Canine recombinant allergen vaccine, preparation method therefor, and use thereof

Abstract

Provided are a canine recombinant allergen vaccine, a pharmaceutical composition thereof, a preparation method therefor, and a use thereof. The canine recombinant allergen vaccine is used for identifying, during diagnosis, an allergen molecule causing an allergic reaction, and is used for performing allergen immunotherapy on a patient during treatment.

Description

Canine recombinant allergen vaccine, its preparation method and application Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a canine recombinant allergen vaccine, its preparation method, and its application. Background Technology

[0002] Dog allergens are a very important type of indoor allergen (also known as allergens), and are a common cause of allergic rhinitis (AR) and bronchial asthma (BA). These allergens are generally produced in dogs' sebaceous glands, salivary glands, and urine. Studies have shown that 9-34.8% of the population in Europe is allergic to dog hair, 36.5% of dog-owning households in the United States are allergic to dog hair, and 14.0% of patients with allergic rhinitis or asthma in my country are allergic to dog dander. Furthermore, the more exposure children under two years old have to dog allergens, the more likely they are to develop allergic diseases caused by dog ​​dander in adulthood.

[0003] Currently, treatments for allergic diseases caused by canine allergens include desensitization therapy and anti-allergy therapy. Anti-allergy therapy involves administering antihistamines to alleviate symptoms such as asthma and allergic rhinitis. This is considered a symptomatic treatment and generally does not address the root cause of the allergy. Specific immunotherapy (SIT), also known as allergen immunotherapy (AIT), involves repeated exposure to the allergen via subcutaneous injection or sublingual administration. This increases the patient's tolerance to the allergen. When the patient is re-exposed to the allergen, the release of inflammatory mediators is significantly reduced, and the severity of the specific allergic reaction is significantly reduced, thus alleviating clinical symptoms and ultimately achieving tolerance or even immune tolerance. SIT is currently the only causal therapy that may influence the natural course of allergic diseases and alter the immune response mechanism. In addition to the treatments mentioned above, other methods include avoiding contact with dogs and immunizing dogs with their own major allergens (mainly Can f1) to induce neutralizing antibodies and reduce the level of endogenous allergens.

[0004] The active ingredient in desensitizing drugs is generally the main allergenic protein, which is an allergen capable of inducing allergic reactions. A clearly defined main allergenic protein can be obtained through direct extraction from allergen products or recombinant expression. The active ingredients in the house dust mite drops and Artemisia annua pollen allergen sublingual drops marketed in China by Wowo Biotech, and the house dust mite allergen preparation marketed in Europe by ALK-Abello, are natural allergen extracts. Currently, there are no marketed desensitizing drugs specifically for canine allergies, either domestically or internationally.

[0005] Natural allergen extracts inevitably suffer from quality issues due to limitations in raw material sources and production methods. These include 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 Association for Allergy and Clinical Immunology (EAACI) Guidelines on allergen immunotherapy for allergic rhinoconjunctivitis (2018) also points out that mixed allergens have many potential drawbacks, including dilution effects, potential allergen degradation due to the enzymatic activity of certain allergens, and difficulty in fully demonstrating the efficacy of allergen combinations. Recombinant major allergens prepared through genetic engineering can achieve high purity, high homogeneity, and immunomodulatory activity comparable to natural allergens, effectively avoiding the drawbacks of naturally extracted mixed allergens. Summary of the Invention

[0006] The applicant hopes to provide a variety of canine recombinant allergens to obtain highly purified and homogeneous allergen proteins with good biological activity, thereby improving the quality control of the products and laying the foundation for desensitization treatment and accurate diagnosis of allergic diseases caused by canine allergens.

[0007] The first object of the present invention is to provide a recombinant canine allergen vaccine for the diagnosis and treatment of allergic diseases caused by canine allergens, comprising one or more of recombinant Can f1 mutants or derivatives thereof, recombinant Can f2 mutants or derivatives thereof, recombinant Can f4 mutants or derivatives thereof, and recombinant Can f5 mutants or derivatives thereof.

[0008] The recombinant Can f 1 mutant or its derivative is characterized by the deletion or substitution of the C at position 100 of the natural Can f 1 mature peptide with another amino acid. When substituting amino acids, one or more of the following amino acids can be used: G, A, V, L, I, Y, S, K, R, H, F, W, M, or T. Preferably, G, A, V, L, I, Y, F, or W is used; more preferably, G, A, V, L, or I is used. Optionally, the mutation further includes the deletion or substitution of the N at position 62, and / or the R at position 83, and / or the K at position 113 of the natural Can f 1 with other amino acids. The amino acid sequence of the natural Can f 1 mature peptide is shown in SEQ ID NO: 1.

