A method for preparing Em-6B protein from Echinococcus multilocularis and its application in the preparation of anti-host inflammatory agents.

Abstract

This invention discloses a multilocular echinococcosis larva. In Preparation method of -6B protein and its application in the preparation of anti-host inflammatory agents. Through protein structure modeling and molecular docking technology, the preparation method was confirmed. In -6B can bind with high affinity to a key subunit of the host 26S proteasome, affecting the assembly of the proteasome in the host and thus interfering with the activity of the NF-κB signaling pathway, thereby regulating the host inflammatory response. Animal experiments have shown that recombinant... In -6B intervention significantly alleviated colonic damage in mice with inflammatory bowel disease, regulated the expression levels of pro-inflammatory and anti-inflammatory factors, and improved the survival rate of septic mice, with no obvious toxic side effects observed. This invention opens up a new pathway for developing anti-inflammatory agents using natural molecules that interact with the parasite's immune system, and provides a novel mechanism of action and biosafety for anti-host inflammatory biological agents, showing great potential for application in the prevention and treatment of inflammatory diseases in humans and livestock.

Description

Technical Field

[0001] This invention relates to the field of protein biopharmaceutical technology, and in particular to a method for preparing Em-6B protein from Echinococcus multilocularis and its application in the preparation of anti-host inflammatory agents. Background Technology

[0002] Inflammation is an important defense mechanism of the host's immune system in response to pathogen invasion and tissue damage. Moderate inflammation can help clear foreign substances and repair tissue damage, while excessive or uncontrolled inflammation can disrupt the body's immune homeostasis and cause a variety of serious diseases such as sepsis, inflammatory bowel disease, and autoimmune diseases. These diseases have complex courses, are difficult to treat clinically, and are difficult to cure with existing treatments. Furthermore, long-term use of drugs can easily lead to side effects and drug resistance, making them a major public health problem that threatens human and animal health. There is an urgent need to develop anti-inflammatory intervention strategies with novel mechanisms of action and high safety.

[0003] In livestock production, inflammation in animals not only directly damages their health but also reduces their productivity, thus having a substantial negative impact on the economic benefits of farming. During livestock production, various factors such as bacterial and viral infections and parasitic infestations can easily induce inflammatory responses in animals. Long-term chronic inflammation can further disrupt the body's immune system, leading to decreased immune defense capabilities, secondary infections causing immune failure, and significantly increasing the risk of disease outbreaks during the farming process. For dairy cows, mastitis directly causes inflammation and damage to mammary gland tissue, resulting in a sharp drop in milk production and a decline in milk quality. It also increases treatment costs for farmers and may even affect the safety of dairy products due to abnormal milk quality, creating a chain reaction of losses across the industry chain. Furthermore, the stress and metabolic disorders caused by inflammation can affect normal growth and development in animals, manifesting as stunted growth and slow weight gain, reducing feed conversion efficiency, severely hindering the healthy and large-scale development of farms, and further increasing farming costs.

[0004] In the course of long-term co-evolution with their hosts, parasites have developed unique mechanisms for precisely regulating the host's immune response and inflammatory response, enabling them to survive long-term within the host while avoiding host death due to excessive inflammation. Recent studies have proposed that parasites can achieve immune evasion by interfering with the host's NF-κB signaling pathway. The 26S proteasome plays a crucial role in the regulation of the NF-κB signaling pathway, and its classical activation pathway relies on the ubiquitination and degradation of the inhibitory factor IκB. The 26S proteasome consists of a 20S core particle and a 19S regulatory particle. The six AAA-ATPase subunits in the 19S regulatory particle's base are responsible for the recognition, unfolding, and transport of protein substrates. Their functional state directly affects the overall activity of the proteasome, thus determining the activation intensity and persistence of the NF-κB signaling pathway, playing a vital role in regulating biological processes such as cell cycle, apoptosis, and inflammatory responses. As the larval stage of Echinococcus tapeworm, the multilocular echinococcosis larva can cause zoonotic alveolar echinococcosis (AE). Within the host, it releases functional protein molecules through various secretory pathways, including vesicular fluid, microvesicles, and exosomes. These molecules can gently and precisely inhibit excessive inflammatory responses in the host, maintaining immune homeostasis. These parasite-derived functional proteins, having evolved to adapt to the host over a long period, have minimal interference with normal physiological functions and possess potential value for development into novel anti-inflammatory active substances. Although some studies have recognized the important role of the proteasome and NF-κB pathway in immune regulation, research and application of "parasite-derived protein molecules that directly act on the host proteasome-related functions and thereby regulate the host immune response" remain very limited. No technical solutions have yet been developed to fully explore natural anti-inflammatory molecules in the parasite-host immune-inflammatory interaction, and there is a lack of ideas and practical solutions for developing novel anti-inflammatory technologies from this mechanism perspective.

