. The application of amphioxus BjIM1 protein as described in claim 2 in lupus erythematosus, characterized in that: The BjIM1 protein can reduce serum creatinine and serum urea concentrations, decrease urinary protein content, and improve lupus-induced renal function damage in systemic lupus erythematosus model mice.

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