Recombinant cat allergen, pharmaceutical composition thereof, preparation method therefor, and use thereof

Abstract

Provided are a recombinant cat allergen, a pharmaceutical composition therefor, a preparation method therefor, and a use thereof. Compared with naturally extracted allergen products, the recombinant cat allergen has multiple advantages in desensitization immunotherapy and allergen diagnosis for cat allergic diseases. For example, high purity and high uniformity are achieved; a pure protein / peptide having specified properties and quality is defined; the allergenicity, immunogenicity, and tolerability of the allergen are predetermined; production is performed according to GMP standards; the production of a specified quantity and concentration in a reproducible manner is enabled; product standardization and quality controllability are achieved; regulatory requirements for modern pharmaceuticals and vaccines are met; and when used for diagnosis, the allergen can identify allergen molecules that truly cause allergic reactions and reveal cross-reactivity, and when used for treatment, the allergen enables targeted allergen immunotherapy for a patient, avoiding the influence of non-allergenic substances in an extract, improving the safety, and facilitating precise diagnosis, precise administration, etc.

Description

Cat recombinant allergen and its pharmaceutical composition, preparation method and application Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals and relates to a recombinant cat allergen and a preparation method and application thereof. Background Art

[0002] Allergic diseases, also known as allergic diseases, are mostly type I allergic reactions mediated by immunoglobulin E (IgE). The development of allergies can be divided into two stages: sensitization and allergy. The sensitization stage is the first exposure to an allergen, triggering B cells to produce allergen-specific IgE antibodies, during which the body does not react. The allergy stage occurs when the same allergen enters the sensitized body again or multiple times. IgE antibodies on the surface of mast cells or basophils cross-link with the allergen, leading to degranulation of mast cells and basophils and the release of a series of bioactive substances, causing allergic reactions (also known as allergy) and related symptoms, such as allergic asthma, hay fever, urticaria, allergic rhinitis, eczema, conjunctivitis, gastrointestinal type I allergic diseases, and severe anaphylaxis. The increasing incidence of allergic diseases has become a global public health concern, with the World Health Organization (WHO) listing them as a disease requiring priority research and prevention in the 21st century.

[0003] Cat allergen is a very important indoor allergen and a common cause of allergic rhinitis (AR) and bronchial asthma (BA). Studies have shown that 30% of AR and BA patients are allergic to cats, and the more children under two years old are exposed to cat allergens, the more likely they are to develop cat allergic diseases in adulthood.

[0004] Current treatments for allergic diseases caused by cat allergens include desensitization therapy (SIT) and anti-allergy therapy. Desensitization therapy, also known as allergen immunotherapy, involves repeatedly exposing patients to the allergen with gradually increasing doses via subcutaneous or sublingual injection, thereby increasing their tolerance to the allergen. Upon further exposure to the allergen, the release of inflammatory mediators is significantly reduced, and the severity of specific allergic reactions is significantly mitigated, thereby alleviating clinical symptoms and ultimately achieving tolerance and even immune tolerance. SIT is currently the only "cause-directed" therapy that can affect the natural course of allergic diseases and alter the immune response. In addition to these treatments, other approaches include avoiding contact with cats and immunizing cats with their own major allergens (primarily Feld 1) to induce neutralizing antibodies and reduce endogenous allergen levels.

[0005] The active ingredient of a desensitizing drug is a major allergenic protein, an allergen that can induce an allergic reaction. Major allergenic proteins with well-defined identities can be obtained through allergen extraction or recombinant expression. The active ingredients of Iwu Bio's dust mite drops and Artemisia annua pollen allergen sublingual drops, both marketed in China, and ALK-Abello's dust mite allergen preparation, marketed in Europe, are natural allergen extracts. Natural allergen extracts inevitably have quality issues due to limitations in raw material sources and production methods, such as the presence of undefined non-allergenic substances, contaminants, and high variability in allergen content and bioactivity (Valenta R, et al. Allergen Extracts for in vivo diagnosis and treatment of allergy: is there a future [J]. Journal of allergy & Clinical immunology in practice, 2018.). The European Academy of Allergy and Clinical Immunology's Guidelines on allergen immunotherapy: allergic rhinoconjunctivitis (2018) also pointed out that mixed allergens have many potential disadvantages, including dilution effects, potential allergen degradation due to the enzymatic activity of some allergens, and difficulties in fully demonstrating the efficacy of allergen combinations. Recombinant allergens produced through genetic engineering can achieve high purity and uniformity. While retaining the immunogenicity of natural allergens, they effectively avoid the drawbacks of mixed allergens extracted from natural sources. For example, pure proteins / peptides of defined properties and quality predetermine the allergenicity, immunogenicity, and tolerability of the allergen. They can be manufactured according to GMP specifications, producing specified quantities and concentrations in a reproducible manner, achieving product standardization and quality control, and meeting the regulatory requirements of modern pharmaceuticals and vaccines. For diagnostic purposes, they can identify the allergen molecules that truly cause allergic reactions and reveal cross-reactivity. For therapeutic purposes, they can target allergen immunotherapy based on the allergenic proteins that truly cause allergies, avoiding the impact of unknown non-allergenic substances in the extracts on diagnosis and treatment. This provides enhanced safety and facilitates accurate diagnosis and precise drug delivery (Valenta R, et al. Allergen Extracts for in vivo diagnosis and treatment of allergy: is there a future [J]. Journal of allergy & Clinical immunology in practice, 2018). There are currently no marketed desensitization drugs for cat allergies in China or abroad. Summary of the Invention

[0006] The applicant hopes to provide a variety of recombinant cat allergens to obtain highly purified and uniform allergen proteins with good biological activity, improve the quality controllability of the products, and lay the foundation for the accurate diagnosis and treatment of allergic diseases caused by cat allergens and the standardization of cat allergen products.

[0007] The first objective of the present invention is to provide a protein for treating allergic diseases caused by feline allergens, which is a recombinant FeI d 1 protein. The recombinant FeI d 1 protein is a fusion protein of FeI d 1 chain 1 (Chain 1) and chain 2 (Chain 2). Native Chain 1 and Chain 2 contain signal peptides, with Genbank accession numbers AAC37318 and AAC41616, respectively. In specific embodiments, the mature peptides lack the signal peptide, and their amino acid sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Natural allergens, influenced by various factors, generally exist as multiple isoallergens or variants. Alloallergens share similar molecular weights, similar or identical biological functions, and amino acid sequence identity >67%. Each allergen may have multiple highly identical sequences (>90% identity, typically differing by only a few amino acids), referred to as variants or isoforms (WHO / IUIS Allergen Nomenclature: providing a common language). The amino acid sequences of Chain 1 and Chain 2 described above may be derived from other alloallergens or variants.

[0008] The recombinant Feld 1 protein is preferably designed to have, from N-terminus to C-terminus, Feld 1 chain 1, followed by Feld 1 chain 2, or, from N-terminus to C-terminus, Feld 1 chain 2, followed by Feld 1 chain 1, with chains 1 and 2 fused directly or via a linker peptide. In specific embodiments, the amino acid sequences of Feld 1-1 and Feld 1-2 are used to illustrate the two aforementioned fusion sequences, wherein chains 1 and 2 are fused directly. It is well known to those skilled in the art that, in most cases, the start codon in prokaryotes is AUG, encoding formylmethionine, while the start codon in eukaryotes is AUG, encoding methionine. Therefore, recombinant proteins expressed in prokaryotes such as Escherichia coli typically contain formylmethionine at the N-terminus in addition to the native amino acid sequence, while recombinant proteins expressed in eukaryotes such as yeast typically contain methionine at the N-terminus in addition to the native amino acid sequence. For proteins with signal peptides, the amino acids encoded by the start codon are removed along with the signal peptide, resulting in the native protein sequence. Furthermore, the initial N-terminal formylmethionine of recombinantly expressed proteins can sometimes be cleaved, but this cleavage is often incomplete, and the cleavage ratio varies depending on the protein type. In the specific examples of the present application, FeI d 1-1 and FeI d 1-2 were expressed in E. coli, and their corresponding amino acid sequences, SEQ ID NO: 1 or SEQ ID NO: 2, have an additional N-terminal formylmethionine. Based on this common knowledge, the amino acid sequence of the recombinant FeI d 1 protein provided herein can also be such that the first N-terminal formylmethionine of the amino acid sequence of SEQ ID NO: 1 or 2 is deleted or replaced with methionine.

[0009] Natural FeI d 1 (nFel d 1) is a type I cat allergen, a secretoglobin, and the major allergenic protein. This protein is primarily found in cat saliva, anal glands, sebaceous glands, and fur. Studies have shown that over 80-95% of cat-allergic patients have Fel d 1-specific IgE antibodies in their sera. Cross-immunoelectrophoresis assays reveal that the majority of IgE antibodies against cat allergens in the sera of allergic patients are directed against Fel d 1, accounting for 69-90% of the total sensitizing activity (Benjamin Bonnet, et al. An update on molecular cat allergen: Fel d 1 and what else? Chapter 1: Fel d 1, the major cat allergen. Allergy Asthma and Clinical Immunology. December 2018). nFel d 1 consists of two chains: Chain 1 and Chain 2. Each chain is expressed from its own gene and undergoes post-translational modification. The two chains assemble through intermolecular disulfide bonds to form the complete Fel d 1. Using recombinant expression techniques to express these two genes separately and then assemble them to form a mature protein similar to the natural one is extremely difficult because the expression levels and assembly of the two genes are independent and difficult to control. The fusion protein obtained by the fusion expression of two chains in the present invention can greatly reduce the difficulty of expression, and the obtained recombinant Feld 1 protein has all the primary structures of chains 1 and 2 of natural Feld 1, has good biological activity, and compared with natural cat allergen extracts, avoids the differences in the content and activity of natural cat major allergens from different sources between batches, and the process and quality are more stable and controllable. It can also avoid the degradation of the major allergens by the interaction of other components in the natural extract and the generation of other allergic reactions, etc., meeting the requirements of modern biological products for safety, effectiveness and controllable quality, and can be used for the treatment and diagnosis of allergic diseases caused by cat allergens such as allergic rhinitis and asthma, improving the precision of desensitization immunotherapy and the accuracy of cat allergy diagnosis, and has good pharmaceutical prospects.

[0010] Preferably, the nucleotide sequence encoding the FeI d 1 protein is SEQ ID NO: 3 or SEQ ID NO: 4, corresponding to the FeI d 1-1 and FeI d 1-2 recombinant proteins, respectively. The chain 1 and chain 2 sequences are codon-optimized for the E. coli expression system, which is more conducive to the expression of the two chains of FeI d 1 in the E. coli expression system.

[0011] A second object of the present invention is to provide a Feld 4 mutant or a derivative thereof for treating allergic diseases caused by cat allergens. The Feld 4 mutant has the C at position 139 (based on the native mature Feld 1 peptide) of native Feld 4 deleted or replaced with another amino acid. When performing the amino acid replacement, any one or more of G, A, V, L, I, Y, S, K, R, H, F, W, M, or T can be selected for replacement, preferably any one of G, A, V, L, I, Y, F, or W, and more preferably any one of G, A, V, L, or I. The native Feld 4 is a native mature peptide, and the amino acid sequence is shown in SEQ ID NO: 7.