[0009] Natural allergens are generally found in multiple isoallergens or variants due to various factors. These isoallergens have similar molecular weights, similar or identical biological functions, and an amino acid sequence identity rate >67%. Each isoallergen may have multiple forms of highly identical sequences (identity >90%, usually differing by only a few amino acids). These sequences are called variants or isoforms (WHO / IUIS Allergen Nomenclature: providing a common language). The aforementioned mature Can f1 peptide sequence can be derived from other isoallergens or variants. Therefore, the Can f1 mutants of this application also include proteins obtained by mutating the aforementioned natural Can f1 isoallergens or variants, with the mutation site corresponding to the mutation site shown in SEQ ID NO: 1.

[0010] As is known to those skilled in the art, in most cases, the start codon for prokaryotes is AUG, encoding formylmethionine; the start codon for eukaryotes is AUG, encoding methionine. Therefore, recombinant proteins expressed in prokaryotes such as *E. coli* typically contain formylmethionine at the N-terminus in addition to the natural amino acid sequence, while recombinant proteins expressed in eukaryotes such as yeast typically contain methionine at the N-terminus in addition to the natural amino acid sequence. For proteins with a signal peptide, the amino acid encoded by the start codon is cleaved along with the signal peptide, forming the natural protein sequence. Furthermore, the N-terminal formylmethionine or methionine in recombinantly expressed proteins can sometimes be cleaved, but this cleavage is often insufficient, and the cleavage ratio varies depending on the protein species. For example, when the recombinant Can f 1 protein (rCan f 1) of this application was prepared by expression in an *E. coli* system, the amino acid coverage results showed that, compared to the sequence shown in SEQ ID NO: 1, rCan f 1 has an additional formylmethionine at the N-terminus. Based on the aforementioned common knowledge, the recombinant Can f1 protein provided in this application may also have an additional formylmethionine or methionine at the N-terminus based on the above mutation.

[0011] The derivatives include the full-length protein of the Can f1 mutant of this application, a portion of the Can f1 mutant of this application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation based on the Can f1 mutant of this application.

[0012] Natural Can f1 is a canine type I allergen and a lipocalin. According to literature reports, over 90% of canine allergy sufferers show IgE binding to Can f1 in the RAST test, and over 90% are positive for Can f1 in the skin prick test (SPT). The ratio of serum IgE antibody reactivity to Can f1 to IgE antibody reactivity to whole canine dander in canine allergy sufferers is 75%. In 15 out of 20 canine allergy sufferers (75%), IgE antibodies were able to bind to rCan f1. Therefore, Can f1 is of great significance for the diagnosis and treatment of canine allergic diseases (Schou C, et al. Purification and characterization of the major dog allergen, Can f1. Clin Exp Allergy. 1991.).

[0013] The present invention also provides the protein primary structure sequence of the mutant with the deletion of cysteine ​​at position 100 of the above-mentioned natural Can f1 mature peptide, as detailed in SEQ ID NO: 2, and also provides the nucleotide sequence encoding the mutant as shown in SEQ ID NO: 3.

[0014] The recombinant Can f 2 mutant or its derivative, for recombinant Can f 2 (rCan f 2), should include a portion of its amino acid sequence that is identical to that of the natural mature Can f 2 peptide. For the recombinant Can f 2 mutant, the mutation site design scheme is as follows: deletion or substitution of amino acids at positions 27 and / or 88 of the natural mature Can f 2 peptide with other amino acids. When performing amino acid substitution mutations, any one or more of the following amino acids can be used: Q, G, A, V, L, I, Y, S, K, R, H, F, W, M, or T. Preferably, any one of Q, G, A, V, L, I, Y, F, or W is used, and more preferably, any one of Q, G, A, V, L, or I is used. The amino acid sequence of the natural mature Can f 2 peptide is shown in SEQ ID NO: 4. The aforementioned natural Can f2 mature peptide sequence can be derived from other allogeneic allergens or variants. Therefore, the Can f2 mutant of this application also includes proteins obtained by mutating the aforementioned natural Can f2 allogeneic allergens or variants, with the mutation site corresponding to the mutation site shown in SEQ ID NO: 4.

[0015] When the recombinant Can f2 protein of this application was prepared by expression in an E. coli system, the amino acid coverage results showed that, compared with the sequence shown in SEQ ID NO: 4, rCan f2 had an additional formylmethionine at the N-terminus. Based on the above common knowledge, the amino acid sequence of the recombinant Can f2 protein provided in this application can also be modified by adding a formylmethionine or methionine at the N-terminus based on the above mutation.

[0016] The derivatives include the full-length protein of the Can f2 mutant of this application, a portion of the Can f2 mutant of this application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation based on the Can f2 mutant of this application.