[0005] Current anti-inflammatory technologies have shortcomings in target selection, intervention strategies, and clinical application, making it difficult to achieve an effective balance between potent inflammation suppression and ensuring host biosafety. Existing anti-inflammatory agents primarily target the host's own inflammatory regulatory molecules, which can easily interfere with the body's normal immune regulatory network and physiological metabolic functions during intervention, leading to a series of side effects such as immune imbalance, increased susceptibility to infection, and organ damage. This fails to simultaneously address both anti-inflammatory efficacy and the protection of normal host physiological functions. Furthermore, current anti-inflammatory research has failed to fully explore the anti-inflammatory molecular resources within natural biological interactions, neglecting the precise inflammatory regulatory mechanisms formed during the long-term co-evolution of parasites and hosts. There is a lack of systematic screening, identification, and application development of functional proteins from parasites that can specifically regulate host inflammatory responses, failing to open up new research pathways for novel anti-inflammatory agents from the perspective of natural biological interactions.

[0006] In addition, traditional anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs and glucocorticoids have limited efficacy, are prone to drug resistance, and have significant side effects. Although targeted biological agents have improved specificity, they have drawbacks such as high price and limited clinical application. There is an urgent need in clinical practice to develop a new type of anti-inflammatory agent with a novel mechanism of action, high safety, and stable efficacy.

[0007] To address the shortcomings of existing technologies, this invention aims to provide a 26S proteasome regulatory subunit 6B (Em-6B) protein derived from *Echinococcus multilocularis* larvae. This protein is homologous to human PSMC4 (Gene ID: 5704, NCBI) and can precisely regulate the host's inflammatory response and immune homeostasis. This invention elucidates the molecular mechanism by which this Em-6B protein regulates the host's inflammatory response, aiming to obtain a novel active substance that can be used to intervene in excessive inflammatory responses in humans and animals. Using this Em-6B protein as the core active ingredient, it is applied to the preparation of anti-host inflammatory agents, thus providing a novel technical solution for anti-inflammatory therapy and offering new ideas and directions for the development of anti-inflammatory agents. This solution combines high specificity and high biosafety, with no obvious toxic side effects. Furthermore, its active ingredient is biodegradable without residue, aligning with the development concept of green biopharmaceuticals and possessing significant application value in the prevention and treatment of inflammatory diseases in humans and livestock. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing Em-6B protein from Echinococcus multilocularis and its application in the preparation of anti-host inflammatory agents.

[0009] This invention addresses the shortcomings of existing anti-inflammatory technologies in terms of target selection, intervention specificity, and biosafety, and mainly solves the following technical problems:

[0010] This invention provides a non-host-derived functional protein molecule that can precisely regulate the host's inflammatory response, overcoming the limitations of existing anti-inflammatory research that targets the host's own molecules, avoiding interference with the host's normal physiological functions during intervention, and improving the biosafety of anti-inflammatory agents.

[0011] To elucidate the molecular mechanism by which the parasite-derived Em-6B protein regulates the host 26S proteasome-NF-κB signaling pathway and inhibits excessive inflammatory response, to establish the basis for precise anti-inflammatory technology based on this protein, and to address the lack of specificity in existing NF-κB pathway intervention methods.

[0012] We will develop an efficient preparation technology for Em-6B protein to obtain high-purity, soluble recombinant Em-6B protein, which can then be converted into an active ingredient for practically applicable anti-inflammatory agents, thus solving the problems of difficulty in obtaining and low yield of proteins from natural parasites.

[0013] To establish an anti-host inflammatory response application protocol based on Em-6B protein, verify its anti-inflammatory effects in different inflammatory disease models, provide a new technical approach for the treatment of excessive inflammatory diseases such as sepsis and inflammatory bowel disease, and solve the problems of limited efficacy and significant side effects of existing anti-inflammatory agents.

[0014] In summary, this invention aims to provide the application of Echinococcus multilocularis Em-6B protein in the preparation of anti-host inflammatory agents, solving the core technical problems of existing anti-inflammatory technologies that are difficult to balance potent anti-inflammatory effects, precise regulation, and biosafety.

[0015] The objective of this invention is achieved through the following technical solution: the application of Echinococcus multilocularis Em-6B protein in the preparation of anti-host inflammatory agents. Em The amino acid sequence of -6B is shown in SEQ ID NO.2.

[0016] Furthermore, Em The cDNA sequence of the -6B gene transcript is shown in SEQ ID NO.1.

[0017] Furthermore, the immunomodulatory protein Em-6B derived from Echinococcus multilocularis in the anti-host inflammatory response agent serves as the core active protein for the anti-host inflammatory response. This protein is present in Echinococcus vesicle fluid, microvesicles, exosomes, and circulating exosomes, and can be recognized by the host and precisely regulate the host's inflammatory response.

[0018] Furthermore, the Em-6B can bind to the host 26S proteasome AAA-ATPase regulatory subunit, regulate the host 26S proteasome activity, and thereby inhibit abnormal activation of the NF-κB signaling pathway. By regulating the NF-κB signaling pathway, it achieves precise anti-inflammatory effects, avoids interference with other physiological pathways of the host, and balances the expression of pro-inflammatory / anti-inflammatory cytokines, thereby inhibiting excessive inflammatory responses.

[0019] Furthermore, the anti-host inflammatory response agent can treat or prevent diseases associated with excessive host inflammatory response, including sepsis and inflammatory bowel disease.

[0020] Furthermore, the anti-host inflammatory response agent modulates the host inflammatory response, thereby limiting the immune escape of parasites within the host and reducing pathological damage and parasite load.