[0012] The FeI d 4 mutant also includes a protein obtained by mutating the allogeneic allergen or variant of the natural FeI d 4, wherein the mutation site is the site corresponding to position 139 of the natural FeI d 4 shown in SEQ ID NO:7.

[0013] In a specific embodiment of the present application, the recombinant FeI d 4 protein was expressed in an E. coli system and had an additional formylmethionine at the N-terminus compared to the amino acid sequence shown in SEQ ID NO: 7. Furthermore, based on the above common knowledge, if expressed in a eukaryotic system, an additional methionine at the N-terminus could also be added compared to the amino acid sequence shown in SEQ ID NO: 7.

[0014] The derivatives include the full-length protein of the FeI d 4 mutant of the present application, partial proteins of the FeI d 4 mutant of the present application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation of the FeI d 4 mutant of the present application.

[0015] Native FeI d 4 (nFel d 4) is a cat type IV allergen and a lipocalin. A study showed that the serum IgE positivity rate for FeI d 4 in patients with cat fur allergy reached 62.96% (17 of 27), a high level, making it of great significance for the diagnosis and treatment of cat allergies (Smith W, et al. Fel d 4, a cat lipocalin allergen. Clin Exp Allergy. 2004 November). The Fel d 4 mutant of the present invention deletes or replaces amino acid 139 of natural Fel d 4 (or the corresponding site of its alloallergen or variant) with other amino acids. Compared with the wild-type Fel d 4 recombinant protein, the molecular weight uniformity is significantly improved (from disordered to highly uniform), and the HPLC-RP purity is significantly improved (from less than 70% to more than 95%). The disulfide bond pairing of the mutant is consistent with the theory, and it has a correct primary structure. It is superior in terms of protein uniformity and purity, and also exhibits good in vitro and in vivo biological activity. Compared with natural cat allergen extracts, the mutant avoids the inter-batch differences in the content and activity of natural cat major allergens from different sources, and the process and quality are more stable and controllable. It can also avoid the interaction of other components in the natural extract to degrade the major allergen and produce other allergic reactions. It meets the requirements of modern biological products for safety, effectiveness, and controllable quality. It can be used for the treatment and diagnosis of allergic diseases caused by cat allergens, such as allergic rhinitis and asthma, and improves the accuracy of desensitization immunotherapy and cat allergy diagnosis. It has good pharmaceutical prospects.

[0016] The present invention also provides a nucleotide sequence encoding the above-mentioned FeI d 4 mutant or its derivatives. The preferred nucleotide base sequence is shown in SEQ ID NO:9.

[0017] A third object of the present invention is to provide a Feld 7 mutant or derivative thereof for treating allergic diseases caused by cat allergens. The Feld 7 mutant comprises a deletion or substitution of the C at position 101 (based on the mature peptide) of native Feld 7 with another amino acid. When performing the amino acid substitution, any one or more of G, A, V, L, I, Y, S, K, R, H, F, W, M, or T can be selected for substitution, preferably with any one of G, A, V, L, I, Y, F, or W, and more preferably with any one of G, A, V, L, or I. The native Feld 7 is a native mature peptide, and the amino acid sequence is shown in SEQ ID NO: 10.

[0018] The FeI d 7 mutant also includes a protein obtained by mutating the allogeneic allergen or variant of the natural FeI d 7, wherein the mutation site is the site corresponding to position 101 of the natural FeI d 7 shown in SEQ ID NO: 10.

[0019] In a specific embodiment of the present application, the recombinant FeI d 7 protein was expressed in an E. coli system and had an additional formylmethionine at the N-terminus compared to the amino acid sequence shown in SEQ ID NO: 10. Furthermore, based on the above common knowledge, if expressed in a eukaryotic system, an additional methionine at the N-terminus could also be added compared to the amino acid sequence shown in SEQ ID NO: 10.

[0020] The derivatives include the full-length protein of the FeI d 7 mutant of the present application, partial protein of the FeI d 7 mutant of the present application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation of the FeI d 7 mutant of the present application.

[0021] Natural Fel d 7 (nFel d 7) is a cat type VII allergen and a lipocalin. It is an important allergen for people with cat allergies in certain areas. It mainly cross-reacts with the dog allergen Can f 1 and the horse allergen Equ c 1. It is of great significance for the diagnosis and treatment of cat allergic diseases or cat-dog cross-allergic diseases (D. Apostolovic, et al. the cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1. Allergy. 2016 October.). The Fel d 7 mutant of the present invention deletes or replaces the 101st amino acid of natural Fel d 7 (or the corresponding site of its alloallergen or variant) with other amino acids. Compared with the wild-type Fel d 7 recombinant protein, the molecular weight is significantly uniform, the disulfide bond pairing is consistent with theory, the amino acid sequence, disulfide bonds and molecular weight are highly similar to the natural protein, and the mutant has good allergen immunoactivity. It is superior in protein homogeneity, purity and quality control. Compared with natural cat allergen extracts, the mutant avoids the differences in the content and activity of natural cat major allergens from different sources between batches, and the process and quality are more stable and controllable. It can also avoid the degradation of the major allergen by the interaction of other components in the natural extract and the generation of other allergic reactions. It meets the requirements of modern biological products for safety, effectiveness and controllable quality. It can be used for the treatment and diagnosis of allergic diseases caused by cat allergens, such as allergic rhinitis and asthma, improves the precision of desensitization immunotherapy and the accuracy of cat allergy diagnosis, and has good pharmaceutical prospects.

[0022] The present invention also provides a nucleotide sequence encoding the above-mentioned FeI d 7 mutant or its derivatives. The preferred nucleotide base sequence is shown in SEQ ID NO: 12.

[0023] The present invention also provides an expression vector comprising the above-mentioned codon-optimized cat recombinant allergen protein encoding gene, including expression vectors based on T7 promoter, such as pET32a, pET26b, pET28a, pDEST14 vectors, etc., and also including expression vectors based on temperature-controlled promoter PL-PR, such as pBV220 vectors, etc.

[0024] The present invention also provides an Escherichia coli host comprising the above-mentioned recombinant expression vector, wherein any Escherichia coli genetically engineered bacteria can be selected for use with the PL-PR promoter-based expression vector, preferably BL21 (DE3), BL21 AI (DE3), Top10, DH5α, JM109, Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys host, etc.; the Escherichia coli genetically engineered bacteria for use with the T7 promoter-based expression vector are preferably BL21 (DE3), BL21 AI (DE3), Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys, etc.

[0025] The present invention also provides a method for expressing a recombinant cat allergen protein, the method comprising the following steps:

[0026] A. constructing a vector containing the gene encoding the above-mentioned FeI d 1, FeI d 4 or FeI d 7 mutant or its derivative;

[0027] B. Linearize the vector from step A and transform it into an E. coli strain, and culture it under appropriate conditions;

[0028] C. Recover and purify the protein.

[0029] The present invention also provides a method for purifying a recombinant cat allergen protein, which is as follows:

[0030] The E. coli cells cultured by the above method are resuspended, broken, and the precipitate or supernatant is collected. The precipitate is the crude extract of the target protein inclusion body;

[0031] If inclusion bodies are collected, they are subjected to crude purification, denaturation, and renaturation treatment to obtain a renaturation solution;

[0032] If the supernatant is collected, it is concentrated by ultrafiltration membrane to obtain the filtrate;

[0033] The refolded solution or filtrate is collected and purified by ion exchange chromatography and hydrophobic chromatography in two steps to obtain the target protein stock solution. In a specific embodiment, Q Sepharose Fast Flow and Phenyl Sepharose High Performance 6 (HS) are used for the two-step purification.

[0034] The present invention also provides the use of the above-mentioned recombinant cat allergen protein in preparing a diagnostic reagent for detecting cat allergies.

[0035] The present invention also provides the use of the above-mentioned recombinant cat allergen protein in preparing medicine for treating cat allergic diseases.

[0036] The present invention also provides a composition for treating allergic diseases caused by cat allergens, comprising FeI d 1, FeI d 4, and FeI d 7. The FeI d 1, FeI d 4, and FeI d 7 are naturally extracted proteins or recombinant proteins. The FeI d 1, FeI d 4, and FeI d 7 are preferably the recombinant proteins described above.

[0037] The cat recombinant allergen protein finally prepared by the present invention meets the requirements of human recombinant DNA products in terms of purity, process impurity residues, molecular characterization, etc., with a purity of >95%, and has an amino acid sequence, disulfide bonds and molecular weight that are completely consistent with natural proteins, thereby improving the quality controllability of cat allergen products. It has good reactivity with clinical allergy-positive serum in vitro and shows good biological activity in vivo. It has the potential to be used in the development of cat allergy desensitization treatment drugs and diagnostic reagents, laying the foundation for the accurate diagnosis and treatment of allergic diseases caused by cat allergens and the standardization of cat allergen products. On the other hand, the recombinant Fel d 1 of the present invention is a fusion protein of Fel d 1Chain 1 and Chain 2, which successfully achieves heterologous expression in Escherichia coli. While retaining the immunogenicity of the natural protein, the difficulty of expression is greatly reduced. The yield of the pure target protein reaches 318 mg / L, which is conducive to the control of the production process and provides a guarantee for the standardized preparation of high-purity and high-uniformity recombinant Fel d 1 protein; the Fel d 4 / Fel d 7 mutant or its derivative recombinant protein of the present invention has obvious advantages over the unmutated wild-type Fel d 4 / Fel d 7 recombinant protein in terms of protein purity, uniformity, molecular characterization, etc., and still retains good allergenic biological activity after mutation of the relevant amino acid sites, which is more conducive to the control of the recombinant expression production process and the standardized and correct preparation of recombinant proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1: HPLC-SEC analysis results of pure recombinant Fel d 1, wherein Figure 1-1 is the HPLC-SEC analysis results of pure Fel d 1-1, and Figure 1-2 is the HPLC-SEC analysis results of pure Fel d 1-2.

[0039] Figure 2: HPLC-RP analysis results of pure recombinant FeI d 1, wherein Figure 2-1 is the HPLC-RP analysis results of pure FeI d 1-1, and Figure 2-2 is the HPLC-RP analysis results of pure FeI d 1-2.

[0040] Figure 3: Deconvolution plot of the molecular weight detection of recombinant Fel d 1, where Figure 3-1 is the deconvolution plot of the molecular weight detection of Fel d 1-1, and Figure 3-2 is the deconvolution plot of the molecular weight detection of Fel d 1-2.

[0041] Figure 4: Fel d 1 amino acid coverage analysis results, where Figure 4-1 is the Fel d 1-1 amino acid coverage analysis results, and Figure 4-2 is the Fel d 1-2 amino acid coverage analysis results.

[0042] Figure 5: Identification and analysis results of disulfide bonds of recombinant FeI d 1, wherein Figure 5-1 shows the identification and analysis results of disulfide bonds of FeI d 1-1, and Figure 5-2 shows the identification and analysis results of disulfide bonds of FeI d 1-2.