[0017] Natural Can f2 is a type II canine allergen and a lipocalin. According to literature reports, the reactivity of serum IgE antibodies against purified Can f2 in canine allergy patients is 23% compared to the reactivity against IgE antibodies against canine dander extract, suggesting that Can f2 is a minor canine allergen (de Groot, H et al. "Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I depleted extract." The journal of allergy and clinical immunology. 1991). Another study indicated that the concentration of Can f2 in canine saliva is higher than that of Can f1 (Kamata, Yoichi, et al. "Characterization of Dog Allergens Can f 1 and Can f 2.2. A comparison of Can f 1 with Can f 2. Regarding Their Biochemical and Immunological Properties." International archives of allergy and immunology. 2007).

[0018] The present invention also provides mutants of the above-mentioned natural Can f2 mature peptide with amino acid substitutions at positions 27 and 88, the corresponding protein primary structure sequence of which is shown in SEQ ID NO: 5, and the corresponding encoding DNA sequence of which is shown in SEQ ID NO: 6.

[0019] The recombinant Can f4 mutant or its derivatives wherein the Can f4 mutant has a deletion or substitution of the N at position 85 of the natural Can f4 mature peptide with another amino acid. When substituting amino acids, one or more of the following amino acids can be used: Q, G, A, V, L, I, Y, S, K, R, H, F, W, M, or T. Preferably, Q, G, A, V, L, I, Y, F, or W is used, and more preferably, Q, G, A, V, L, or I is used. The amino acid sequence of the natural Can f4 mature peptide is shown in SEQ ID NO: 7. The above-mentioned natural Can f4 mature peptide sequence can be derived from other allogeneic allergens or variants. Therefore, the Can f4 mutant of this application also includes proteins obtained by mutating the above-mentioned allogeneic allergens or variants of natural Can f4, with the mutation site corresponding to the mutation site shown in SEQ ID NO: 7.

[0020] When the recombinant Can f4 (rCan f4) protein of this application was prepared by expression in an E. coli system, the amino acid coverage results showed that, compared with the sequence shown in SEQ ID NO: 7, rCan f4 has an additional formylmethionine at the N-terminus. Based on the above common knowledge, the amino acid sequence of the recombinant Can f4 protein provided in this application can also be modified by adding a formylmethionine or methionine at the N-terminus based on the above mutation.

[0021] The derivatives include the full-length protein of the Can f4 mutant of this application, a portion of the Can f4 mutant of this application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation based on the Can f4 mutant of this application.

[0022] Natural Can f4 is a canine type IV allergen, a lipocalin protein found in dog dander. One study, using ImmunoCAP assays, showed that 13 out of 37 canine allergy sufferers (35%) had IgE antibodies against naturally purified Can f4 in their serum (Mattsson, L et al. "Molecular and immunological characterization of Can f4: a dog dander allergen cross-reactive with a 23kD odorant-binding protein in cow dander." Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology. 2010).

[0023] The present invention also provides a mutant of the above-mentioned natural Can f4 mature peptide with an 85th amino acid substitution, the corresponding protein primary structure sequence of which is shown in SEQ ID NO: 8, and the corresponding encoding DNA sequence of which is shown in SEQ ID NO: 9.

[0024] The recombinant Can f 5 mutant or its derivatives wherein the Can f 5 mutant has a deletion or substitution of amino acids at positions 83, 47, and 55 of the natural Can f 5, with other amino acids. When performing amino acid substitution mutations, one or more of the following amino acids can be used: G, Q, A, V, L, I, Y, S, H, F, W, M, or T. Preferably, G, Q, A, V, L, I, Y, F, or W can be substituted, and more preferably, G, Q, A, V, L, or I can be substituted. The amino acid sequence of the natural Can f 5 mature peptide is shown in SEQ ID NO: 10. The above-mentioned natural Can f 5 mature peptide sequence can be derived from other allogeneic allergens or variants; therefore, the Can f 5 mutants of this application also include proteins obtained by mutating the above-mentioned allogeneic allergens or variants of the natural Can f 5, with the mutation site corresponding to the mutation site shown in SEQ ID NO: 10.

[0025] When the recombinant Can f 5 (rCan f 5N) protein of this application was prepared by expression in an E. coli system, the amino acid coverage results showed that, compared with the sequence shown in SEQ ID NO: 10, rCan f 5 has an additional formylmethionine at the N-terminus. Based on the above common knowledge, the amino acid sequence of the recombinant Can f 5 protein provided in this application can also be modified by adding a formylmethionine or methionine at the N-terminus based on the above mutation.

[0026] The derivatives include the full-length protein of the Can f 5 mutant of this application, a portion of the Can f 5 mutant of this application, and proteins, fusion proteins, and various forms of modifications obtained by further mutation based on the Can f 5 mutant of this application.