[0021] Furthermore, the Em-6B protein product can be prepared into Em-6B protein-derived molecules by artificially synthesizing the core binding domain of the Em-6B protein to obtain Em-6B-derived polypeptides; or by performing site-directed mutagenesis and structural modification on Em-6B through protein engineering to obtain mutant proteins with stronger anti-inflammatory activity and higher stability, which can be used as active ingredients in anti-host inflammatory response agents.

[0022] Furthermore, the Em-6B protein can be combined with nanocarriers or targeting carriers to prepare anti-host inflammatory response agents with stronger targeting and higher bioavailability, thereby enriching the Em-6B protein at the site of inflammation and enhancing the anti-inflammatory effect.

[0023] Furthermore, the Em-6B gene is constructed into expression vectors of lentiviruses and adeno-associated viruses. The vectors are then introduced into host cells via local or systemic administration, enabling the host cells to express the Em-6B protein in vivo. This achieves long-term regulation of the inflammatory response and is used for the long-term treatment of chronic inflammatory diseases.

[0024] Furthermore, Echinococcus multilocularis Em The preparation method of -6B protein includes the following steps: (1) Total RNA was extracted from the prostomium of Echinococcus multilocularis and reverse transcribed to obtain Em -6B gene sequence; (2) Em The -6B gene sequence was seamlessly cloned into the pET-32a prokaryotic expression vector and induced to express in Rosetta competent cells; (3) Optimize induction conditions and induce recombination based on isopropyl-β-D-thiogalactoside IPTG. Em -6B expression levels reached their optimal level; (4) Obtained by Ni-NTA affinity chromatography and ultrafiltration concentration Em -6B protein products.

[0025] Further, in step (1), multilocular echinococcosis cysts adhering to the abdominal cavity and tissues were removed from the passaged long-clawed gerbils, filtered and ground to obtain a protoscolex suspension, and after washing, collecting the precipitate and filtering, a clear protoscolex mixture precipitate was obtained; RNA was extracted from the protoscolex and reverse transcribed into cDNA using a reverse transcription kit, and nested PCR was performed using the cDNA as a template; the pET-32a prokaryotic expression vector was double-digested to obtain... Em-6B Gene sequence.

[0026] The beneficial effects of this invention are:

[0027] This invention is the first to develop Em-6B protein derived from Echinococcus multilocularis as an active ingredient against host inflammatory responses, opening up a new pathway for developing novel anti-inflammatory agents using natural molecules involved in parasite-host immune interactions. Compared with existing anti-inflammatory technologies, it has the following advantages:

[0028] The target is novel and highly specific. Existing technologies mainly focus on mechanistic explanations, lacking systematic exploration of key proteins in parasites that regulate host immunity. This invention uses the parasite-derived Em-6B protein as its anti-inflammatory active ingredient. Em-6B specifically binds to a specific subunit of the host's 26S proteasome to regulate the NF-κB signaling pathway, inhibiting overactivated inflammatory responses with minimal impact on normal host immune function and physiological metabolism. This solves the problems of immune imbalance and significant side effects caused by existing anti-inflammatory technologies targeting host molecules.

[0029] It exhibits significant anti-inflammatory effects and has a wide range of applications. In two classic models of sepsis induced by LPS (systemic acute inflammation) and inflammatory bowel disease induced by DSS (local chronic inflammation), Em-6B protein showed significant anti-inflammatory effects: it could significantly improve the survival rate of sepsis mice, alleviate the pathological damage of intestinal inflammatory tissues, and effectively balance the expression of pro-inflammatory / anti-inflammatory factors. It also has a regulatory effect on acute, chronic, local, and systemic excessive inflammatory responses, and its application range is far superior to some traditional anti-inflammatory agents.

[0030] It exhibits good biocompatibility with no significant toxic side effects. In animal experiments, mice did not show significant weight loss or organ damage after intraperitoneal injection of recombinant Em-6B protein. Furthermore, it does not affect the host's normal immune defense function after inflammation is repaired, overcoming the drawbacks of traditional preparations such as glucocorticoids and nonsteroidal anti-inflammatory drugs, including liver and kidney damage, gastrointestinal irritation, and immunosuppression.

[0031] The preparation process is simple and can be scaled up. This invention constructs a prokaryotic expression system to prepare recombinant Em-6B protein. By optimizing the induction conditions, the protein is expressed in a soluble and efficient manner. Combined with Ni-NTA affinity chromatography and ultrafiltration concentration, high-purity protein products can be obtained. The preparation process is simple, low-cost, and easy to scale up, solving the problems of difficult acquisition and low yield of natural anti-inflammatory molecules.

[0032] Environmentally friendly and in line with green ecological requirements. The core of this solution is Em-6B, a protein derived from Echinococcus multilocularis and its intervention preparations. It can be naturally degraded in the body and in the environment, with no chemical drug residues or environmental pollution risks. Compared with chemically synthesized anti-inflammatory drugs, it is more in line with the development trend of green biopharmaceuticals and meets the urgent needs of modern animal husbandry and public health for green and safe prevention and control technologies.