[0043] Figure 6: Penh detection results under stimulation with different concentrations of methacholine (Mch) in Example 6.

[0044] Figure 7: Serum antibody ELISA test results of Example 6.

[0045] Figure 8: Bronchoalveolar lavage fluid (BALF) cell staining in Example 6, where Figure 8-1 shows the white blood cell count results of the negative group and the recombinant FeI d 1 group, Figure 8-2 shows the microscopic field of view of BALF cell staining in the negative group, and Figure 8-3 shows the microscopic field of view of BALF cell staining in the recombinant FeI d 1 group.

[0046] Figure 9: Cytokine detection results of the supernatant of spleen cells stimulated in vitro in Example 6.

[0047] Figure 10: HPLC-RP analysis results, wherein Figure 10-1 is the HPLC-RP analysis results of the pure wild-type FeI d 4.

[0048] Figure 10-2 shows the HPLC-RP analysis results of pure Fel d 4 mutant 2.

[0049] FIG11 : HPLC-SEC analysis results of pure Fel d 4 mutant 2.

[0050] Figure 12: Non-reducing SDS-PAGE analysis results of pure FeI d 4, wherein Lane 1-2 is the non-reducing electrophoresis analysis of pure FeI d 4 wild type (5, 10 μl), and Lane 3-4 is the non-reducing electrophoresis analysis of pure FeI d 4 mutant 2 (10, 5 μl).

[0051] Figure 13: Deconvolution plot of molecular weight detection of FeI d 4 mutant 2.

[0052] FIG14 : Amino acid coverage analysis results of Fel d 4 mutant 2.

[0053] FIG15 : Results of disulfide bond identification analysis of Fel d 4 mutant 2.

[0054] Figure 16: Serum antibody ELISA test results of Example 12.

[0055] Figure 17: Bronchoalveolar lavage fluid (BALF) cell staining in Example 12, wherein Figure 17-1 shows the microscopic view of BALF cell staining in the negative group, and Figure 17-2 shows the microscopic view of BALF cell staining in the Fel d 4 mutant group.

[0056] Figure 18: Cytokine detection results of the supernatant of spleen cells stimulated in vitro in Example 12.

[0057] Figure 19: HPLC-RP analysis results, wherein Figure 19-1 is the HPLC-RP analysis results of the pure wild-type FeI d 7.

[0058] Figure 19-2 shows the HPLC-RP analysis results of pure Fel d 7 mutant 1.

[0059] FIG20 : HPLC-SEC analysis results of pure Fel d 7 mutant 1.

[0060] Figure 21: Non-reducing SDS-PAGE analysis results of pure Fel d 7 mutant 1 and wild-type Fel d 7, wherein Lane 1: non-reducing electrophoresis analysis of pure Fel d 7 wild-type (10 μl); Lane 2: non-reducing electrophoresis analysis of pure Fel d 7 mutant 1 (10 μl).

[0061] Figure 22: Molecular weight detection deconvolution diagram, wherein Figure 22-1 is the molecular weight detection deconvolution diagram of Fel d 7 wild type.

[0062] Figure 22-2 is a deconvolution diagram of the molecular weight detection of Fel d 7 mutant 1.

[0063] FIG23 : Amino acid coverage analysis results of Fel d 7 mutant 1.

[0064] FIG24 shows the results of disulfide bond identification analysis of Fel d 7 mutant 1.

[0065] Figure 25: Penh detection results under stimulation of different concentrations of methacholine (Mch) in Example 17.

[0066] Figure 26: Serum-specific IgE ELISA test results of Example 17.

[0067] Figure 27: Bronchoalveolar lavage fluid (BALF) cell staining in Example 17, wherein Figure 27-1 is the microscopic view of BALF cell staining in the negative group, and Figure 27-2 is the microscopic view of BALF cell staining in the Fel d 7 group.

[0068] Figure 28: Cytokine detection results of the supernatant of spleen cells stimulated in vitro in Example 17.

[0069] FIG29 : Penh detection results under stimulation of different concentrations of methacholine (Mch) in Example 18. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to specific examples. It should be understood that the examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0071] Example 1: Design of recombinant FeI d 1 and construction of expression strain

[0072] The mature peptide sequences of FeI d 1, Chain 1, and Chain 2 were obtained according to Genbank accession numbers AAC37318 and AAC41616, respectively. FeI d 1-1 was designed according to the sequence N-Chain 1 mature peptide-Chain 2 mature peptide-C. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to perform codon optimization of the gene using an E. coli expression system, and the coding DNA sequence for FeI d 1-1 was synthesized. Based on the synthetic FeI d 1-1 coding DNA sequence, the coding DNA sequence for FeI d 1-2 (constructed as N-Chain 2-Chain 1-C) was obtained by PCR. The FeI d 1-1 and FeI d 1-2 coding DNA sequences had an NdeI restriction site introduced at the 5' end and an XhoI restriction site introduced at the 3' end.

[0073] The recombinant expression of FeI d 1 in the present application can be based on an expression vector of a T7 promoter, such as pET32a, pET26b, pET28a, pDEST14 vector, etc., and can also be based on an expression vector of a temperature-controlled promoter PL-PR, such as pBV220 vector, etc. Among them, any Escherichia coli genetically engineered bacteria can be selected for use with an expression vector based on a PL-PR promoter, such as BL21 (DE3), BL21 AI (DE3), Top10, DH5α, JM109, Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys host, etc.; Escherichia coli genetically engineered bacteria based on a T7 promoter expression vector can be selected from BL21 (DE3), BL21 AI (DE3), Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys, etc. This embodiment uses pET26b vector and BL21 (DE3) host as construction examples.

[0074] The DNA sequences of Fel d 1-1 and Fel d 1-2 were cloned into the pET26b vector to form pET26b-Fel d 1-1 and pET26b-Fel d 1-2 expression plasmids, respectively. The two recombinant expression plasmids were transformed into BL21 (DE3) hosts to form BL21 (DE3)-pET26b-Fel d 1-1 and BL21 (DE3)-pET26b-Fel d 1-2 expression strains, respectively.

[0075] Table 1

[0076] Example 2: Recombinant expression of recombinant FeI d 1

[0077] The expression of FeI d 1-1 in pET26b vector and BL21(DE3) host is shown below. The same method was used to express FeI d 1-2.

[0078] 1. A glycerol tube of BL21(DE3)-pET26b-Fel d 1-1 strain was streaked onto an LB-agar plate containing 50 μg / ml and cultured at 37°C for 12 h.

[0079] 2. Pick a colony from the agar plate and inoculate it into 10 ml of TB liquid medium at 37°C, 220 rpm, and 8 hours. This is the primary seed medium.

[0080] 3. Inoculate 5% of the primary seeds into 10 bottles of 500 ml TB liquid culture medium at a ratio of 5%. Culture conditions: 37°C, 220 rpm. When the OD of the culture medium reaches 0.8, add 0.5 mM IPTG to induce expression. After 6 hours of induction, collect the bacteria by centrifugation for later use.

[0081] Example 3: Extraction and purification of recombinant FeI d 1

[0082] The extraction and purification of FeI d 1-1 are shown below, and the same process was used to obtain the purified sample of FeI d 1-2.

[0083] 1. The fermented bacteria were resuspended to 100 g / L in 10 mM PB + 150 mM sodium chloride (pH 7.4) buffer and evenly dispersed; crushed by a high-pressure homogenizer with a crushing pressure of 900 bar ± 50 bar, and the crushing cycle was repeated three times. The material temperature was controlled not to exceed 25 ° C during the crushing process; after the crushing, the precipitate was collected by centrifugation. The precipitate is the crude extract of the target protein inclusion body;

[0084] 2. The obtained inclusion bodies were resuspended in 10 mM PB + 1.5 M urea + 0.5% Triton X-100 + 150 mM sodium chloride, pH 7.4, at a ratio of 1:100 (mass / volume), dispersed evenly, stirred at 2-8°C for 8 h, and the inclusion body precipitate was collected by centrifugation. Repeat this process twice to obtain crude and pure inclusion bodies.

[0085] 3. After crude purification, resuspend the inclusion bodies in denaturation solution at a ratio of 1g / 100ml and stir at room temperature for at least 1 hour until basically clear. Denaturation solution: 10mM PB + 6M guanidine hydrochloride + 10mM DTT, pH 8.0. After denaturation, centrifuge and collect the supernatant.

[0086] 4. Renaturation: Slowly add the denaturing solution to the renaturing solution at a ratio of 1 / 10, stirring rapidly to disperse the protein, to prevent the local protein concentration in the renaturing solution from being too high. Control the addition time of the denaturing solution to 10 hours; control the temperature of the renaturing solution at 15°C during the addition process; after the denaturing solution is added, maintain the renaturation for 36 hours; Renaturing solution: 20mM Tris + 0.5M urea + 3mM reduced glutathione + 1mM oxidized glutathione, pH 8.0;

[0087] 5. After renaturation, centrifuge the renaturation solution, filter to remove insoluble matter, and ultrafilter using a 5 kDa ultrafiltration membrane to concentrate the solution to 10 mM PB, pH 8.0. Filter and set aside.

[0088] 6. First purification step: After refolding the buffer, the sample was purified using Q Sepharose Fast Flow, with the sodium chloride concentration linearly reaching 0.5 M within 10 column volumes, and the target protein peak was collected; purification binding buffer: 10 mM PB, pH 8.0; purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 8.0;

[0089] 7. Second purification step: Supplement the purified sample from the first step with ammonium sulfate to a final concentration of 0.5 M and adjust the pH to 7.5. Purify using a Phenyl Sepharose High Performance 6 (HS) column, ensuring that the elution buffer reaches 100% linearity within 10 column volumes, and collect the target protein peak. Purification elution buffer: 10 mM PB, pH 7.4; purification binding buffer: 10 mM PB + 0.5 M ammonium sulfate, pH 7.4.

[0090] 8. The target protein peak collected from the two-step chromatographic purification was ultrafiltered into 20 mM PB buffer at pH 7.0. After filtration and sterilization, the target protein stock solution was obtained. After the content of the stock solution was determined, the inclusion bodies produced in a single batch in a 3L fermenter were renatured and purified to obtain 954 mg of the target protein, equivalent to a pure target protein yield of 318 mg / L.

[0091] 9. The final samples were analyzed using HPLC-SEC and HPLC-RP, and the results are shown in Figures 1 and 2, respectively. Figures 1 and 2 show that both FeI d 1-1 and FeI d 1-2 achieved purity exceeding 95% using the two different detection methods, meeting general purity requirements for recombinant proteins for pharmaceutical use.

[0092] Example 4: Primary structure analysis and molecular weight of recombinant FeI d 1

[0093] LC-MS molecular weight can accurately reflect whether the primary sequence of biological macromolecules is correct, including whether the N- and C-terminal sequences are missing, and whether there are post-translational modifications such as glycosylation, oxidation, and deamidation. It is one of the most important analytical methods for biological macromolecules.

[0094] Using our Thermo Scientific TM The purified recombinant Fel d 1-1 and Fel d 1-2 proteins were analyzed by LC-MS molecular weight and primary structure using a high-resolution mass spectrometry system. The results are shown in Figures 3 to 5.