[0027] Naturally occurring Can f5 is a canine type V allergen, a prostatic kallikrein found in both dog urine and dander. One study, using ImmunoCAP assays, showed that 28 out of 37 patients (76%) with canine allergies had IgE antibodies against naturally purified Can f5 protein in their serum, suggesting that Can f5 is a major canine allergen (Mattsson, L et al. "Prostatic kallikrein: a new major dog allergen." The journal of allergy and clinical immunology. 2009.).

[0028] The present invention also provides a mutant of the above-mentioned natural Can f5 mature peptide with amino acid substitutions at positions 47 and 83, the corresponding protein primary structure sequence of which is shown in SEQ ID NO: 11 and the corresponding encoding DNA sequence of which is shown in SEQ ID NO: 12.

[0029] Another object of the present invention is to provide an expression vector containing the nucleotide gene encoding the above-mentioned canine recombinant allergen protein; wherein, for prokaryotic expression, there are expression vectors based on the T7 promoter, such as pET32a, pET26b, pET28a, pDEST14 vectors, etc., and expression vectors based on the temperature-controlled promoter PL-PR, such as pBV220 vectors, etc.; for eukaryotic expression, there are secretory expression vectors, such as pPIC9K, pPIC9, pGAPZαA, pPICZαA, pH1L-S1, pYAM75P vectors, etc., and intracellular expression vectors, such as pH2L-D2, pAO815, pPIC3K, pHWO10, pPIC3.5K, etc.

[0030] Another object of the present invention is to provide an *E. coli* host or a *Pichia pastoris* host containing the recombinant expression vector described above; wherein, the *E. coli* host based on the PL-PR promoter expression vector can be any *E. coli* genetically engineered bacteria, preferably BL21(DE3), BL21 AI(DE3), Top10, DH5α, JM109, Rosetta(DE3), Rosetta gamiB(DE3), BL21(DE3)plys, etc.; the *E. coli* host based on the T7 promoter expression vector is preferably BL21(DE3), BL21 AI(DE3), Rosetta(DE3), Rosetta gamiB(DE3), BL21(DE3)plys, etc.; the *Pichia pastoris* host is preferably GS115, X33, KM71H, SMD1168, Y11430, MG1003, etc.

[0031] This invention also provides a method for expressing canine recombinant allergen protein in a prokaryotic expression system (such as Escherichia coli), the method comprising the following steps:

[0032] A. Construct a vector containing the gene encoding a canine allergen mutant or its derivative as described above;

[0033] B. The recombinant plasmid from step A was transformed into an Escherichia coli strain using a heat shock transformation method and cultured under suitable conditions;

[0034] C. Recover and purify the protein.

[0035] This invention also provides a method for purifying canine recombinant allergen protein, the purification method being as follows:

[0036] The E. coli bacteria cultured by the above method are resuspended, lysed, and the precipitate or supernatant is collected. The precipitate is the crude extract of the target protein inclusion bodies.

[0037] If inclusion bodies are collected, they are subjected to crude purification, denaturation, and refolding treatments to obtain a refolded solution.

[0038] If the collected liquid is the supernatant, it is concentrated by ultrafiltration to obtain the filtrate.

[0039] Collect the refolding solution or filtrate, and perform three-step purification using Sepharose Q Fast Flow, Sepharose SP High Performance, and Sepharose Phenyl High Performance 6 (HS) to obtain the target protein stock solution.

[0040] This invention also provides a method for expressing canine recombinant allergen protein in a eukaryotic expression system (such as Pichia pastoris X33), the method comprising the following steps:

[0041] A. Construct a vector containing the gene encoding a canine allergen mutant or its derivative as described above;

[0042] B. The recombinant plasmid from step A is transformed into the Escherichia coli cloning host strain by heat shock transformation and cultured under suitable conditions;

[0043] C. Perform plasmid extraction on the recombinant strain from step B to obtain high-purity and high-concentration recombinant plasmids; linearize the recombinant plasmids using appropriate restriction endonucleases and recover the linearization product;

[0044] D. Electroporate the linearized product of the recombinant plasmid from step C into a Pichia pastoris (e.g., X33) host, and obtain recombinants by culturing under suitable conditions and pressure screening;

[0045] E. Recover and purify proteins.

[0046] This invention also provides a method for purifying canine recombinant allergen protein, the purification method being as follows:

[0047] The above-mentioned recombinant Pichia pastoris strain was fermented and expressed, and the target protein secretion product was collected. The secretion product was desalted to obtain a crude extract.

[0048] The crude extract was purified in three steps using Sepharose Q Fast Flow, Sepharose SP High Performance, and Sepharose Phenyl High Performance 6 (HS) to obtain the target protein stock solution.