[0033] The anti-inflammatory agents and applications related to the Em-6B protein of *Echinococcus multilocularis* involved in this invention differ fundamentally from existing anti-inflammatory patents and products in core technologies such as protein source, mechanism of action, and regulatory pathways. Furthermore, compared to publicly disclosed patents related to parasite-derived anti-inflammatory proteins, this invention uncovers a novel type of anti-inflammatory active protein and constructs a unique anti-inflammatory system. Specific differences are reflected in:

[0034] Firstly, compared with traditional chemical anti-inflammatory drugs and host-derived biological anti-inflammatory agents, existing products mostly target the host's own inflammatory regulatory molecules or achieve anti-inflammatory effects by non-specifically inhibiting inflammatory factors and blocking inflammatory pathway nodes, which can easily interfere with the host's normal immune physiological functions. This invention uses Em-6B protein derived from Echinococcus multilocularis as the core of anti-inflammatory action, and explores active substances from the natural immune interaction mechanism co-evolved between parasites and hosts. The target is a functional protein of non-host origin, which has higher biosafety.

[0035] Secondly, compared with the publicly disclosed anti-inflammatory protein patents derived from parasites, the active proteins used in the prior art are serine protease inhibitors derived from nematodes. The core of these proteins is to achieve anti-inflammatory effects by directly inhibiting the activity of host serine proteases. In contrast, the Em-6B protein of this invention is derived from Echinococcus multilocularis (tapeworm) and is a proteasome regulatory subunit protein. It belongs to a different protein family than the aforementioned serine protease inhibitors. It regulates the NF-κB signaling pathway, a core signaling pathway of the host inflammatory response, by binding with a high affinity to the host 26S proteasome regulatory subunit. By blocking the assembly and synthesis of the host proteasome upstream, it achieves precise inhibition of the inflammatory response, rather than simply inhibiting the activity of a certain type of protease. Its mechanism of action is more unique.

[0036] In summary, this invention differs from all existing anti-inflammatory patents / products in terms of the species source, protein family type, core mechanism of action, and regulatory pathway of the anti-inflammatory active protein, opening up a new direction for the research and development of parasite-derived anti-inflammatory proteins. The technical solutions involved have significant advantages in anti-inflammatory precision, biosafety, and application potential. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 A schematic diagram of the molecular docking between Em-6B and the host 26S proteasome regulatory subunit;

[0039] Figure 2A schematic diagram illustrating the expression characteristics and solubility of recombinant Em-6B;

[0040] Figure 3 This diagram illustrates the purification and ultrafiltration concentration of recombinant Em-6B protein; where M: marker; A: flow-through buffer after column loading; 10, 20, 40, 60, 250: refer to the fractions eluted with 10mM, 20mM, 40mM, 60mM, and 250mM imidazole elution buffers, respectively; C: fractions obtained by ultrafiltration concentration.

[0041] Figure 4 A schematic diagram illustrating the effect of Em-6B on the survival rate of septic mice;

[0042] Figure 5 A schematic diagram illustrating the effect of Em-6B on colon length in mice with colitis;

[0043] Figure 6 This diagram illustrates the regulatory effect of Em-6B on immune factors; where (A) represents the expression levels of anti-inflammatory factors TGF-β1 and IL-10; and (B) represents the expression levels of pro-inflammatory factors TNF-α and IL-1β. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the invention.

[0045] The core of this invention lies in providing a method for preparing Em-6B (26S proteasome regulatory subunit 6B, hereinafter referred to as Em-6B), an immunomodulatory target protein derived from Echinococcus multilocularis, and its application in the preparation of anti-host inflammatory agents. Utilizing the protein's ability to precisely regulate the host's 26S proteasome-NF-κB signaling pathway, inhibit excessive inflammatory responses, and without significantly interfering with normal host immune function, it is developed as a core active ingredient in anti-host inflammatory agents. This invention constructs a complete technical solution through four core steps: target screening and identification, molecular mechanism elucidation, recombinant protein preparation, and in vivo functional verification, as detailed below:

[0046] (1) Target Screening and Identification: To achieve precise targeting of key factors in parasitic immune regulation, this invention employs a multi-source proteomics data integration and analysis combined with mass spectrometry verification. By systematically reviewing publicly available proteomics data on Echinococcus cyst fluid, microvesicles, exosomes, and circulating exosomes, candidate proteins simultaneously present in multiple secretory systems were screened. Further verification was achieved through mass spectrometry analysis of whole Echinococcus cyst proteins, confirming that Em-6B is an immune regulatory molecule stably secreted by the parasite at different developmental stages and directly perceptible to the host. Specifically:

[0047] Based on the systematic integration and analysis of existing research on Echinococcus larvae proteomics, this invention summarizes and classifies reported Echinococcus larvae-related protein data into four categories: vesicular fluid, microvesicles, exosomes, and circulating exosomes. Cross-comparison analysis revealed that some proteins coexist in multiple secretory systems. Em-6B was detected in all four categories, suggesting that this protein is a core functional molecule in which Echinococcus multilocularis actively transmits immune regulatory signals to the host through multiple secretory pathways at different developmental stages, and can be effectively recognized and exerted by the host (as shown in Table 1). Furthermore, mass spectrometry analysis of whole Echinococcus multilocularis proteins directly confirmed the stable presence of Em-6B in the larvae, laying a solid foundation for its screening and identification as an anti-inflammatory protein.

