A leucocytozoon caulleryi allergen recombinant protein vaccine and a preparation method and application thereof

Abstract

The application relates to the technical field of immunization and insect allergy prevention and treatment, and specifically discloses a blood-sucking midge allergen recombinant protein vaccine and a preparation method and application thereof. The blood-sucking midge allergen recombinant protein vaccine comprises a Cul t 1 recombinant protein and an immune adjuvant; the preparation method comprises the following steps: S1, constructing a recombinant expression vector containing a Cul t 1 gene; S2, transfecting the carrier obtained in S1 into mammalian cells for expression; S3, collecting the expression cells in S2 and purifying to obtain the Cul t 1 recombinant protein; and S4, mixing the protein obtained in S3 after purification with the immune adjuvant to prepare the vaccine; and the blood-sucking midge allergen recombinant protein vaccine prepared by the preparation method is applied to the preparation of a medicine for preventing or treating allergic reactions caused by bites of the blood-sucking midge. The recombinant protein vaccine can be used for preventing or treating allergic reactions caused by bites of the blood-sucking midge, and has the advantages of clear components, high safety and good immunization effect.

Description

Technical Field

[0001] This application relates to the fields of immunology and insect allergy prevention and control, and more specifically, it relates to a recombinant protein vaccine for halophilic midge allergens, its preparation method, and its application. Background Technology

[0002] The application of immunotherapy in sensitization to midges mainly manifests in allergen-specific immunotherapy. By gradually increasing the dosage of allergen extracts, the patient's immune system develops tolerance, thereby reducing or eliminating allergic reactions after the bite. Insect allergy prevention and control technology focuses on comprehensive management, including the use of repellents, protective clothing, and environmental management to reduce contact, as well as the use of antihistamines, adrenaline, and other drugs for intervention during acute reactions. The advantages of the two are complementary: immunotherapy can provide long-term, fundamental immune regulation, with the potential to achieve a cure; while prevention and control technology provides immediate and flexible prevention and emergency measures. The combination of the two can build a multi-level, full-cycle effective prevention and control system for midge allergy patients, from short-term protection to long-term cure, from prevention of contact and emergency treatment to long-term desensitization.

[0003] Related midge allergies can be addressed through specific immunotherapy based on natural allergen extracts. However, these extracts are often complex in composition and have low standardization. Furthermore, the adjuvants they commonly use tend to induce Th2 immune responses, thereby enhancing the sensitization rather than the protective effect of the allergen. This leads to an imbalance in immune regulation, insufficient production of blocking antibodies, and difficulty in achieving stable and lasting tolerability. Summary of the Invention

[0004] To address the problem of immune dysregulation and insufficient production of blocking antibodies caused by intervention with specific immunotherapy based on natural allergen extracts for midge allergies, this application provides a recombinant protein vaccine for midge allergens, its preparation method, and its application.

[0005] In the first aspect, this application provides a recombinant protein vaccine against halophilic midge allergens, employing the following technical solution: A recombinant protein vaccine against vampire allergens, comprising Cul t 1 recombinant protein and an immune adjuvant.

[0006] By adopting the above technical solution, since the antigen component of the vaccine is a recombinant Cult 1 protein prepared through genetic engineering technology, it can present the antigenic epitope of the natural allergen. At the same time, the addition of the immune adjuvant can non-specifically enhance the strength and persistence of the body's immune response to the antigen. After immunization, this combination can effectively stimulate the body to produce high titers of specific neutralizing antibodies and guide the immune response to shift towards the Th1 type. Thus, when exposed to midge saliva antigen, it can block the binding of allergens to specific IgE on the surface of effector cells and inhibit the excessive activation and degranulation of effector cells such as mast cells. Therefore, it can safely prevent or reduce type I hypersensitivity reactions caused by midge bites.

[0007] Preferably, the amino acid sequence of the Cult 1 recombinant protein is as shown in SEQ ID NO:1, or a variant that has at least 90% homology with it and retains immunogenicity.

[0008] By adopting the above technical solution, using the standard Cult 1 protein of SEQ ID NO:1 or its highly homologous variant as the antigen, the high consistency between the vaccine antigen and the natural allergen on major B cell and T cell epitopes is ensured. This is the basis for inducing cross-protective immunity. Allowing no more than 10% amino acid difference provides space for improving protein solubility, expression level, or stability through site-directed mutagenesis. At the same time, the immunogenicity preservation requirement ensures that sequence modifications do not impair its function of eliciting a protective immune response. Therefore, the effect of enhancing the controllability of the production process and the stability of the product is achieved while ensuring vaccine efficacy. SEQ ID NO:1: .