[0049] This invention also provides the application of the above-mentioned canine recombinant allergen protein in the preparation of diagnostic reagents for detecting canine allergies. This invention further provides the application of the above-mentioned canine recombinant allergen protein in the preparation of medicaments for treating canine allergic diseases.

[0050] The present invention also provides a composition for treating allergic diseases in dogs caused by allergens, comprising Can f1, Can f2, Can f4, and Can f5. Can f1, Can f2, Can f4, and Can f5 are naturally extracted proteins or recombinant proteins. Preferably, Can f1, Can f2, Can f4, and Can f5 are the aforementioned recombinant proteins.

[0051] The canine recombinant allergen mutant finally prepared by this invention meets the requirements for human recombinant DNA products in terms of purity, residual process impurities, and molecular characterization. It has a purity >95%, an amino acid sequence highly similar to the natural protein, disulfide bond pairing consistent with theory, and uniform molecular weight. Its in vitro reactivity with clinically positive allergy serum correlates well with clinically reported values, demonstrating good in vivo activity. It can induce an allergic immune response in animals, showing a clear desensitization therapeutic effect and good safety profile in vivo, and has the potential to be applied to the development of canine allergy desensitization treatment drugs and diagnostic reagents. Furthermore, compared with the unmutated wild-type recombinant protein, the mutant recombinant protein of this invention has significant advantages in protein purity, uniformity, and molecular characterization. By only slightly modifying the primary structure of the natural allergen protein, it solves the problems of the inability to express and purify the natural canine allergen sequence, as well as the disordered molecular weight. Even after mutation at relevant amino acid sites, it retains good allergen biological activity, which is more conducive to the control of the recombinant expression production process and the correct preparation of the recombinant protein. Compared to natural canine allergen extracts, the recombinant canine allergen protein of this invention avoids batch-to-batch variations in the content and activity of major canine allergens from different sources. The process and quality are more stable and controllable. It also avoids the degradation of major allergens and the generation of other sensitizing reactions caused by interactions between other components in natural extracts. This meets the requirements of modern biological products for safety, efficacy, and quality control. It can be used for the treatment and diagnosis of allergic diseases caused by canine allergens, such as allergic rhinitis and asthma, improving the precision of desensitization immunotherapy and the accuracy of canine allergy diagnosis, and has promising pharmaceutical prospects. Attached Figure Description

[0052] Figure 1: Unconvolution plot of molecular weight determination of wild-type Can f1 (expressed by Pichia pastoris)

[0053] Figure 2: Unconvolution plot of molecular weight detection for wild-type Can f1 (expressed in E. coli)

[0054] Figure 3: Deconvolution plot of molecular weight detection for Can f1 mutant 1 (expressed in E. coli)

[0055] Figure 4: Deconvolution plot of molecular weight detection for Can f2 mutant 2 (expressed in E. coli)

[0056] Figure 5: Unconvolution plot of molecular weight detection for Can f4 wild-type (expressed in E. coli)

[0057] Figure 6: Unconvolution plot of molecular weight detection for Can f4 wild-type (Pichia pastoris expression)

[0058] Figure 7: Deconvolution plot of molecular weight determination of Can f4 mutant 1 (expressed in E. coli)

[0059] Figure 8: Electrophoretic analysis of Can f5 mutant 2 in reduction and non-reduction forms

[0060] Figure 9: Deconvolution plot of molecular weight detection for sample 1 in lane 1 of Figure 8

[0061] Figure 10: Deconvolution plot of molecular weight detection for sample 8 in lane 8 of Figure 8

[0062] Figure 11: Amino acid coverage of the sample marked ① in lane 9 of Figure 8

[0063] Figure 12: SDS-PAGE analysis results of pure, non-reducing Can f5 mutant 1

[0064] Figure 13: Deconvolution plot of molecular weight detection for Can f5 mutant 1

[0065] Figure 14: Penh detection results under stimulation with different concentrations of methacholine (Mch) in Example 18

[0066] Figure 15: Results of serum antibody ELISA detection in Example 18

[0067] Figure 16: Results of antibody ELISA detection in bronchoalveolar lavage fluid (BALF) supernatant in Example 18

[0068] Figure 17: White blood cell count results in bronchoalveolar lavage fluid (BALF) in Example 18

[0069] Figure 18: Results of cytokine detection in supernatant of spleen cells stimulated in vitro in Example 18

[0070] Figure 19: Penh detection results under stimulation with different concentrations of methacholine (Mch) in Example 19

[0071] Figure 20: Serum IgE and IgG1 detection results in Example 19

[0072] Figure 21: EOS% and white blood cell count in bronchoalveolar lavage fluid (BALF) of Example 19

[0073] Figure 22: Results of cytokine detection in supernatant of spleen cells stimulated in vitro in Example 19 Detailed Implementation