[0048] Table 1. Common proteins in some Echinococcus larval vesicle fluid and extracellular vesicles

[0049]

[0050] (2) Elucidation of Molecular Mechanisms: After identifying the candidate target proteins, this invention explores the potential mechanisms of action of Em-6B at the molecular level. To elucidate the molecular basis of Em-6B's immunomodulatory role, this invention employs a structural biology modeling approach combined with molecular docking analysis. The structure of Em-6B-related proteins was predicted in three dimensions using AlphaFold 3, and the interaction between Em-6B and the 26S proteasome-related subunit was simulated using Schrödinger molecular docking software. This technically reveals that Em-6B possesses the ability to bind with high affinity to the host proteasome system, providing quantifiable and verifiable structural evidence for Em-6B's potential intervention in host inflammation-related signaling pathways. The specific process is described in Example 1 below.

[0051] (3) Recombinant protein preparation: After clarifying the target and its potential mechanism of action, this invention further employs a prokaryotic expression system to construct a recombinant protein biopharmaceutical. By cloning the Em-6B gene and constructing the pET-32a prokaryotic expression vector, and optimizing the induction conditions, efficient and soluble expression of Em-6B protein in Escherichia coli was achieved. Subsequently, high-purity recombinant Em-6B protein was obtained through Ni-NTA affinity chromatography and ultrafiltration concentration, transforming the abstract immune regulatory target into a prepareable, dose-controllable, and reusable biopharmaceutical entity, thus providing a basis for practical application. Details are as follows:

[0052] To address the difficulty in obtaining natural Em-6B protein, this invention constructs a prokaryotic expression system to achieve efficient and soluble preparation of recombinant Em-6B protein. Total RNA was extracted from the prostomium of *Echinococcus multilocularis* isolated from passaged long-clawed gerbils and reverse transcribed to obtain the cDNA sequence of the Em-6B gene transcript (EmuJ_000223000, WormBaseParaSite), as shown in SEQ ID NO.1: ATGGAAGAGATTGGTCTGTCCCCTAATCCTCATGAGCAGGGAGAGGTGGATGTTCCCGACTTTGCTCCAATCACTCCTGT AGATCCACTTCGACTTAATGATGAGGATTTGTATCAAAAGCTTAAATCCCTCAAGAAGCAGCTGGAGTTTATCCAGGTTC AAGAGGATTACATTAAAGACGAGCAAAAGAATCTCAAAAAAGAATACATGCACGCCCAGGAGGAAGTGAAGCGCATCAAA AGTGTCCCCTTAGTGATTGGTCAATTTTTGGAGGCTGTGGATCAGACCACTGGCATTGTTGGTTCCACAACAGGCTCCAA CTATTACGTTCGTATCCTTTCTACAATTGACCGTGAACTCCTTAAGCCAAGTGCTAGTGTTGCTCTCCACAAGCACAGCA ACGCACTCGTGGATGTTCTTCCACCTGAGGCTGACAGCAGTATCACAATGCTGCAGGCAGACGAAAAACCAGATGTCTCT TATGCTGATATTGGTGGAATGGATCTTCAGAAGCAAGAGGTACGCGAGGCAGTCGAGTTGCCATTGACACACTTTGACCT CTATAAACAAATCGGAATTGACCCACCTCGTGGTGTCCTAATGTTCGGTCCTCCCGGGTGCGGGAAAACTATGCTTGCCA AGGCTGTTGCTCATCACACCACCGCTGCATTCATCCGCGTTGTTGGGTCAGAATTTGTGCAAAAGTACCTTGGTGAGGGT CCTCGCATGGTGCGTGATGTCTTTCGGTTGGCAAAGGAGAATGCACCTGCTATTATCTTCATTGATGAAATTGATGCGAT CGCAACGAAACGATTTGATGCTCAGACTGGAGCTGACAGAGAAGTTCAGAGGATTCTTCTGGAACTTCTTAACCAAATGG ACGGGTTTGATCAGGCCGTTAATGTCAAGGTCATAATGGCTACGAATCGAGCTGACACCTTGGATCCAGCCCTTCTGCGT CCTGGCCGTCTGGACCGAAAGATTGAATTCCCCCTCCCAGATCGTCGTCAAAAGCGCCTCATTTTCTCCACCATCACCAG CAAGATGAATTTAAGTGAGGAAGTCGATCTTGAGGATTATGTGGCTCGTCCAGACAAAATCTCCGGTGCTGATATCAATG CCATTTGCCAAGAGGCTGGTATGCAGGCAGTGCGTGAGAATCGGTATGTTGTTTTGGCCAAGGATTTTGAGAAAGGCTAC AAAAATAATATCAAAAAGGGTAACCAGGAACTCGATTTCTACAAGTAA; EmThe amino acid sequence of -6B is as SEQ ID Shown in NO.2: MEEIGLSPNPHEQGEVDVPDFAPITPVDPLRLNDEDLYQKLKSLKKQLEFIQVQEDYIKDEQKNLKKEYMHAQEEVKRIKSVPLVIGQFLEAVDQTTG IVGSTTGSNYYVRILSTIDRELLKPSASVALHKHSNALVDVLPPEADSSITMLQADEKPDVSYADIGGMDLQKQEVREAVELPLTHFDLYKQIGIDPPRGVLMFGP PGCGKTMLAKAVAHHTTAAFIRVVGSEFVQKYLGEGPRMVRDVFRLAKENAPAIIFIDEIDAIATKRFDAQTGADREVQRILLELLNQMDGFDQAVNVKVIMATNR ADTLDPALLRPGRLDRKIEFPLPDRRQKRLIFSTITSKMNLSEEVDLEDYVARPDKISGADINAICQEAGMQAVRENRYVVLAKDFEKGYKNNIKKGNQELDFYK.