[0095] As shown in Figures 3 to 5, the molecular weights of Fel d 1-1 and Fel d 1-2 are consistent with the expected molecular weight (theoretical molecular weight of 18,100 Da), and the amino acid coverage of both proteins reaches 100%, thus confirming that the primary structures of both proteins are correct. Simultaneously, identification of disulfide bond pairings in Fel d 1-1 and Fel d 1-2 revealed that both designs identified disulfide bonds consistent with the expected theoretical disulfide bonds, laying the foundation for the formation of higher-order structures.

[0096] Example 5: Serological reactivity detection of recombinant FeI d 1

[0097] In this example, clinical cat allergy serum (collected from Jiangsu Provincial People's Hospital, March-May 2023) was used to perform reactivity detection with the recombinant FeI d 1 prepared in this application. The detection method is as follows:

[0098] 1. Coating: Dilute the recombinant protein Fel d 1 to 1 μg / mL in 0.05 M carbonate coating buffer (pH 9.6), add 100 μL / well to the ELISA plate, and place at 2-8°C overnight;

[0099] 2. Blocking: The next day, remove the ELISA plate, wash once with PBST, add 2% BSA in PBST at 200 μl / well, and incubate at 37°C for 2 h.

[0100] 3. Sample preparation: Dilute clinical cat allergy-positive and -negative sera with 2% BSA in PBST (dilution multiples are shown in Tables 2 and 3). 2% BSA in PBST is used as a blank background. Discard the liquid in the ELISA plate and add 100 μL / well of each dilution sample to the ELISA plate. Incubate at 37°C, 300 rpm, for 2 h.

[0101] 4. Detection: Wash the ELISA plate three times with PBST, add 1:1500 diluted mouse anti-human IgE-HRP secondary antibody at 100 μl / well, and incubate at 37°C at 300 rpm for 1 hour.

[0102] 5. Color development, termination, and reading: Wash the plate three times with PBST, add 100 μl / well of TMB VII colorimetric solution, react at 37°C for 15 min, then add 50 μl / well of 2 M sulfuric acid, and immediately read at 450 nm (microplate reader: Thermo MultiSKANGO).

[0103] 6. Result Interpretation and Analysis: Microplate reader readings reflect the level of binding of Fel d 1 to specific IgE in serum. Table 2 shows the results of the reactions of Fel d 1-1 and Fel d 1-2 with different clinical cat allergy sera. Both Fel d 1-1 and Fel d 1-2 exhibit varying degrees of reactivity with cat allergy-positive sera. Their reactivity with different cat allergy sera at the same dilution factor is similar. The reactivity shows a positive correlation with the clinically reported values ​​of positive sera, consistent with the conclusion that Fel d 1 is the primary allergen in cat allergies.

[0104] Table 3 uses 1.5 times the blank background value as the cutoff value, and reports the maximum dilution factor above the cutoff value as the reaction result. Higher dilutions indicate higher levels of specific IgE reactive with Fel d 1 in clinical cat allergic sera. The results show that recombinant Fel d 1-1 reacted to varying degrees with 11 clinical cat allergy-positive sera but not with negative sera, consistent with the conclusion that Fel d 1 is the primary allergen in cat allergies. Furthermore, the results showed that three clinical sera (RY179, RY187, and RY195) did not react with recombinant Fel d 1-1, yet the clinically reported results were positive, suggesting that these three cat allergic sera may not contain Fel d 1-specific IgE but rather specific IgE other than Fel d 1, suggesting that the patients may have reacted to cat allergens other than Fel d 1. The reaction results for recombinant Fel d 1-2 also align with the above conclusions.

[0105] In summary, the recombinant FeI d 1 of the present application is reactive with clinical cat allergy-positive serum, can bind to specific IgE in the serum, and the reactivity is correlated with the clinical report value. The recombinant FeI d 1 of different construction forms of the present application has similar reactivity with clinical serum.

[0106] Table 2

[0107] Table 3

[0108] Example 6: Evaluation of in vivo sensitization and desensitization efficacy of recombinant FeI d 1

[0109] 1. Experimental Materials:

[0110] BALB / c female mice, 6-7 weeks (age upon arrival), were provided by Changzhou Cavens Laboratory Animal Co., Ltd.

[0111] The recombinant FeI d 1 solution prepared in this application (taking FeI d 1-1 as an example).

[0112] Aluminum hydroxide adjuvant was provided by Thermo Fisher Scientific.

[0113] 2. Allergen preparation:

[0114] Sensitization sample: Prepare a 0.0067 mg / ml FeI d 1-1 solution with aluminum hydroxide adjuvant at a volume ratio of 3:1. Shake well before injection.

[0115] Stimulation sample: A FeI d 1-1 solution with a concentration of 0.05 mg / ml was used as the stimulation sample.

[0116] 3. Drug delivery system:

[0117] 3.1 Sensitization stage:

[0118] Sixteen mice were randomly divided into two groups based on body weight, with 8 mice in each group. Group and dosing details are shown in Table 4. The recombinant Fel d 1 group received a subcutaneous injection of 1 μg of the sensitized sample per mouse, while the negative group received an equal amount of blank sample once a week for a total of three times.

[0119] Table 4 Grouping and drug administration

[0120] 3.2 Excitation stage:

[0121] One week after the final sensitization, the recombinant Fel d 1 group was challenged with the challenge sample (40 μl / animal / time) once daily for 7 consecutive days. The negative group was administered an equal volume of blank sample. See Table 4 for details.

[0122] 4. Evaluation indicators:

[0123] 4.1 Penh:

[0124] Twenty-four hours after the final challenge, all mice were monitored using a noninvasive whole-body plethysmography (WBP) system at methacholine (Mch) concentrations of 0, 6.25, 12.5, 25, and 50 mg / ml to measure Penh values. Penh values ​​reflect airway resistance; higher Penh values ​​indicate increased airway reactivity and more severe airway symptoms.

[0125] 4.2 Serum antibodies

[0126] 48 hours after the last challenge, whole blood was collected by enucleation of the eyeball, allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 10 minutes to prepare serum. Serum antibodies were detected by ELISA (antigen coated with FeI d 1-1).

[0127] 4.3 White blood cell count in BALF:

[0128] After collecting bronchoalveolar lavage fluid (BALF), three BALF fluids were combined and centrifuged, the supernatant was discarded, and the precipitated cells were resuspended in 1× PBS buffer and mixed well. After staining according to the "Wright-Giemsa Staining Solution Operating Instructions", three fields of view were randomly selected under an upright microscope at 200 times magnification, and the stained cells were counted using Image J software to calculate the average value.

[0129] 4.5 Cytokines in the supernatant of spleen cells stimulated in vitro:

[0130] Splenocytes were isolated from mice in each group under sterile conditions and stimulated with the challenge sample. Supernatants were collected after 144 hours of co-culture with the challenge sample. Cytokines IL-4, IL-5, IL-10, and IL-13 in the supernatants of splenocytes stimulated in vitro were assayed using CBA.

[0131] 5. Experimental results:

[0132] 5.1 Penh:

[0133] As shown in Table 5 and Figure 6, the Penh of the recombinant FeI d 1 group mice was higher than that of the negative group under stimulation with different concentrations of MCh. When the MCh concentration was 6.25 mg / mL and 12.5 mg / mL, the Penh was significantly higher than that of the negative group (p < 0.05).

[0134] Table 5 Penh values ​​of mice (Mean ± SEM) Note: * indicates p < 0.05 compared with the negative group

[0135] 5.2 Serum antibodies

[0136] As shown in Table 6 and Figure 7, the serum antibody test results of each group are

[0137] IgE: The recombinant FeI d 1 group was significantly higher than the negative group (p<0.01).

[0138] IgG1: The expression of recombinant FeI d 1 group was significantly higher than that of negative group (p<0.01).

[0139] IgG2a: The level of recombinant FeI d 1 group was significantly higher than that of negative group (p<0.01).

[0140] Table 6 Serum antibody values ​​(Mean ± SEM) Note: ** Compared with the negative group, P < 0.01

[0141] 5.3 WBC count in BALF

[0142] As shown in Table 7, Figure 8-1 and Figures 8-2 and 8-3, which show that the WBC count in the BALF of mice in the recombinant Fel d 1 group was significantly higher than that in the negative group (p < 0.05).

[0143] Table 7 WBC count in BALF (Mean ± SEM) Note: * indicates p < 0.05 compared with the negative group

[0144] 5.4 Cytokines in the supernatant of spleen cells stimulated in vitro

[0145] As shown in Table 8 and Figure 9, the detection results of TH2 cytokines IL4, IL5, IL10, and IL13 showed that the recombinant Fel d 1 group increased significantly, and IL-4, IL-5, and IL-10 were significantly different from those in the negative group (p < 0.05).

[0146] Table 8 TH2 cytokines in the supernatant of spleen cells stimulated in vitro (Mean ± SEM) Note: * indicates p < 0.05 compared with the negative group

[0147] 6.1 Experimental Conclusions

[0148] The results of this study showed that in an allergic airway inflammation model constructed by sensitization and stimulation with recombinant FeI d 1 protein, mice in the recombinant FeI d 1 group showed elevated levels of various indicators, including airway symptoms, antibody changes, target organ inflammation, and cytokine production. This indicates that the recombinant FeI d 1 protein of this application is immunogenic in vivo, capable of producing allergic reactions, increasing allergen-specific antibodies in the body, and stimulating the body's immune response. Therefore, the recombinant FeI d 1 protein of this application has the potential to be used as an active ingredient in cat desensitization drugs or as a component of cat allergen detection reagents for the diagnosis and treatment of feline allergic diseases.

[0149] Example 7: Design of FeI d 4 mutant and construction of expression strain

[0150] Natural FeI d 4 may have a variety of alloallergens or variants. This example takes the nFel d 4 sequence with Genbank accession number AAS77253 as an example.

[0151] The mature peptide sequence of nFel d 4 was obtained based on the Genbank accession number, as shown in SEQ ID NO:7. This sequence was reverse-translated into a DNA sequence and codon-optimized using an E. coli expression system at Nanjing GenScript Biotechnology Co., Ltd. The coding DNA sequence for nFel d 4 was synthesized artificially, as shown in SEQ ID NO:8. A deletion or amino acid substitution mutation was introduced at position 139 of nFel d 4 by PCR (mutation information is shown in Table 9). The coding DNA sequence of the mutants had an NdeI restriction site introduced at the 5' end and an XhoI restriction site introduced at the 3' end. For mutant 2, its DNA sequence is shown in SEQ ID NO:9.

[0152] The recombinant expression of FeI d 4 in the present application can be based on an expression vector of a T7 promoter, such as pET32a, pET26b, pET28a, pDEST14 vector, etc., and can also be based on an expression vector of a temperature-controlled promoter PL-PR, such as a pBV220 vector. Among them, any Escherichia coli genetically engineered bacteria can be selected based on the PL-PR promoter expression vector, such as BL21 (DE3), BL21 AI (DE3), Top10, DH5α, JM109, Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys host, etc.; Escherichia coli genetically engineered bacteria based on the T7 promoter expression vector can be selected from BL21 (DE3), BL21 AI (DE3), Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys, etc. This embodiment is described using pET26b vector and BL21 (DE3) host as examples.