[0074] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the embodiments are used only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0075] Example 1: Design and construction of expression strains for the Can f1 mutant

[0076] The mature peptide sequence of Can f1 was obtained from the Genbank accession number, as shown in SEQ ID NO: 1. It was reverse-translated into a DNA sequence, and Nanjing GenScript Biotech Co., Ltd. was commissioned to optimize the codons and synthesize the artificially encoded DNA sequence according to the *E. coli* expression system. Mutations were performed on some sites using PCR (mutation information is shown in Table 1). The amino acid sequence of mutant 1 is shown in SEQ ID NO: 2, and the encoding DNA sequence is shown in SEQ ID NO: 3. During the PCR stage, when the mutant was expressed using *E. coli*, an NdeI restriction site was introduced at the 5' end and an XhoI restriction site was introduced at the 3' end of its encoding DNA sequence. When the mutant was expressed using *Pichia pastoris*, an XhoI restriction site was introduced at the 5' end and an XbaI restriction site was introduced at the 3' end of its encoding DNA sequence.

[0077] This application's canine recombinant allergen can be used for recombinant expression in two systems: the *E. coli* prokaryotic expression system and / or the *Pichia pastoris* eukaryotic expression system. For the prokaryotic expression system, it can be used with expression vectors based on the T7 promoter, such as pET32a, pET26b, pET28a, and pDEST14, and can be used with *E. coli* genetically engineered bacteria such as BL21(DE3), BL21AI(DE3), Rosetta(DE3), Rosetta gamiB(DE3), and BL21(DE3)plys. Alternatively, it can be used with expression vectors based on the temperature-controlled promoter PL-PR, such as pBV220, and can be used with any *E. coli* genetically engineered bacteria, such as BL21(DE3), BL21AI(DE3), Top10, DH5α, JM109, Rosetta(DE3), Rosetta gamiB(DE3), and BL21(DE3)plys hosts. For eukaryotic expression systems, secretory expression vectors such as pPIC9K, pPIC9, pGAPZαA, pPICZαA, pH1L-S1, and pYAM75P can be used; intracellular expression vectors such as pH2L-D2, pAO815, pPIC3K, pHWO10, and pPIC3.5K can also be used. Based on the auxotrophic markers of the vectors, Pichia pastoris hosts such as GS115, X33, KM71H, SMD1168, Y11430, and MG1003 can be selected. In the examples, the pET26b vector and BL21(DE3) host were used for prokaryotic expression, and the pGAPZαA vector and X33 host were used for eukaryotic expression.

[0078] Construction of prokaryotic expression strains: The DNA sequences of the above Can f1 mutants were cloned into the pET26b vector to form the pET26b-Can f1 mutant expression plasmid, and transformed into BL21(DE3) Escherichia coli host to form BL21(DE3)-pET26b-Can f1 mutant expression strain;

[0079] Construction of eukaryotic expression strains: The DNA sequences of the above Can f1 mutants were cloned into the pGAPZαA vector to form the pGAPZαA-Can f1 mutant plasmid, and then electroporated into the X33 yeast host to form the X33-pGAPZαA-Can f1 mutant expression strain.

[0080] Table 1 Summary of mutation information for each sample

[0081] Example 2: Recombinant expression of Can f1 and its mutants

[0082] The Can f1 and its mutants can be prepared in prokaryotic or eukaryotic expression systems using the following expression process. For prokaryotic expression systems, the expression of Can f1 mutant 1 in pET26b vector and BL21(DE3) host is taken as an example. For eukaryotic expression systems, the expression of Can f1 mutant 5 in pGAPZαA vector and X33 host is taken as an example.

[0083] Prokaryotic system expression:

[0084] 1. BL21(DE3)-pET26b-Can f 1 mutant strain 1 was streaked into LB agar plates containing 50 μg / mL of glycerol and incubated at 37°C for 12 h.

[0085] 2. Pick colonies from the agar plate and inoculate them into 10 mL of TB liquid medium. Culture conditions: 37℃, 220 rpm, culture time: 8 h; this is the primary seed culture.

[0086] 3. Inoculate the primary seed at a ratio of 1-2% into 10 bottles of 500mL TB liquid culture medium. Culture conditions: 37℃, 220rpm. When the OD of the culture medium reaches 0.8, add 0.5mM IPTG to induce expression. After induction for 4 hours, centrifuge to collect the bacterial cells for later use.

[0087] Eukaryotic system representation:

[0088] 1. Five mutant strains of X33-pGAPZαA-Can f1 were streaked into YPD-agar plates containing 0.1 mg / mL of glycerol and incubated at 30°C for 72 h.

[0089] 2. Pick colonies from the agar plate and inoculate them into 10 mL of YPD liquid medium. Culture conditions: 30℃, 220 rpm, culture time: 12 h; this is the primary seed culture.