[0053] Will Em Em-6B was seamlessly cloned into the pET-32a prokaryotic expression vector and induced to express in Rosetta competent cells. By optimizing the induction conditions, the expression level of recombinant Em-6B reached its optimal level after 6 h of induction with IPTG (Isopropyl-beta-D-thiogalactopyranoside) at 37°C. Furthermore, Em-6B was soluble in the supernatant of bacterial lysates and was a soluble protein (e.g., ...). Figure 2 (As shown). High-purity protein products (such as...) were obtained by Ni-NTA affinity chromatography and ultrafiltration concentration. Figure 3 As shown in the figure, this provides a stable and reliable biological agent basis for subsequent in vitro and in vivo functional verification, and provides a stable active ingredient for the preparation of anti-inflammatory agents. The specific process is described in Examples 2-3 below.

[0054] (4) In vivo functional verification: To verify whether the above technical solution truly achieves the purpose of the invention, this invention utilizes animal inflammation models combined with immune indicator detection to verify the immunomodulatory function of Em-6B and its application potential. Through DSS-induced inflammatory bowel disease models and LPS-induced sepsis models, the immunomodulatory effect of recombinant Em-6B protein was systematically evaluated. Changes in key pro-inflammatory and anti-inflammatory factors were detected by ELISA. Simultaneously, a comprehensive analysis combining survival rate, tissue damage, and other indicators demonstrated that intervention with Em-6B biological agents can effectively regulate excessive inflammatory responses in the host. Specifically:

[0055] In a lipopolysaccharide (LPS, Solarbio)-induced lethal sepsis model in C57BL / 6 mice, Em-6B was pretreated with a protein endotoxin removal kit (Beyotime) followed by intraperitoneal injection of 50 μg Em-6B protein. Four days later, acute inflammation was induced by intraperitoneal injection of 50 mg / kg LPS. The results showed that Em-6B pretreatment significantly improved the survival rate of mice, indicating that it can effectively inhibit systemic cytokine storm and has significant protective effects against severe acute inflammation and in maintaining immune homeostasis (e.g., Figure 4 As shown, ρ < 0.0001 (***), hazard ratio (log-rank) = 0.06033). In a C57BL / 6 mouse model of inflammatory bowel disease induced by 2.5% (w / v) dextran sulfate sodium (DSS, Solarbio), after treatment with protein endotoxin (Beyotime), mice were allowed to drink water naturally during the observation period and were treated with daily intraperitoneal injections of 50 μg Em-6B protein. The results showed that Em-6B intervention could significantly alleviate inflammatory phenotypes such as shortened colon length, and had no obvious side effects (e.g., Figure 5 As shown), it can effectively regulate the expression levels of pro-inflammatory and anti-inflammatory cytokines in serum. The decrease in anti-inflammatory factors TGF-β1 and IL-10 caused by DSS was significantly reversed, and the expression levels of pro-inflammatory factors TNF-α and IL-1β were significantly decreased. No obvious toxic side effects were observed, confirming that it can precisely regulate the local inflammatory response in the intestine and repair inflammatory tissue damage by balancing the expression of pro-inflammatory / anti-inflammatory factors (such as...). Figure 6 (As shown). For the specific process, please refer to Examples 4-5 below.

[0056] In summary, this invention achieves the goal of intervening in the anti-host inflammatory response with Em-6B protein derived from Echinococcus multilocularis as the core active ingredient through the synergistic implementation of a series of specific technical means, including target screening and identification, molecular mechanism verification, recombinant protein preparation, and in vivo functional verification in animals. This invention focuses on the mechanism of inflammatory regulation in the interaction between parasites and the host immune system. A parasite-derived immunomodulatory target protein, Em-6B, was systematically screened and identified. The molecular mechanism by which Em-6B competitively binds to the host's 26S proteasome, inhibiting the host's proteasome assembly process, and precisely suppressing excessive inflammatory responses by regulating the NF-κB signaling pathway was clarified. A highly efficient preparation process for recombinant Em-6B protein was successfully constructed. The anti-inflammatory activity and biosafety of this protein were comprehensively verified using two classic animal models of excessive host inflammatory response: sepsis and inflammatory bowel disease. Finally, a technical solution for anti-host inflammatory response formulations with Em-6B protein as the core active ingredient was developed. This invention prepares recombinant Em-6B protein into anti-inflammatory agents in different dosage forms, such as injections (intraperitoneal, intravenous), oral formulations (microencapsulation), and topical formulations (gels, ointments), suitable for the treatment of different types and locations of inflammatory diseases. Furthermore, this invention can combine Em-6B protein with low-dose traditional anti-inflammatory agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to achieve synergistic anti-inflammatory effects, reduce the dosage of traditional anti-inflammatory agents, and decrease their side effects. This invention overcomes the limitations of existing anti-inflammatory technologies, which often target host molecules and are prone to causing immune imbalances and toxic side effects. It opens up a new pathway for developing novel anti-inflammatory agents using parasite-host innate immune interaction molecules, providing a novel, highly specific, safe, and promising biotechnological solution for the treatment of sepsis, inflammatory bowel disease, and other excessively inflammatory diseases, with excellent clinical translation prospects.