[0153] The DNA sequences of the above-mentioned FeI d 4 mutants were cloned into the pET26b vector to form the pET26b-Fel d 4 mutant expression plasmid, and then transformed into the BL21 (DE3) host to form the BL21 (DE3)-pET26b-Fel d 4 mutant expression strain.

[0154] Table 9 Summary of mutation information of each sample

[0155] Example 8: Recombinant expression of FeI d 4 and its mutants

[0156] The expression of FeI d 4 mutant 2 in pET26b vector and BL21 (DE3) host is shown below. The same method was used to express nFel d 4 and other mutants.

[0157] 1. A glycerol tube of BL21(DE3)-pET26b-Fel d 4 mutant 2 was streaked onto an LB-agar plate containing 50 μg / ml and cultured at 37°C for 12 h.

[0158] 2. Pick a colony from the agar plate and inoculate it into 10 ml of TB liquid medium at 37°C, 220 rpm, and 8 hours. This is the primary seed medium.

[0159] 3. Inoculate 10 bottles of 500 ml TB liquid culture medium with 1% of the first-level seeds at a ratio of 1-2%. Culture conditions: 37°C, 220 rpm. When the OD of the culture medium reaches 0.8, add 0.5 mM IPTG to induce expression. After 4 hours of induction, collect the bacteria by centrifugation for later use.

[0160] Example 9: Extraction and purification of FeI d 4 and its mutants

[0161] The extraction and purification of Fel d 4 mutant 2 are shown below. nFel d 4 and other mutants were obtained using the same process.

[0162] 1. The fermented bacteria were resuspended to 100 g / L in 10 mM PB + 150 mM sodium chloride (pH 7.4) buffer and evenly dispersed; crushed by a high-pressure homogenizer with a crushing pressure of 900 bar ± 50 bar, and the crushing cycle was repeated three times. The material temperature was controlled not to exceed 25 ° C during the crushing process; after the crushing, the precipitate was collected by centrifugation. The precipitate is the crude extract of the target protein inclusion body;

[0163] 2. The obtained inclusion bodies were resuspended in 10 mM PB + 1.5 M urea + 0.5% Triton X-100 + 150 mM sodium chloride, pH 7.4, at a ratio of 1:100 (mass / volume), dispersed evenly, stirred at 2-8°C for 10 h, and the inclusion body precipitate was collected by centrifugation. Repeat the above operation twice to obtain crude and pure inclusion bodies.

[0164] 3. After crude purification, resuspend the inclusion bodies in denaturation solution at a ratio of 1g / 100ml and stir at room temperature for at least 1 hour until basically clear. Denaturation solution: 10mM PB 6M guanidine hydrochloride + 10mM DTT, pH 8.0. After denaturation, centrifuge and collect the supernatant.

[0165] 4. Renaturation: Slowly add the denaturing solution to the renaturing solution at a ratio of 1 / 10, stirring rapidly to disperse the protein, to prevent the local protein concentration in the renaturing solution from being too high. Control the addition time of the denaturing solution to 10 hours; control the temperature of the renaturing solution at 15°C during the addition process; after the denaturing solution is added, maintain the renaturation for 36 hours; Renaturing solution: 20mM Tris + 0.5M urea + 3mM reduced glutathione + 1mM oxidized glutathione, pH 8.0;

[0166] 5. After renaturation, centrifuge or filter the renaturation solution to remove insoluble matter, and ultrafilter and concentrate using a 10 kDa ultrafiltration membrane to replace the solution with 10 mM PB, pH 8.0. Filter and set aside.

[0167] 6. First purification step: After refolding the buffer, the sample was purified using Q Sepharose Fast Flow, with the sodium chloride concentration linearly reaching 0.5 M within 10 column volumes, and the target protein peak was collected; purification binding buffer: 10 mM PB, pH 8.0; purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 8.0;

[0168] 7. Second purification step: Supplement the purified sample from the first step with ammonium sulfate to a final concentration of 0.5 M and adjust the pH to 7.4-8.0. Purify using a Phenyl Sepharose High Performan Ce 6 (HS) column, ensuring that the elution buffer reaches 100% linearity within 10 column volumes, and collect the target protein peak. Purification elution buffer: 10 mM PB, pH 7.4; purification binding buffer: 10 mM PB + 0.5 M ammonium sulfate, pH 7.4.

[0169] 8. The target protein peak collected from the two-step chromatography purification was ultrafiltered and replaced with a 20 mM PB buffer at pH 7.0; after filtration and sterilization, the target protein stock solution was obtained;

[0170] 9. The final samples were analyzed using HPLC-SEC, HPLC-RP, and non-reduced SDS-PAGE. The results are shown in Figures 10 to 12.

[0171] The test results indicate that both wild-type Fel d 4 and mutant 2 achieved the same electrophoretic purity under the same process conditions, with no visible impurities observed by non-reducing electrophoresis. However, the HPLC-RP purity of wild-type Fel d 4 exhibited a distinct, difficult-to-separate impurity peak, while the mutant did not. This suggests that the wild-type Fel d 4 pure product contains related proteins with similar molecular weights to the target protein but significantly different hydrophobic properties. This suggests that mutant 2 offers advantages over wild-type Fel d 4 in terms of recombinant protein purity and homogeneity.

[0172] Further HPLC-RP purity analysis of each mutant (the results are shown in Table 10) revealed that under the same process conditions, the purity of each mutant reached over 95%. Since each mutant mutated amino acid 139 of nFel d 4, the mutation of amino acid at position 139 should be the key to improving the purity of Fel d 4 RP. Compared with wild-type Fel d 4, the Fel d 4 mutant of the present invention has greater advantages in recombinant protein purity and homogeneity, and is more conducive to recombinant expression and production.

[0173] Table 10 Summary of HPLC-RP purity of each sample

[0174] Example 10: Primary structure analysis and molecular weight of Fel d 4 and its mutants

[0175] LC-MS molecular weight can accurately reflect whether the primary sequence of biological macromolecules is correct, including whether the N- and C-terminal sequences are missing, and whether there are post-translational modifications such as glycosylation, oxidation, and deamidation. It is one of the most important analytical methods for biological macromolecules.

[0176] Using our company's Thermo SCientifiC TM A high-resolution mass spectrometry system was used to perform LC-MS molecular weight and primary structure analysis on the purified wild-type FeI d 4 and its mutant recombinant proteins. The results are shown in Figures 13-15 and Table 11. Recombinant proteins expressed in E. coli usually contain an additional formylmethionine at the N-terminus, encoded by a start codon, based on the native amino acid sequence. In the specific examples of the present application, recombinant FeI d 4 (rFel d 4) was expressed and prepared in an E. coli system. Therefore, compared to the nFel d 4 mature peptide sequence, as shown in Figure 14, rFel d 4 has one more amino acid, the first at the N-terminus being formylmethionine, and the rest of the primary structure is consistent with nFel d 4. In addition, the nFel d 4 mature peptide forms a theoretical disulfide bond at C66 / C156. Since the rFel d 4 expressed in E. coli in the present application has an additional M at the N-terminus, a disulfide bond is formed at C67 / C157, which is consistent with the theory.

[0177] The test results show that the wild-type FeI d 4 has a chaotic molecular weight, with many components of similar molecular weights. However, the molecular weights of the mutants of the present invention after mutation of amino acid position 139 are significantly uniform. This further confirms that the FeI d 4 mutants of the present invention have greater advantages in terms of recombinant protein purity and uniformity, are more conducive to the correct preparation of FeI d 4, and improve the quality controllability of FeI d 4 recombinant production.

[0178] Table 11

[0179] Example 11: Activity detection of Fel d 4 mutants

[0180] In this example, clinical cat allergy serum (collected from Jiangsu Provincial People's Hospital, March-May 2023) was used to perform reactivity detection with the recombinant Fel d 4 mutant prepared in this application. The detection method is as follows:

[0181] 1. Coating: Dilute the recombinant protein Fel d 4 to 1 μg / mL using 0.05 M carbonate coating buffer (pH 9.6), add 100 μL / well to the ELISA plate, and incubate at 2-8°C overnight.

[0182] 2. Blocking: The next day, remove the ELISA plate, wash once with PBST, add 2% BSA in PBST at 200 μl / well, and incubate at 37°C for 2 h.

[0183] 3. Sample preparation: Dilute clinical cat allergy-positive and -negative serum with 2% BSA in PBST (dilution multiples are shown in Table 12). 2% BSA in PBST is used as a blank background. Discard the liquid in the ELISA plate and add 100 μL / well of each dilution sample to the ELISA plate. Incubate at 37°C, 300 rpm, for 2 h.

[0184] 4. Detection: Wash the ELISA plate three times with PBST, add 1:1500 diluted mouse anti-human IgE-HRP secondary antibody at 100 μl / well, and incubate at 37°C at 300 rpm for 1 hour.

[0185] 5. Color development, termination, and reading: Wash the plate three times with PBST, add 100 μl / well of TMB VII colorimetric solution, react at 37°C for 15 min, then add 50 μl / well of 2 M sulfuric acid. Immediately read the plate at a wavelength of 450 nm (microplate reader: Thermo MultiSKANGO). The reading on the microplate reader reflects the binding level of Fel d 4 to specific IgE in the serum.

[0186] Table 12 shows the results (microplate reader readings) of the reactions of the Fel d 4 mutant 2 and mutant 3 recombinant proteins prepared in this application with different clinical cat allergy sera. The results show that the majority of the 11 positive sera reacted with the two mutant recombinant proteins, and the two mutants showed similar reactivity with different cat positive sera. Serum samples SZ20, 1363, and SZ28 showed high reactivity with the two mutant recombinant proteins, but most samples showed low reactivity. The specific IgE binding levels reflected in some results (such as SZ19) were inconsistent with the clinically reported values. In other words, the clinically reported values ​​were higher, while the test results in this example only showed lower specific IgE binding levels. This suggests that the corresponding patients were not allergic to Fel d 4 or had a weak allergy, but were more sensitive to other cat allergens, such as Fel d 1. In addition, the inventors of the present application also found that for the clinical serum samples listed in Table 12, the reactivity of the Fel d 4 mutant was generally lower than that of the cat type I allergen Fel d 1. Among them, the reactivity of rFel d 4 was higher than or similar to that of Fel d 1 in only a few clinical allergic sera. It is well known in the art that Fel d 1 is the major cat allergen with a higher sensitization rate, and patients allergic to Fel d 1 are more common clinically.

[0187] Table 12

[0188] Example 12: Evaluation of in vivo sensitization and desensitization efficacy of Fel d 4 mutants

[0189] 1. Experimental Materials:

[0190] BALB / c female mice, 6-7 weeks (age upon arrival), were provided by Changzhou Cavens Laboratory Animal Co., Ltd.

[0191] The FeI d 4 mutant solution prepared in this application (taking mutant 2 as an example).

[0192] Aluminum hydroxide adjuvant was provided by Thermo Fisher Scientific.