[0009] Preferably, the immune adjuvant comprises CpG ODN1826 and Alum.

[0010] By employing the above-mentioned technical solution, using Alum and CpG ODN1826 as a combined adjuvant system, Alum adsorbs antigens to form an antigen reservoir at the injection site and promotes phagocytosis and presentation by antigen-presenting cells, while simultaneously inducing a strong Th2 humoral immune response to generate antibodies. CpG ODN1826, on the other hand, is a TLR9 agonist that can activate immune cells such as plasmacytoid dendritic cells, secreting large amounts of cytokines such as type I interferon, driving the immune response toward Th1 polarization. The combined use of the two produces a synergistic effect, not only increasing the specific antibody titer against Cult1 protein but also balancing the Th1 / Th2 immune response and reducing the risks associated with pure Th2 dominance.

[0011] Preferably, the dose of Cult 1 recombinant protein in the vaccine is 25 μg, 50 μg, or 100 μg.

[0012] By adopting the above technical solution, three gradient antigen doses of 25μg, 50μg, and 100μg are set. The low dose of 25μg is used for basic or booster immunization, minimizing the amount of antigen used while ensuring immunogenicity. The 50μg dose can induce a robust and long-lasting antibody response and is the preferred dose for routine immunization. The high dose of 100μg is suitable for special cases requiring rapid establishment of strong immune protection or for individuals with low responses. Multiple dose options provide choices for different individual differences, ensuring that an antigen dose that can induce effective immune protection is found within a safe range. Therefore, the effect of ensuring that vaccine use can balance immunization efficacy, safety, and economy is achieved.

[0013] Secondly, this application provides a method for preparing a recombinant protein vaccine containing halophilic midge sensitizers, employing the following technical solution: A method for preparing a recombinant protein vaccine containing halophilic midge allergens includes the following steps: S1. Construct a recombinant expression vector containing the Cult1 gene; S2. Transfect the vector obtained in S1 into mammalian cells for expression; S3. Collect the expression cells from S2 and purify them to obtain the Cul t 1 recombinant protein; S4. The purified protein obtained in S3 is mixed with an immune adjuvant to prepare a vaccine.

[0014] By adopting the above-mentioned technical solution, the process flow from gene cloning to protein expression, purification, and finally adjuvant preparation is as follows: First, molecular cloning technology is used to ensure the accurate insertion and regulated expression of the target gene Cult1, laying the foundation for subsequent production; then, a mammalian expression system is used to achieve the correct folding and post-translational modification of the recombinant protein, ensuring its native conformation and immunogenicity; then, an efficient chromatographic purification step is used to remove impurities such as host cell proteins and nucleic acids to obtain high-purity antigen proteins; finally, the purified antigen is physically mixed with a specific ratio of adjuvant to form a stable vaccine formulation. Therefore, an efficient, stable, and scalable vaccine antigen preparation process is obtained.

[0015] Preferably, the mammalian cell is a HEK293F cell.

[0016] By adopting the above technical solution and using HEK293F cells in suspension culture as the expression host, this cell line has the characteristics of fast growth rate, easy high-density suspension culture, high transfection efficiency and high protein expression level. Its expression system can complete complex protein folding and disulfide bond formation, ensuring that the secreted Cul t1 recombinant protein has a conformation and immunological properties similar to the natural protein. The use of serum-free suspension culture system not only reduces the risk of introducing exogenous animal-derived components and improves product safety, but also facilitates subsequent purification processing. Therefore, the effect of increasing antigen yield and meeting the requirements of industrial production is achieved while ensuring protein quality.

[0017] Preferably, the purification step employs nickel column affinity chromatography, specifically as follows: equilibrate the nickel column with equilibration buffer, load cell lysate or culture supernatant containing recombinant protein, remove non-specifically bound contaminating proteins with washing buffer, and finally elute the target protein with elution buffer.

[0018] By employing the above technical solution, the specific and reversible binding between the histidine tag carried by the recombinant Cult1 protein and nickel ions is achieved. The equilibration buffer creates a suitable pH and ionic strength environment for protein binding. After loading, impurities are removed by flow-through due to non-specific binding. Subsequently, a washing buffer containing a low concentration of imidazole is used to further elute weakly non-specifically adsorbed impurities, improving the purity of the intermediate product. Finally, a competitive elution buffer containing a high concentration of imidazole is used to obtain the high-purity target protein. This method is highly selective, has a large loading capacity, and is easy to operate. It can efficiently purify high-purity Cult1 recombinant protein from complex samples, providing high-quality antigen raw materials for subsequent vaccine formulation. Therefore, it achieves efficient, stable, and easily scalable results.