[0057] Example 1: Verification of the binding of Em-6B multilocular echinococcosis to the 26S proteasome regulatory subunit

[0058] Em-6B docking screening with the 26S proteasome receptor: The three-dimensional structures of Em-6B (GeneID: EmuJ_000223000, WormBase ParaSite) and the six AAA-ATPase regulatory subunits of the 26S proteasome (GeneID: 5700, 5701, 5702, 5704, 5705, 5706, NCBI) were predicted using AlphaFold 3. The interaction between Em-6B and the AAA-ATPase regulatory subunits of the 26S proteasome was simulated using the Schrödinger small molecule docking software. Using the human homolog PSMC4 (homological to Em-6B, Gene ID: 5704, NCBI) as a reference, it was replaced with Em-6B for structural matching with the remaining five AAA-ATPase regulatory subunits. The results are as follows: Figure 1 As shown, Em-6B can bind with high affinity to multiple AAA-ATPase regulatory subunits of the host 26S proteasome, with an RMSD value of 1.074. This indicates that the structure is highly similar after replacing PSMC4 with Em-6B, suggesting that it has the potential to competitively or functionally interfere with the activity of the host proteasome, affecting the assembly of the host proteasome and thus affecting the activation state of the NF-κB signaling pathway. This provides a reasonable structural basis for its immune regulatory function by blocking excessive inflammatory responses at the molecular level.

[0059] Example 2: Obtaining and identifying the Em-6B gene from Echinococcus multilocularis larvae

[0060] (1) Isolation, purification and in vitro culture of protoscolex

[0061] Echinococcus cysts adhering to the abdominal cavity and tissues were harvested from passaged long-clawed gerbils. The cyst tissue was minced and placed on a 180 μm filter screen. The cells were continuously washed with PBS buffer containing 1% (v / v) penicillin and antibiotics, and then continuously ground. The protoscolex suspension obtained from the filter screen was collected in beakers, washed repeatedly with PBS solution, and allowed to stand. The supernatant was discarded, and the precipitate was collected. This filtration and collection process was repeated five times until a clear protoscolex mixture precipitate was visible. The protoscolex was aliquoted into T25 culture flasks, and an appropriate amount of DMEM medium containing 1% (v / v) penicillin and antibiotics was added. The flasks were then cultured in vitro at 37°C with 5% CO2.

[0062] (2) Acquisition of the Em-6B gene from Echinococcus multilocularis and construction of the pET-32a-6B vector

[0063] In vitro activation of protostomes was achieved by incubating them in 0.05% (w / v) pepsin solution (pH = 2) at 37°C for 30 min. The reaction was terminated with PBS and the cells were washed three times. RNA was extracted from 3000 protostomes using the RNeasy® Plus Mini Kit (Qiagen). The precipitate was dissolved in DEPC water and verified by a nano-nucleic acid analyzer and gel electrophoresis. 1 μg of RNA was then reverse transcribed into cDNA using the FSK-101 reverse transcription kit. Nested PCR was performed using this cDNA as a template. The pET-32a prokaryotic expression vector was double-digested with enzymes. The obtained Em-6B gene sequence (Gene ID: EmuJ_000223000, WormBase ParaSite) was seamlessly cloned (Novizan Seamless Cloning Kit) into the digested pET-32a prokaryotic expression vector. The vector was then transformed into Rosseta competent expression single strains for PCR amplification and gel electrophoresis identification.

[0064] Example 3: Induced expression, purification, and immunization of recombinant Em-6B protein

[0065] (1) Expression and solubility characteristics of recombinant Em-6B protein

[0066] Positive bacterial cultures were inoculated at a 1:100 (v / v) ratio into 10 mL of LB liquid medium containing ampicillin (50 ng / μL) and cultured at 37°C with shaking until the OD600 reached 0.6. Then, 1 M IPTG inducer was added at a 1:5000 (v / v) ratio, and induction was performed at 37°C. Bacterial cultures were collected at 0 h, 2 h, 4 h, 6 h, and 8 h of induction, and the medium was removed by centrifugation. Another 8 h of bacterial culture was centrifuged, added to PBS buffer, and sonicated. The supernatant and precipitate were collected separately for SDS-PAGE analysis to determine the optimal induction time and the solubility characteristics of the recombinant protein. Results are as follows: Figure 2 As shown, Em-6B showed the best effect when induced at 37℃ for 6 h, and was expressed in the supernatant as a soluble protein.

[0067] (2) Purification of recombinant Em-6B protein

[0068] Positive bacterial culture was inoculated at a ratio of 1:100 (v / v) into 300 mL of LB medium containing ampicillin (50 ng / μL) and cultured to the logarithmic growth phase. IPTG was added to induce expression. The induced bacterial culture was collected and aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the bacterial pellet was resuspended in 50 mM PBS buffer (pH = 7.4). The pellet was thoroughly mixed by pipetting and then sonicated (2 s working, 2 s rest, 45 min total). The pellet was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. The supernatant was purified by Ni-NTA agarose resin and eluted with 10 mM, 20 mM, 40 mM, 60 mM, and 250 mM imidazole PBS elution buffers. The elution buffers of different concentrations were collected and SDS-PAGE electrophoresis was performed to detect the purification effect. The liquid eluted with 250 mM imidazole PBS was ultrafiltered using Millipore Amicon® Ultra-4 ultrafiltration centrifuge tubes at 4000 rpm for 20 min. The results are as follows: Figure 3 As shown, M is the marker, A is the flow-through solution after column loading, and 10, 20, 40, 60, and 250 refer to the fractions eluted with 10 mM, 20 mM, 40 mM, 60 mM, and 250 mM imidazole PBS elution buffers, respectively. C is the fraction obtained by ultrafiltration concentration. The results show that 250 mM imidazole PBS elution buffer can elute Em-6B and can be enriched in large quantities after ultrafiltration.