[0193] 2. Allergen preparation:

[0194] Sensitization sample: Prepare a 0.0067 mg / ml solution of the Fel d 4 mutant with aluminum hydroxide adjuvant at a volume ratio of 3:1. Shake well before injection.

[0195] Stimulation sample: A FeI d 4 mutant solution with a concentration of 0.05 mg / ml was used as the stimulation sample.

[0196] 3. Drug delivery system:

[0197] 3.1 Sensitization stage:

[0198] Sixteen mice were randomly divided into two groups based on body weight, with 8 mice per group. Group and dosing details are shown in Table 13. The Fel d 4 mutant group received a subcutaneous injection of 1 μg of the sensitized sample per mouse, while the negative group received an equal amount of blank sample once weekly for a total of three times.

[0199] Table 13 Grouping and Dosage

[0200] 3.2 Excitation stage:

[0201] One week after the final sensitization, the mutant group was challenged intranasally with the challenge sample (40 μl / mouse / time) once daily for 7 consecutive days. The negative group was administered an equal volume of blank sample. See Table 13 for details.

[0202] 4. Evaluation indicators:

[0203] 4.1 Serum antibodies

[0204] 48 hours after the last challenge, whole blood was collected by enucleation of the eyeball, allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 10 minutes to prepare serum. Serum antibodies were detected by ELISA (antigen coated with mutant 2).

[0205] 4.2 White blood cell count (WBC) in BALF:

[0206] After collecting bronchoalveolar lavage fluid (BALF), three BALF fluids were combined and centrifuged, the supernatant was discarded, and the precipitated cells were resuspended in 1× PBS buffer. After mixing, they were stained according to the "Wright-Giemsa Staining Solution Operating Instructions" and the number of stained white blood cells was observed under an upright microscope at 200 times.

[0207] 4.3 Cytokines in the supernatant of spleen cells stimulated in vitro:

[0208] Splenocytes were isolated from mice in each group under sterile conditions and stimulated with the challenge sample. Supernatants were collected after 144 hours of co-culture with the challenge sample. Cytokines IL-4, IL-5, IL-10, and IL-13 in the supernatants of splenocytes stimulated in vitro were assayed using CBA.

[0209] 5. Experimental results:

[0210] 5.1 Serum antibodies

[0211] As shown in Table 14 and Figure 16, the serum antibody test results of each group are

[0212] IgE: The level of IgE in the Fel d 4 mutant group was significantly higher than that in the negative group (p<0.01).

[0213] The expression of IgG1:Fel d 4 mutant group was significantly higher than that of negative group (p<0.01).

[0214] IgG2a: The level of Fel d 4 mutant group showed an increasing trend compared with the negative group.

[0215] Table 14 Serum antibody values ​​(Mean ± SEM) Note: ** Indicates P < 0.01 compared with the negative group.

[0216] 5.2 WBC count in BALF

[0217] Observation of BALF cell staining under a microscope (Figure 17-1, Figure 17-2) showed that the number of white blood cells in the Fel d 4 mutant group was significantly increased compared with the negative group.

[0218] 5.3 Cytokines in the supernatant of spleen cells stimulated in vitro

[0219] As shown in Table 15 and Figure 18 , the results of TH2 cytokines IL4, IL5, IL10, and IL13 showed that the Fel d 4 mutant group had a significant increase, and IL5, IL10, and IL13 were significantly different from those in the negative group (p < 0.05, p < 0.01), and IL-4 showed an increasing trend compared with the negative group.

[0220] Table 15 TH2 cytokines in the supernatant of splenocytes stimulated in vitro (Mean ± SEM) Note: * indicates p < 0.05 compared with the negative group; ** indicates p < 0.01 compared with the negative group

[0221] 6. Experimental conclusion:

[0222] The results of this study showed that in an allergic airway inflammation model constructed by sensitization and stimulation with the Fel d 4 mutant protein of this application, mice in the Fel d 4 mutant group showed elevated levels of various indicators, including antibody changes, target organ inflammation, and cytokine levels. This indicates that the Fel d 4 mutant protein of this application is immunogenic in vivo, capable of producing allergic reactions, increasing allergen-specific antibodies in the body, and stimulating the body's immune response. Therefore, the Fel d 4 mutant protein of this application has the potential to be used as an active ingredient in cat desensitization drugs or as a component of cat allergen detection reagents for the diagnosis and treatment of feline allergic diseases.

[0223] Example 13: Design of FeI d 7 mutants and construction of expression strains

[0224] Natural FeI d 7 may have a variety of alloallergens or variants. This example takes the nFel d 7 sequence with Genbank accession number ADK56160 as an example.

[0225] The mature peptide sequence of nFel d 7 was obtained based on the Genbank accession number, as shown in SEQ ID NO:10. This sequence was reverse-translated into a DNA sequence and codon-optimized using an E. coli expression system at Nanjing GenScript Biotechnology Co., Ltd. The coding DNA sequence for nFel d 7 was synthesized artificially, as shown in SEQ ID NO:11. A deletion or amino acid substitution mutation was introduced at position 101 of nFel d 7 by PCR (mutation information is shown in Table 16). The coding DNA sequence of the mutants had an NdeI restriction site at the 5' end and an XhoI restriction site at the 3' end. For mutant 1, its DNA sequence is shown in SEQ ID NO:12.

[0226] The recombinant expression of FeI d 7 in this application can be based on an expression vector of a T7 promoter, such as pET32a, pET26b, pET28a, pDEST14 vector, etc., and can also be based on an expression vector of a temperature-controlled promoter PL-PR, such as a pBV220 vector. Among them, any Escherichia coli genetically engineered bacteria can be selected based on an expression vector based on a PL-PR promoter, such as BL21 (DE3), BL21 AI (DE3), Top10, DH5α, JM109, Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys host, etc.; Escherichia coli genetically engineered bacteria based on a T7 promoter expression vector can be selected from BL21 (DE3), BL21 AI (DE3), Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys, etc. This embodiment is described using pET26b vector and BL21 (DE3) host as examples.

[0227] The DNA sequences of the above-mentioned FeI d 7 mutants were cloned into the pET26b vector to form the pET26b-Fel d 7 mutant expression plasmid, and then transformed into the BL21 (DE3) host to form the BL21 (DE3)-pET26b-Fel d 7 mutant expression strain.

[0228] Table 16 Summary of mutation information of each sample

[0229] Example 14: Recombinant expression of FeI d 7 and its mutants

[0230] The expression of FeI d 7 mutant 1 in pET26b vector and BL21 (DE3) host is shown below. The same method was used to express nFel d 7 and other mutants.

[0231] 1. A glycerol tube of BL21(DE3)-pET26b-Fel d 7 mutant 1 was streaked onto an LB-agar plate containing 50 μg / ml and cultured at 37°C for 12 h.

[0232] 2. Pick a colony from the agar plate and inoculate it into 10 ml of TB liquid medium at 37°C, 220 rpm, and 8 hours. This is the primary seed medium.

[0233] 3. Inoculate 5% of the primary seeds into 10 bottles of 500 ml TB liquid culture medium at a ratio of 5%. Culture conditions: 37°C, 220 rpm. When the OD of the culture medium reaches 0.8, add 0.5 mM IPTG to induce expression. After 4 hours of induction, collect the bacteria by centrifugation for later use.

[0234] Example 15: Extraction and purification of FeI d 7 and its mutants

[0235] The extraction and purification of Fel d 7 mutant 1 are as follows. nFel d 7 and other mutants were obtained using the same process.

[0236] 1. Resuspend the fermented bacteria in 10mM PB pH 7.4 buffer to 80g / L and disperse evenly. Use a high-pressure homogenizer to crush the bacteria at a crushing pressure of 900bar±50bar, and repeat the crushing process three times. Keep the material temperature below 25℃ during the crushing process. After the crushing is completed, centrifuge and collect the supernatant.

[0237] 2. The supernatant was filtered using a 0.45 μm membrane. The resulting supernatant was concentrated using a 100 kDa ultrafiltration membrane with a molecular weight cutoff. The solution was concentrated and replaced with 10 mM PB, pH 8.0, and the filtrate was collected.

[0238] 3. First step purification: The filtrate was purified using Q Sepharose Fast Flow, with the sodium chloride concentration linearly reaching 0.5 M within 10 column volumes, and the target protein peak was collected; purification binding buffer: 10 mM PB, pH 8.0; purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 8.0;

[0239] 4. Second purification step: Supplement the purified sample from the first step with ammonium sulfate to a final concentration of 2 M and adjust the pH to 7.4. Purify using a Phenyl Sepharose High Performance 6 (HS) column, ensuring that the elution buffer reaches 100% linearity within 10 column volumes, and collect the target protein peak. Purification elution buffer: 10 mM PB, pH 7.4; purification binding buffer: 10 mM PB + 2 M ammonium sulfate, pH 7.4.

[0240] 5. The target protein peak collected from the two-step chromatography purification was ultrafiltered and replaced with a 20 mM PB buffer at pH 7.0; after filtration and sterilization, the target protein stock solution was obtained;

[0241] 6. The final samples were analyzed using HPLC-SEC, HPLC-RP, and non-reduced SDS-PAGE. The results are shown in Figures 19 to 21.

[0242] The test results indicate that both the wild-type and mutant 1 of Fel d 7 achieved the same electrophoretic purity under the same process conditions, with no visible impurities observed by non-reducing electrophoresis. However, the wild-type HPLC-RP purity exhibited a distinct, difficult-to-separate impurity peak, while the mutant did not. This suggests that the wild-type pure product contains related proteins with similar molecular weights to the target protein but with significantly different hydrophobic properties.

[0243] Further analysis of the HPLC-RP purity of each mutant (the results are shown in Table 17) revealed that under the same process conditions, the purity of each mutant reached over 95%. Since each mutant mutated the amino acid at position 101 of nFel d 7, the mutation of the amino acid at position 101 should be the key to improving the purity of Fel d 7 RP. Compared with wild-type Fel d 7, the Fel d 7 mutant of the present invention has greater advantages in the purity and homogeneity of the recombinant protein, and is more conducive to recombinant expression and production.

[0244] Table 17 Summary of HPLC-RP purity of each sample

[0245] Example 16: Primary structure analysis and molecular weight of FeI d 7 and its variants

[0246] LC-MS molecular weight can accurately reflect whether the primary sequence of biological macromolecules is correct, including whether the N- and C-terminal sequences are missing, and whether there are post-translational modifications such as glycosylation, oxidation, and deamidation. It is one of the most important analytical methods for biological macromolecules.

[0247] Using our Thermo Scientific TMA high-resolution mass spectrometry system was used to analyze the molecular weight and primary structure of the purified wild-type FeI d 7 and its mutant recombinant proteins using LC-MS. The results are shown in Figures 22-24 and Table 18. Recombinant proteins expressed in E. coli typically contain an additional formylmethionine at the N-terminus, encoded by a start codon, in addition to the native amino acid sequence. In the specific examples of this application, recombinant FeI d 7 (rFel d 7) was produced by expression in an E. coli system. Therefore, compared to the nFel d 7 mature peptide sequence, the rFel d 7 shown in Figure 23 has one additional amino acid, a formylmethionine at the N-terminus. The primary structure, excluding the remaining mutation sites, is consistent with that of nFel d 7. Furthermore, the nFel d 7 mature peptide forms a theoretical disulfide bond at C62 / C152. Since the E. coli-expressed rFel d 7 in this application has an additional M at the N-terminus and a missing C101 compared to nFel d 7, the disulfide bond at C62 / C152 was detected, consistent with the theory.