[0019] Thirdly, this application provides an application of a recombinant protein vaccine against halophilic midge sensitizers, employing the following technical solution: Application of a recombinant protein vaccine containing a midge allergen in the preparation of drugs for the prevention or treatment of allergic reactions to bites of Culicoides stripe midge.

[0020] By employing the above-mentioned technical solution, a recombinant vaccine specifically targeting the Cult 1 protein, the main allergen of Cult 1, is applied to prevent or treat allergic reactions to Cult 1 bites. Its mechanism of action is to induce the body to produce high levels of specific IgG antibodies against the Cult 1 antigen through active immunization. When an individual is bitten by a Cult 1 again, the natural Cult 1 protein in their saliva enters the body, and the specific IgG antibodies induced by the vaccine bind to these allergens first, forming immune complexes. This blocks the cross-linking of allergens with specific IgE receptors on the surface of sensitized mast cells or basophils, fundamentally inhibiting the release of inflammatory mediators such as histamine and leukotrienes.

[0021] Preferably, the immunization dose of the vaccine is 23-27 μg per dose.

[0022] By adopting the above technical solution, the immunization dose of each vaccine is limited to the range of 23-27 μg. A dose below 23 μg is insufficient to induce a sufficiently strong protective immune response in all immunized individuals; while a dose above 27 μg can further enhance the response, but the improvement in immune effect has reached a plateau, and increasing the amount of antigen will increase costs and the risk of immune tolerance; a dose of about 25 μg per dose has been proven to induce stable and efficient protective antibody levels in the vast majority of individuals, while also having high safety. Therefore, a balance is achieved between efficacy and risk and cost.

[0023] Preferably, the vaccine is administered via intramuscular injection.

[0024] By employing the above-mentioned technical solution, intramuscular injection is used as the route of vaccination, selecting muscle tissues such as the deltoid or vastus lateralis for injection. Muscle tissue is rich in blood supply and contains antigen-presenting cells. When the vaccine is injected into the muscle, the adjuvant forms an antigen reservoir, slowly releasing the antigen. Simultaneously, the antigen and adjuvant are effectively taken up, processed, and presented by local antigen-presenting cells, subsequently migrating to the draining lymph nodes to initiate an adaptive immune response. Compared to subcutaneous injection, intramuscular injection is generally less painful and has fewer local reactions. Compared to intravenous injection, it is safer and easier to operate and control antigen release. Therefore, it achieves the effect of ensuring vaccine safety, efficacy, and good compliance.

[0025] In summary, this application has the following beneficial effects: 1. Since this application uses a complex adjuvant system composed of recombinant Cult 1 protein, CpG ODN1826 and Alum to construct a vaccine, the recombinant protein can mimic the epitope of natural allergens, while Alum provides sustained antigen release and enhances antibody response, and CpG ODN1826 drives Th1 immune response to achieve balance. Through comprehensive and balanced immune function, the two can achieve the effect of efficiently inducing blocking antibodies and preventing or reducing allergic reactions caused by midge bites.

[0026] 2. In this application, the preferred preparation process is expression in HEK293F cells combined with nickel column affinity chromatography purification. Since the mammalian cell system ensures the correct folding and high immunogenicity of the recombinant protein, this lays the foundation for efficient capture of high-quality antigens in subsequent purification processes, thereby achieving the effect of stably obtaining high-purity and high-activity antigens in large-scale production, ensuring the consistency and effectiveness of vaccine batches.

[0027] 3. The method of this application allows reasonable variants of the Cult1 recombinant protein sequence to exist and combine with a specific immune adjuvant system. Due to the adaptive improvement within a certain range of the sequence, the high expression level and stability of the antigen are ensured. This provides a material basis for efficient compatibility with the adjuvant system composed of Alum and CpG ODN1826, enabling the vaccine to induce a robust and durable Th1 / Th2 balanced immune response in different individuals. Therefore, it achieves the effects of improving vaccine universality, addressing herd immunity differences, and achieving long-term protective effects. Attached Figure Description