[0090] 3. Inoculate the primary seeds into 10 bottles of 500mL BMGY liquid medium at a ratio of 1-2%. Culture conditions: 30℃, 220rpm. When the OD of the culture medium reaches 2.0, add glucose to a final concentration of 0.5% every 24 hours for nutrient supplementation. After 72 hours of expression, centrifuge and collect the supernatant for later use.

[0091] Example 3: Extraction and purification of Can f1 and its mutants

[0092] The Can f1 and its mutants can be extracted and purified in prokaryotic or eukaryotic expression systems using the following process. This embodiment takes Can f1 mutant 1 expressed in a prokaryotic system and Can f1 mutant 5 expressed in a eukaryotic system as examples.

[0093] The main treatment method for Can f1 mutant 1 expressed in the prokaryotic expression system is as follows:

[0094] 1. The fermentation cells were resuspended in 10mM PB (pH 7.4) buffer to 100g / L and dispersed evenly. The cells were then homogenized in a high-pressure homogenizer at a pressure of 900±50 bar and homogenized 3 times. The material temperature was controlled not to exceed 25℃ during the homogenization process. After the homogenization was completed, the supernatant was collected by centrifugation. The supernatant is the crude extract of the target protein.

[0095] 2. The crude protein extract was salted out using ammonium sulfate at a ratio of 150g / 1000mL. The supernatant was collected, and the supernatant was salted out again using 100g / L ammonium sulfate for a second stage of salting out. The supernatant and precipitate were collected by centrifugation.

[0096] 3. Redissolution of precipitate: The salt-precipitate was redissolved using 10mM PB at a ratio of 500mL / g at pH 8.0. The salt-precipitate was dissolved by stirring at room temperature for 1 hour. After final dissolution, the sample was filtered through a 0.8μM membrane.

[0097] 4. First step purification: After changing the refolding buffer, the sample was purified using Sepharose Q Fast Flow. The sodium chloride concentration passing through the column bed was linearly reduced to 0.5M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM PB, pH 8.0; Purification elution buffer: 10mM PB + 1M sodium chloride, pH 8.0.

[0098] 5. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1.5M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 0.5M ammonium sulfate, pH 7.4.

[0099] 6. Third step: Chromatographic purification: The sample purified in the second step was desalted using ultrafiltration until the conductivity was less than 5 mS / cm. The pH was adjusted to 5.0 with phosphate. The sample was then purified using a Sepharose SP High Performance chromatography column. Within 10 column volumes, the proportion of elution buffer passing through the column bed was linearly achieved to 20%. The target protein peak was collected. Purification binding buffer: 10 mM PB, pH 5.0; Purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 5.0.

[0100] 7. Replace the target protein peak collected by the chromatography purification in step 3 with ultrafiltration buffer of 20mM PB, pH 7.0; after filtration and sterilization, the target protein stock solution is obtained.

[0101] 8. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0102] The main processing method for Can f1 mutant 5 expressed in the eukaryotic expression system is as follows:

[0103] 1. Separate fermentation cells by centrifugation, collect the fermentation supernatant, filter with 0.8 μM solution until clear, and then concentrate using an ultrafiltration system to replace the solution with 10 mM acetate-sodium acetate at pH 5.0;

[0104] 2. First step purification: After changing the buffer, the sample was purified using Sepharose SP High Performance. The sodium chloride concentration passing through the column bed was linearly reduced to 0.3M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM acetate-sodium acetate, pH 6.0; Purification elution buffer: 10mM acetate-sodium acetate 1M sodium chloride, pH 6.0.

[0105] 3. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 1M ammonium sulfate, pH 7.4.

[0106] 4. Third step: Chromatographic purification: The ultrafiltration buffer for the sample purified in the second step was changed to 10 mM PB, pH 8.0. Purification was performed using a Sepharose Q Fast Flow column. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly maintained at 20%. 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.

[0107] 5. Replace the target protein peak collected by the chromatography purification in step 3 with ultrafiltration buffer of 20mM PB, pH 7.0; after filtration and sterilization, the target protein stock solution is obtained.

[0108] 6. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0109] Table 2 Summary of HPLC-RP and HPLC-SEC purity of each sample

[0110] Can f1 is an apolipoprotein with a typical "barrel" structure formed by β-sheets in its higher-order structure. This barrel structure can bind lipid molecules such as sterols and retinol, and participate in lipid transport in the body.

[0111] Can f1 or its mutants, whether expressed in an E. coli system or a Pichia pastoris system, can be purified to a high purity of over 95% by the corresponding purification methods described in the above examples.