[0248] The test results show that the wild-type FeI d 7 has a chaotic molecular weight, with many components of similar molecular weights. However, the molecular weights of the mutants of the present invention after the amino acid 101 mutation are significantly uniform. This further confirms that the FeI d 7 mutants of the present invention have greater advantages in terms of recombinant protein purity and uniformity, are more conducive to the correct preparation of FeI d 7, and improve the quality controllability of FeI d 7 recombinant production.

[0249] Table 18 Summary of LC-MS analysis results of each sample

[0250] Example 17: Evaluation of the in vivo sensitization and desensitization efficacy of Fel d 7 mutants

[0251] 1. Experimental Materials:

[0252] BALB / c female mice, 6-7 weeks (age upon arrival), were provided by Changzhou Cavens Laboratory Animal Co., Ltd.

[0253] The recombinant FeI d 7 mutant solution prepared in this application (taking mutant 1 as an example).

[0254] Aluminum hydroxide adjuvant was provided by Thermo Fisher Scientific.

[0255] 2. Allergen preparation:

[0256] Sensitized sample: Prepare a 0.0067 mg / ml solution of the Fel d 7 mutant with aluminum hydroxide adjuvant at a volume ratio of 3:1. Shake thoroughly before injection.

[0257] Stimulation sample: A FeI d 7 mutant solution with a concentration of 0.05 mg / ml was used as the stimulation sample.

[0258] 3. Drug delivery system:

[0259] 3.1 Sensitization stage:

[0260] Sixteen mice were randomly divided into two groups of 8 mice each based on body weight. Group and dosing details are shown in the table below. Each group received a subcutaneous injection of 1 μg of the sensitized sample, while the negative group received an equal amount of blank sample. This was repeated once a week for a total of three times.

[0261] Table 19 Grouping and Dosage

[0262] 3.2 Excitation stage:

[0263] One week after the final sensitization, the mutant group was challenged intranasally with the challenge sample (40 μl / mouse / time) once daily for 7 consecutive days. The negative group was administered an equal volume of blank sample. See Table 19 for details.

[0264] 4. Evaluation indicators:

[0265] 4.1 Penh:

[0266] Twenty-four hours after the last challenge, all mice were tested for Penh values ​​using a noninvasive whole-body plethysmography (WBP) system at methacholine (Mch) concentrations of 0, 6.25, 12.5, 25, and 50 mg / ml. Penh values ​​reflect airway resistance; higher Penh values ​​indicate increased airway reactivity and more severe airway symptoms.

[0267] 4.2 Serum-specific IgE antibodies

[0268] 48 hours after the last challenge, whole blood was collected by enucleation, allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 10 minutes to prepare serum. Serum antibodies were detected by ELISA (antigen coated with mutant 1).

[0269] 4.3 White blood cell count (WBC) in BALF:

[0270] After collecting bronchoalveolar lavage fluid (BALF), three BALF fluids were combined and centrifuged, the supernatant was discarded, and the precipitated cells were resuspended in 1× PBS buffer. After mixing, they were stained according to the "Wright-Giemsa Staining Solution Operating Instructions" and the number of stained white blood cells was observed under an upright microscope at 200 times.

[0271] 4.4 Cytokines in the supernatant of spleen cells stimulated in vitro:

[0272] Splenocytes were isolated from mice in each group under sterile conditions and stimulated with the challenge sample. Supernatants were collected after 144 hours of co-culture with the challenge sample. Cytokines IL-4, IL-5, IL-10, and IL-13 in the supernatants of splenocytes stimulated in vitro were assayed using CBA.

[0273] 5. Experimental results:

[0274] 5.1 Penh:

[0275] As shown in the test results in Table 20 and Figure 25, the Penh of the FeI d 7 mutant group mice was higher than that of the negative group under different MCh stimulations.

[0276] Table 20 Mouse Penh value (Mean ± SEM)

[0277] 5.2 Serum-specific IgE antibodies

[0278] As shown in Table 21 and Figure 26 , the serum specific IgE antibody test results of each group were significantly higher in the FeI d 7 mutant group than in the negative group (p < 0.01).

[0279] Table 21 Serum antibody values ​​(Mean ± SEM) Note: ** Indicates P < 0.01 compared with the negative group (mutant 1)

[0280] 5.3 WBC count in BALF

[0281] Observation of BALF cell staining under a microscope (Figure 27-1, Figure 27-2) showed that the number of white blood cells in the Fel d 7 mutant group was significantly increased compared with the negative group.

[0282] 5.4 Cytokines in the supernatant of spleen cells stimulated in vitro

[0283] As shown in Table 22 and Figure 28, the TH2 cytokines IL4, IL5, IL10, and IL13 were detected in the Fel d 7 mutant group, with all cytokines showing an elevated trend, with IL-10 showing a significant difference compared to the negative group (p < 0.05). After adjusting the rFel d 7 sensitization and challenge doses, the cytokines IL-4, IL-5, and IL-13 in the supernatant of splenocytes stimulated in vitro in the Fel d 7 mutant group were also significantly elevated compared to the negative group.

[0284] Table 22 TH2 cytokines in the supernatant of spleen cells stimulated in vitro (Mean ± SEM) Note: * indicates p < 0.05 compared with the negative group

[0285] 6. Experimental conclusion:

[0286] The results of this study showed that in the allergic airway inflammation model constructed by sensitization and stimulation with the Fel d 7 mutant protein of the present application, the airway symptoms, antibody changes, target organ inflammation, cytokines and other indicators of the Fel d 7 mutant group mice all showed high levels. These indicators further increased at higher concentrations of sensitization and stimulation doses, indicating that the Fel d 7 mutant protein of the present application is immunogenic in the body, can cause an increase in allergen-specific antibodies in the body, produce allergic reactions, and stimulate the body's immune response. The Fel d 7 mutant protein of the present application has the potential to be used as an effective ingredient in cat desensitization drugs or a component of cat allergen detection reagents for the diagnosis and treatment of cat allergic diseases.

[0287] Example 18: Study on the Effect of Feline Recombinant Allergen Protein Mixture on Inducing Allergic Airway Inflammation in Mice

[0288] 1. Experimental Materials:

[0289] BALB / c female mice, 8-9 weeks old (used in weeks), were provided by Shanghai Slake Laboratory Animal Co., Ltd.

[0290] Cat dander extract, total protein content 1 mg / ml, batch number: F2124A, provided by Wokawei (Beijing) Biotechnology Co., Ltd.

[0291] Aluminum hydroxide adjuvant, batch number: 230017, was provided by SERVA.

[0292] 2. Sample preparation:

[0293] 2.1 Sensitized samples:

[0294] Use normal saline (NS) for injection to prepare dilutions of recombinant FeI d 1, recombinant FeI d 4, and recombinant FeI d 7 at a concentration of 0.04 mg / ml each (the allergen recombinant proteins used were the samples prepared in the above examples, using FeI d 1-1, FeI d 4 mutant 2, and FeI d 7 mutant 1 as examples, the same below). Equal volumes of the three were thoroughly mixed to prepare a recombinant protein solution. Separately, use NS to prepare a dilution of cat dander extract solution at a concentration of 0.1 mg / ml. Take equal volumes of the above recombinant protein solution and cat dander extract solution and mix thoroughly. Add 3 volumes of aluminum hydroxide adjuvant dropwise and mix thoroughly for 30 minutes using a 3D mixer. Prepare immediately before use, shake well before use, and inject immediately after extraction to prevent precipitation.

[0295] 2.2 Excite the sample:

[0296] Recombinant FeI d 1, FeI d 4, and FeI d 7 were diluted to a concentration of 0.2 mg / ml using NS. Equal volumes of the three solutions were thoroughly mixed to prepare a recombinant protein solution. Separately, a cat dander extract solution was diluted to a concentration of 0.25 mg / ml using NS. Equal volumes of the recombinant protein solution and cat dander extract solution were taken and thoroughly mixed to prepare a cat mixed allergen challenge sample solution.

[0297] 2.3 Recombinant protein mixture:

[0298] High-dose recombinant protein group: Weigh 0.7446 mg of the recombinant protein mixed powder (recombinant Fel d 1, recombinant Fel d 4, and recombinant Fel d 7 mixed at a mass ratio of 2:1:1), add 621 μL of ultrapure water, and use a pipette to mix thoroughly to prepare a recombinant protein solution with a concentration of 1.2 mg / mL (calculated based on the main allergen content) for administration to the high-dose recombinant protein group.

[0299] Medium-dose recombinant protein group: 311 μL of the high-dose recombinant protein solution with a concentration of 1.2 mg / mL was aspirated and 311 μL of ultrapure water was added to prepare a recombinant protein solution with a concentration of 0.6 mg / mL (calculated based on the main allergen content) for administration to the medium-dose recombinant protein group.

[0300] Low-dose recombinant protein group: 311 μL of the medium-dose recombinant protein solution with a concentration of 0.6 mg / mL was aspirated, and 311 μL of ultrapure water was added to prepare a recombinant protein solution with a concentration of 0.3 mg / mL (calculated based on the main allergen content) for administration to the low-dose recombinant protein group.

[0301] 3. Drug delivery system:

[0302] 3.1 Modeling stage:

[0303] 96 female BALB / c mice were randomly divided into two groups according to body weight: negative group and model group.

[0304] Sensitization: After grouping, the modeling group was intraperitoneally injected with 0.2 ml of cat mixed allergen sensitization sample solution per rat once a week for 3 consecutive weeks; the negative group was given NS solution in the same way.

[0305] Challenge: 7 days after the last sensitization, the mice in the modeling group were given 40 μL of cat mixed allergen challenge sample solution per mouse per nasal drop, once a day for 7 consecutive days; the negative group was given NS in the same way.

[0306] Grouping and dosing information are detailed in Table 23.

[0307] Table 23 Mouse grouping and medication information during modeling period Note: ① The dosage in the table, such as "1+2.5", means 1ug / animal / time of recombinant sample and 2.5ug / animal / time of extract (CD). Other dosages are not mentioned here. ② The animals were tested for serum-specific antibodies, and the animals were selected for inclusion in the study groups based on the test results.

[0308] 3.2 Treatment phase:

[0309] Of the 80 mice in the modeling group, 48 mice with high serum specific IgE and IgG1 levels and good general condition were screened and randomly divided into four groups: model group, low-dose recombinant agent group, medium-dose recombinant agent group, and high-dose recombinant agent group, with 12 mice in each group. The negative group consisted of 12 mice, the same as those in the negative group during the modeling phase. See Table 24 for details.