[0028] Figure 1 This is a schematic flowchart of a method for preparing a recombinant protein vaccine containing halophilic midge allergens proposed in this application; Figure 2 Electrophoresis diagram of PCR amplification of the Cult1 gene and double enzyme digestion verification of the recombinant plasmid in this application; Figure 3 This image shows the expression and purification verification of the Cul t 1 recombinant protein in HEK293F cells. Figure 4 This is a schematic diagram of the mouse immunization and challenge experiment of this application; Figure 5 Comparison of H&E staining and immunohistochemical results of mouse skin tissue from different groups in this application; Figure 6 This is a graph showing the results of the detection of mouse serum-specific IgE and IgG2a antibody levels at different time points in this application; Figure 7 This is a statistical graph showing the number of scratching behaviors in mice within 30 minutes after local stimulation according to this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Technical concept: Related midge allergies can be addressed through specific immunotherapy based on natural allergen extracts. However, these extracts are often complex in composition and have low standardization. Furthermore, the adjuvants they commonly use tend to induce Th2 immune responses, thereby enhancing the sensitization rather than the protective effect of the allergen. This leads to an imbalance in immune regulation, insufficient production of blocking antibodies, and difficulty in achieving stable and lasting tolerability.

[0031] This application discloses a recombinant protein vaccine containing cuneus allergens, its preparation method, and its application. The cuneus allergen recombinant protein vaccine comprises Cult 1 recombinant protein and an immune adjuvant; its preparation method includes: S1, constructing a recombinant expression vector containing the Cult 1 gene; S2, transfecting the vector obtained in S1 into mammalian cells for expression; S3, collecting the expression cells from S2 and purifying the Cult 1 recombinant protein; S4, mixing the purified protein obtained in S3 with the immune adjuvant to prepare a vaccine; the cuneus allergen recombinant protein vaccine prepared by this method is used in the preparation of drugs for the prevention or treatment of allergic reactions to cuneus bites.

[0032] This application employs a complex adjuvant system composed of recombinant Cult 1 protein, CpG ODN1826, and Alum to construct a vaccine. The recombinant protein can mimic the epitopes of natural allergens, while Alum provides sustained antigen release and enhances antibody responses. CpG ODN1826 drives a balanced Th1 immune response. Through comprehensive and balanced immune functions, both achieve the effect of efficiently inducing blocking antibodies and preventing or reducing allergic reactions caused by midge bites.

[0033] Example 1: Construction and Expression of Cult1 Recombinant Protein Vaccine: The Cult1 gene was synthesized through codon optimization and cloned into the pCI-neo vector to construct the recombinant plasmid pCI-neo-Cult1-6His; subsequently, the recombinant plasmid was transfected into HEK293F cells for large-scale transient expression; after expression, the protein was purified by nickel column affinity chromatography, eluted with buffers containing 20 mM and 250 mM imidazole; finally, the protein concentration was determined by the BCA method, obtaining Cult1 protein with a concentration of 0.505 mg / mL; Figure 1 It can be seen that the entire process, starting from gene synthesis, through vector construction, cell transfection, protein expression and purification, is clear and systematic, ensuring the clarity and reproducibility of vaccine components; Figure 2 The PCR product and the size of the bands after enzyme digestion met expectations, indicating successful gene cloning and correct vector construction, laying the foundation for subsequent expression. Figure 3 Western blotting and SDS-PAGE analysis showed clear bands at the expected molecular weight and high protein purity after purification, demonstrating that the eukaryotic expression system can produce high-purity recombinant protein, supporting the reliability and stability of the vaccine.

[0034] Example 2: Vaccine preparation: See Figure 1It is known that the purified Cult1 protein was mixed with the immune adjuvant, namely 50 μg CpG ODN1826 and 500 μg Alum, to prepare vaccine formulations with doses of 25 μg, 50 μg and 100 μg, respectively.

[0035] Example 3: Mouse Immunoprotection Experiment: Mice were first divided into a blank control group (PBS), an adjuvant control group (VO), and low, medium, and high dose vaccine groups (V25, V50, V100). A three-stage model of "preventive immunization—systemic sensitization—local challenge" was then used for the experiment. After immunization, the vaccine efficacy was systematically evaluated from three levels: histopathology, humoral immunity, and behavior. Specifically, H&E staining and immunohistochemistry were used to analyze local tissue inflammatory responses, ELISA was used to detect serum specific antibody levels to assess humoral immune responses, and the number of scratching behaviors was recorded to quantify allergy-related behavioral changes. See Figure 4 , Figure 4 The detailed timeline and operational steps of the three stages, including immunization injection, sensitization challenge, and sample collection, demonstrate that the experimental design is rigorous and can simulate real allergic reactions; Figure 5 The results showed that inflammatory cell infiltration in the skin tissue of mice in the V25 group was reduced, and CD4... + and CD8 + The low level of T-cell infiltration supports the vaccine's ability to suppress local allergic inflammatory responses; while the blank control group and adjuvant control group showed cell infiltration, highlighting the vaccine's protective effect; see below. Figure 6 The bar chart revealed that the V25 group showed a decrease in specific IgE levels and an increase in protective antibody IgG2a levels after sensitization, with better results than other dose groups. This indicates that the vaccine can induce a Th1 immune response, balance the Th2 allergic reaction, and thus alleviate systemic allergy symptoms.