[0069] Example 4: Inhibitory effect of Em-6B protein on acute systemic inflammation

[0070] Sepsis was induced in mice by injection of lipopolysaccharide (LPS, Solarbio). C57BL / 6 mice (n = 20) were divided into two groups. Experimental group 1 received intraperitoneal injections of 50 μg Em-6B protein daily for 3 days, followed by a tail vein injection of 150 μg Em-6B protein on day 4, and an intraperitoneal injection of 50 mg / kg LPS. Control group 2 received no pretreatment, but received the same volume of PBS via tail vein injection on day 4, followed by an intraperitoneal injection of 50 mg / kg LPS. Mortality was recorded every 24 hours in both groups until all mice in both groups had died. Results are as follows: Figure 4 As shown, Em-6B can significantly improve the survival rate of septic mice.

[0071] Example 5: Inhibitory effect of Em-6B protein on local chronic inflammation

[0072] Colonic inflammation was induced in mice using sodium dextran sulfate (DSS, Solarbio). C57BL / 6 mice (n=20) were randomly divided into 4 groups, with one group serving as a blank control group, which received drinking water (…). Figure 5The blank control group); two groups were Em-6B-only treatment groups, receiving daily intraperitoneal injections of 50 μg Em-6B protein (…). Figure 5 The three groups were DSS modeling groups, given drinking water containing 2.5% (w / v) DSS (Solarbio). Figure 5 The four groups (DSS + Em-6B intervention group) were given drinking water containing 2.5% DSS and injected with 50 μg Em-6B protein. Figure 5 (DSS + Em-6B in the sample). Seven days later, the colon length of each mouse was measured at dissection. Results are as follows. Figure 5 Em-6B effectively alleviated the impact on colon length in mice with colitis without significant side effects. Serum samples were collected from each group, and the expression levels of anti-inflammatory factors TGF-β1 and IL-10, and pro-inflammatory factors TNF-α and IL-1β in the serum of the four groups of mice were detected by ELISA. Results are as follows: Figure 6 As shown, after Em-6B intervention, the decrease in IL-10 and TGF-β1 concentrations caused by DSS modeling was significantly reversed, and the expression levels of pro-inflammatory factors TNF-α and IL-1β were significantly decreased.

[0073] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A multilocular echinococcosis larva. Em The application of -6B protein in the preparation of anti-host inflammatory agents, characterized in that, Em The amino acid sequence of 6B is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Em The cDNA sequence of the -6B gene transcript is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The anti-host inflammatory response agent contains immunomodulatory proteins derived from Echinococcus multilocularis. Em -6B is a core active protein against host inflammatory response. This protein is present in Echinococcus vesicle fluid, microvesicles, exosomes and circulating exosomes. It can be recognized by the host and precisely regulate the host's inflammatory response.

4. The application according to claim 1, characterized in that, The Em -6B can bind to the host 26S proteasome AAA-ATPase regulatory subunit, regulate the host 26S proteasome activity, and thereby inhibit abnormal activation of the NF-κB signaling pathway. By regulating the NF-κB signaling pathway, it achieves precise anti-inflammatory effects, avoids interference with other physiological pathways of the host, and balances the expression of pro-inflammatory / anti-inflammatory cytokines, thus inhibiting excessive inflammatory responses.

5. The application according to claim 1, characterized in that, The anti-host inflammatory response agents can treat or prevent diseases associated with excessive host inflammatory response, including sepsis and inflammatory bowel disease.

6. The application according to claim 1, characterized in that, The anti-host inflammatory response agent modulates the host's inflammatory response, thereby limiting the immune escape of parasites within the host and reducing pathological damage and parasite load.

7. The application according to claim 1, characterized in that, The Em -6B protein products can be prepared into Em -6B protein-derived molecules, Em The core binding domain of the -6B protein was artificially synthesized to obtain... Em -6B-derived polypeptide; Or through protein engineering Em -6B was subjected to site-directed mutagenesis and structural modification to obtain mutant proteins with stronger anti-inflammatory activity and higher stability, which were then used as active ingredients in anti-host inflammatory response agents.

8. The application according to claim 1, characterized in that, The Em -6B protein can bind to nanocarriers or targeting carriers to prepare more targeted and bioavailable anti-host inflammatory agents, achieving... Em -6B protein accumulates at sites of inflammation, enhancing anti-inflammatory effects.

9. The application according to claim 1, characterized in that, Will Em The -6B gene is constructed into expression vectors for lentiviruses and adeno-associated viruses. These vectors are then introduced into host cells via local or systemic administration, enabling the host cells to express the gene in vivo. Em -6B protein enables long-term regulation of inflammatory responses and is used for the long-term treatment of chronic inflammatory diseases.

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