[0310] Table 24 Grouping and dosing information

[0311] Each group was administered the corresponding test solution according to the information in "Table 24." Sublingual administration procedure: Hold the mouse and use a pipette to accurately pipette 20 μL of the corresponding test solution listed in Table 24 and drip it sublingually. Continue holding the mouse for 20 seconds to prevent it from swallowing the test solution immediately. Dosing duration and frequency: 5 times per week for 8 consecutive weeks. Within 3 days of the last sublingual administration, each group of mice received a cat allergen challenge solution (40 μL / mouse / time) intranasally once daily for 7 consecutive days. The negative group was administered NS in the same manner.

[0312] 4. Evaluation indicators:

[0313] 4.1 Penh:

[0314] 24 hours after the last challenge, the Penh values ​​of all mice were measured using a non-invasive whole body plethysmography (WBP) system at methacholine (Mch) concentrations of 0, 6.25, 12.5, 25, and 50 mg / ml.

[0315] 4.2 Serum IgE levels

[0316] 48 hours after the last challenge, whole blood was collected by enucleation of the eyeball, allowed to stand at room temperature for 2–3 hours, and then centrifuged at 3500 rpm for 10 minutes to prepare serum. Serum IgE levels were measured by ELISA.

[0317] 4.3 WBC and IgE levels in BALF:

[0318] After collecting bronchoalveolar lavage fluid (BALF), three BALF aliquots were combined and centrifuged at 800 rpm for 10 min. The supernatant was discarded, and the pelleted cells were resuspended in 0.5 mL of pre-chilled, sterile 1× PBS buffer containing 1% BSA and mixed thoroughly. EOS in the BALF suspension was measured using a Siemens ADVIA2120i hematology analyzer. The remaining BALF suspension was diluted 10-fold with 1% BSA in 1× PBS buffer and centrifuged at 800 rpm for 5 min. Slides were prepared and stained according to the Wright-Giemsa staining instructions. Three randomly selected fields of view were counted under an upright microscope at 200x magnification. The stained cells were counted using Image J software and the average value was calculated.

[0319] The supernatant was taken and the IgE level in BALF was detected by ELISA.

[0320] 4.4 Cytokines in the supernatant of spleen cells stimulated in vitro:

[0321] Splenocytes were isolated from mice in each group under sterile conditions and stimulated with the challenge sample. Supernatants were collected after 144 hours of co-culture with the challenge sample. Cytokines IL-4, IL-5, IL-10, and IL-13 in the supernatants of splenocytes stimulated in vitro were assayed using CBA.

[0322] 5. Experimental results:

[0323] 5.1 Penh:

[0324] After sublingual administration, the Penh values ​​of the model group mice at all MCh concentrations were significantly higher than those of the negative group (P < 0.05, P < 0.01). The Penh values ​​of the low-, medium-, and high-dose groups of the recombinant protein mixture at all MCh concentrations were lower than those of the model group. Specifically, the Penh values ​​of the medium- and high-dose groups at 6.25 mg / mL MCh were significantly lower than those of the model group (P < 0.05, P < 0.01). These results indicate that sublingual administration of the recombinant protein mixture can improve airway hyperresponsiveness in the mouse allergic airway inflammation model.

[0325] Table 25 Mouse Penh value (Mean ± SEM) Note: * indicates P < 0.05, ** indicates P < 0.01, compared with the model group

[0326] 5.2 IgE in serum and BALF

[0327] Serum IgE levels were lower in the medium and high-dose recombinant protein mixture groups than in the model group, with the high-dose group significantly lower than the model group (p < 0.05). BALF IgE levels were lower in the low, medium, and high-dose recombinant protein mixture groups than in the model group, with the medium and high-dose groups significantly lower than the model group (p < 0.05). These results indicate that local and systemic allergen immune responses decreased after treatment with the recombinant protein mixture.

[0328] Table 26 Antibody values ​​(Mean ± SEM) Note: * indicates P < 0.05, ** indicates P < 0.01, compared with the model group

[0329] 5.3 WBC and EOS counts in BALF

[0330] The number of EOS in BALF was significantly lower in the recombinant protein mixture-treated groups than in the model group (p < 0.01). The number of WBCs in the recombinant protein mixture-treated groups was lower than in the model group, with the high-dose group showing a significant decrease (p < 0.01). These results indicate that treatment with the recombinant protein mixture significantly improved lung inflammation in mice.

[0331] Table 27 WBC count in BALF (Mean ± SEM) Note: ** indicates p < 0.01 compared with the model group

[0332] 5.4 Cytokines in the supernatant of spleen cells stimulated in vitro

[0333] Results for TH2 cytokines IL-4, IL-5, IL-10, and IL-13 showed a decrease in each indicator in the medium and high-dose groups. IL-4 levels in the medium and high-dose groups were significantly different from those in the model group (p < 0.01), while IL-5 and IL-10 levels in the high-dose group were significantly different from those in the model group (p < 0.05). These results indicate that treatment with the recombinant protein mixture downregulated Th2 cytokines in the model animals, a finding closely associated with improved allergic reactions.

[0334] Table 28 TH2 cytokines in the supernatant of spleen cells stimulated in vitro (Mean ± SEM) Note: * indicates p < 0.05 compared with the model group; ** indicates p < 0.01 compared with the model group

[0335] 6. Experimental conclusion:

[0336] The results of this study showed that in the mouse model of allergic airway inflammation, the feline recombinant allergen protein mixture of the present invention has a clear sublingual immunotherapy effect. It can effectively reduce allergic airway inflammation by downregulating Th2 cytokine production and IgE, and inhibiting the infiltration and activation of inflammatory cells.

Claims

1. A recombinant Fel d 1 protein, which is a fusion protein of Fel d 1 chain 1 and Fel d 1 chain 2.

2. The feline type I recombinant allergen protein according to claim 1, wherein the amino acid sequence of Fel d 1 chain 1 has more than 67% identity with the sequence shown in SEQ ID NO: 5; the amino acid sequence of Fel d 1 chain 2 has more than 67% identity with the sequence shown in SEQ ID NO:

6.

3. The recombinant Fel d 1 protein according to claim 1, wherein Fel d 1 chain 1 and Fel d 1 chain 2 are directly fused or fused through a linker peptide; Fel d 1 chain 1 and Fel d 1 chain 2 are respectively at the N-terminus and C-terminus of the fusion protein or respectively at the C-terminus and N-terminus of the fusion protein.

4. The recombinant Fel d 1 protein according to claim 1, wherein the amino acid sequence of Fel d 1 is as shown in SEQ ID NO: 1 or as shown in SEQ ID NO: 2, or the N-terminal first formylmethionine of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is deleted or replaced with methionine.

5. The nucleotide encoding the recombinant Fel d 1 protein according to claim 4, with the base sequence being SEQ ID NO: 3 or SEQ ID NO:

4.

6. A Fel d 4 mutant or its derivative, which mutates the protein sequence of natural Fel d 4 or an allogeneic allergen or variant of natural Fel d 4; the amino acid sequence of the natural Fel d 4 is as shown in SEQ ID NO: 7, and the mutation site is the 139th position of the natural Fel d 4 protein sequence; the mutation site of the allogeneic allergen or variant of the natural Fel d 4 is the site corresponding to the 139th position of SEQ ID NO: 7; the mutation method is deletion or replacement with other amino acids.

7. The Fel d 4 mutant or its derivative according to claim 6, wherein the mutation site of the Fel d 4 mutant is replaced with any one or several amino acids of G or A or V or L or I or Y or S or K or R or H or F or W or M or T.

8. The Fel d 4 mutant or its derivative of claim 6, with one more methionine or formylmethionine at the N-terminus.

9. The nucleotide encoding the Fel d 4 mutant or its derivative according to claim 6.

10. A Fel d 7 mutant or its derivative, which mutates the protein sequence of natural Fel d 7 or an allogeneic allergen or variant of natural Fel d 7; the amino acid sequence of the natural Fel d 7 is as shown in SEQ ID NO: 10, and the mutation site is the 101st position of the natural Fel d 7 protein sequence; the mutation site of the allogeneic allergen or variant of the natural Fel d 7 is the site corresponding to the 101st position of SEQ ID NO: 10; the mutation method is deletion or replacement with other amino acids.

11. The Fel d 7 mutant or its derivative according to claim 10, wherein the mutation site of the Fel d 7 mutant is replaced with any one or several amino acids selected from G, A, V, L, I, Y, S, K, R, H, F, W, M, and T.

12. The Fel d 7 mutant or its derivative according to claim 10, having one additional methionine or formylmethionine at the N-terminus.

13. A nucleotide encoding the Fel d 7 mutant or its derivative according to claim 10.

14. A vector containing the nucleotide according to claim 5, 9, or 13, wherein the vector is an expression vector based on the T7 promoter: pET32a, pET26b, pET28a, pDEST14, or an expression vector based on the temperature-controlled promoter PL-PR: pBV220.

15. An Escherichia coli strain containing the vector according to claim 14, which is BL21(DE3), BL21 AI(DE3), Top10, DH5α, JM109, Rosetta(DE3), Rosetta gamiB(DE3), BL21(DE3)plys host compatible with an expression vector based on the PL-PR promoter; or BL21(DE3), BL21 AI(DE3), Rosetta(DE3), Rosetta gamiB(DE3), BL21(DE3)plys host compatible with an expression vector based on the T7 promoter.

16. A method for expressing a recombinant Fel d 1 protein, a Fel d 4 mutant or its derivative, or a Fel d 7 mutant or its derivative, the method comprising the following steps: A. Construct a vector containing the vector according to claim 14; B. Linearize the vector in step A and transfer it into an Escherichia coli strain, and culture it under appropriate conditions; C. Recover and purify the protein.

17. A method for purifying a recombinant Fel d 1 protein, a Fel d 4 mutant or its derivative, or a Fel d 7 mutant or its derivative, the purification method is as follows: A. Resuspend, disrupt the Escherichia coli cells obtained by culturing in claim 16, and collect the precipitate or supernatant, and the precipitate is the crude extract of the target protein inclusion body; B. If the collected is inclusion body, perform crude purification, denaturation, and renaturation treatment on it to obtain a renaturation solution; If the collected is supernatant, filter and concentrate it with an ultrafiltration membrane to obtain a filtrate; C. Collect the above renaturation solution or filtrate, and perform two-step purification by ion exchange chromatography and hydrophobic chromatography to obtain the stock solution of the target protein.

18. Use of the recombinant Fel d 1 protein according to any one of claims 1-4, the Fel d 4 mutant or its derivative according to any one of claims 6-8, or the Fel d 7 mutant or its derivative according to any one of claims 10-12 in the preparation of a drug for treating feline allergic diseases or a diagnostic reagent for detecting feline allergens.

19. A composition for treating allergic diseases caused by feline allergens, containing Fel d 1, Fel d 4, and Fel d 7.

20. A composition for treating allergic diseases caused by cat allergens as described in claim 19, wherein: The Fel d 1 is the recombinant Fel d 1 protein as described in any one of claims 1-4; And / or, the Fel d 4 is the Fel d 4 mutant or its derivative as described in any one of claims 6-8; And / or, the Fel d 7 is the Fel d 4 mutant or its derivative as described in any one of claims 10-12.

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