[0036] Example 4: Optimization of vaccine immunization dosage: By comparing the immunoprotective effects of different dosage groups of V25, V50, and V100, among which... Figure 6 The antibody level results support the conclusion of dose optimization: the V25 dose showed the best performance in inhibiting IgE and promoting IgG2a, and its effect remained stable over time; and combined with Figure 5 Histological results showed that the V25 group exhibited significant inflammation suppression, while Figure 7 The statistical analysis of the number of scratching behaviors in mice indirectly confirmed that the 25μg dose was the most effective, indicating that a low dose can achieve high-efficiency protection, which is beneficial to the safety and economy of the vaccine.

[0037] Example Conclusion: Example 1: High-purity Cul t1 recombinant protein was efficiently expressed in HEK293F cells using codon-optimized gene and mammalian expression systems. The correctness of the gene cloning was verified by... Figure 2 The PCR and enzyme digestion verification bands shown were confirmed, and the quality of protein expression and purification was verified by... Figure 3 Western blotting and SDS-PAGE results confirmed a clear, single target band, providing a well-defined and highly immunogenic antigenic basis for the vaccine. Example 2 involved combining the purified protein with CpGODN1826 and Alum adjuvant to prepare vaccine formulations at different doses. Example 3's mouse immunoprotection experiment used... Figure 4 The three-stage model shown indicates that the vaccine can effectively induce a protective immune response: the 25 μg dose group showed the highest efficacy. Figure 5 It showed that it could reduce inflammatory cell infiltration and CD4 in local skin tissue. + / CD8 + T cell aggregation, Figure 6 Further studies confirmed that this dose could reduce allergy-specific IgE levels and increase protective IgG2a antibody titers, thereby balancing the Th1 / Th2 immune response. Figure 7 A reduction in recorded scratching behavior reflects the relief of allergy symptoms; the dose optimization comparison in Example 4 ultimately determined 25 μg as the optimal immunization dose, which ensures the immune protection effect while also having advantages in safety and economy; in summary, this vaccine, through the synergistic effect of recombinant protein and compound adjuvant, can effectively prevent or reduce allergic reactions caused by midge bites, and the preparation process is stable and the dosage is reasonable.

[0038] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A recombinant protein vaccine containing midge allergens, characterized in that, It contains Cul t 1 recombinant protein and immune adjuvant.

2. The recombinant protein vaccine against midge sensitizers according to claim 1, characterized in that, The amino acid sequence of the Cult1 recombinant protein is shown in SEQ ID NO:1, or a variant that has at least 90% homology with it and retains immunogenicity.

3. The recombinant protein vaccine against midge sensitizers according to claim 1, characterized in that, The immune adjuvant comprises CpG ODN1826 and Alum.

4. The recombinant protein vaccine against midge allergens according to claim 1, characterized in that, The dose of Cul t 1 recombinant protein in the vaccine is 25 μg, 50 μg, or 100 μg.

5. A method for preparing a recombinant protein vaccine containing schistosome allergens, characterized in that, A recombinant protein vaccine against a midge allergen as described in any one of claims 1-4, comprising the following steps: S1. Construct a recombinant expression vector containing the Cult1 gene; S2. Transfect the vector obtained in S1 into mammalian cells for expression; S3. Collect the expression cells from S2 and purify them to obtain the Cul t 1 recombinant protein; S4. The purified protein obtained in S3 is mixed with an immune adjuvant to prepare a vaccine.

6. The method for preparing a recombinant protein vaccine against midge sensitizers according to claim 5, characterized in that, The mammalian cells in question are HEK293F cells.

7. The method for preparing a recombinant protein vaccine against midge sensitizers according to claim 5, characterized in that, The purification step employs nickel column affinity chromatography, specifically: equilibrate the nickel column with equilibration buffer, load cell lysate containing recombinant protein onto the column, remove non-specifically bound contaminating proteins with washing buffer, and finally elute the target protein with elution buffer.

8. The use of a recombinant protein vaccine according to any one of claims 1-4 in the preparation of a medicament for the prevention or treatment of allergic reactions to bites of Culicoides stripe midge.

9. The application of the recombinant protein vaccine against midge allergens according to claim 8, characterized in that, The immunization dose of the vaccine is 23-27 μg per dose.

10. The application of the recombinant protein vaccine containing midge sensitizer according to claim 8, characterized in that, The vaccine is administered via intramuscular injection.

Images