[0112] In HPLC-RP analysis, wild-type Can f1 expressed in the Pichia pastoris system, or its mutants, showed significant differences. Mutant 5 achieved a purity of over 95%, while wild-type, mutant 1, mutant 2, mutant 3, and mutant 4 exhibited multi-peak patterns with indistinct main peaks in HPLC-RP analysis. Combined with HPLC-SEC analysis results, it can be inferred that the five purified proteins are not homogeneous, containing multiple molecular weights of similar forms. These molecules with similar molecular weights exhibit differences in hydrophobicity, resulting in multi-peak patterns on HPLC-RP that are difficult to integrate. The LC-MS molecular weight analysis results of wild-type Can f1 below show that the pure product contains numerous molecular weight forms with similar molecular weights. Therefore, methods like HPLC-SEC, which rely on differences in molecular weight and shape to determine purity, cannot distinguish between them. In contrast, methods like HPLC-RP (which separates molecules based on differences in hydrophobic properties) that do not rely on molecular weight differences can identify these differences to some extent. For wild-type, mutant 1, mutant 2, mutant 3, and mutant 4, their HPLC-RP peaks were disordered, and LC-MS molecular weight analysis showed the presence of many similar molecular weight impurities. One possible hypothesis is that these molecules bound to small lipid metabolites from Pichia pastoris during expression in the Pichia pastoris system, and the type and number of lipid molecules bound to each protein molecule varied, ultimately leading to molecular heterogeneity in the pure product and thus significant differences in LC-MS and HPLC-RP detection.

[0113] Based on the above speculation, the design of mutants revolved around the principle of minimizing changes to the primary structure of the Can f1 protein while disrupting its ability to bind lipid molecules. Significant differences in HPLC-RP purity were observed between mutants 1, 3, 4, and 5 expressed in *E. coli* and *Pichia pastoris*. One possible explanation is that the lipid metabolism profiles of the *Pichia pastoris* and *E. coli* systems are different. These mutants could bind different types and numbers of lipid molecules in the two systems. Overall, in the *E. coli* system, the HPLC-RP and HPLC-SEC purities of mutants 1, 3, 4, and 5 all reached over 95%, indicating that each mutant was more advantageous than wild-type Can f1 in obtaining recombinant Can f1 with uniform molecular weight in the *E. coli* system. In the *Pichia pastoris* system, the HPLC-RP purity of mutant 5 was significantly better than the other mutants, indicating that the design of mutant 5 was beneficial for expressing recombinant Can f1 with uniform molecular weight in the *Pichia pastoris* system. Based on the above analysis, the Can f1 mutant of this application solves the problems of poor HPLC-RP purity and non-uniform molecular weight of wild-type Can f1 in Escherichia coli and Pichia pastoris systems. It may impair the lipid binding ability of Can f1 in the expression system, thus making it more conducive to the uniformity control of expression products and the scale-up of recombinant expression production in Escherichia coli and / or Pichia pastoris systems.

[0114] Example 4: Primary structure analysis and molecular weight of Can f1 and its variants

[0115] LC-MS molecular weight analysis can accurately reflect whether the primary sequence of biological macromolecules is correct, including whether 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.

[0116] Using our Thermo Scientific TM A high-resolution mass spectrometry analysis system was used to perform LC-MS molecular weight and primary structure analysis on the purified wild-type Can f1 and its mutant recombinant proteins. The mutants successfully expressed and purified in the above examples all showed uniform molecular weight, with the target molecule accounting for more than 90% and the disulfide bonds being correctly paired, as shown in Figures 1-3 and the table below.

[0117] Table 3 Summary of LC-MS molecular weight and disulfide bond analysis of Can f1 and its mutants

[0118] Example 5: Design and construction of expression strains for Can f2 mutants

[0119] The mature peptide sequence of Can f2 was obtained from the GenBank accession number, as shown in SEQ ID NO: 4. It was reverse-translated into a DNA sequence, and Nanjing GenScript Biotech Co., Ltd. was commissioned to optimize the codons and synthesize the artificially encoded DNA sequence according to the *E. coli* expression system. Mutations were performed on some sites using PCR (mutation information is shown in Table 4). The amino acid sequence of mutant 2 is shown in SEQ ID NO: 5, and the encoding DNA sequence is shown in SEQ ID NO: 6. During the PCR stage, when the mutant was expressed using *E. coli*, an NdeI restriction site was introduced at the 5' end and an XhoI restriction site was introduced at the 3' end of its encoding DNA sequence. When the mutant was expressed using *Pichia pastoris*, an XhoI restriction site was introduced at the 5' end and an XbaI restriction site was introduced at the 3' end of its encoding DNA sequence.

[0120] Construction of prokaryotic expression strains: The DNA sequences of the above Can f2 mutants were cloned into the pET26b vector to form the pET26b-Can f2 mutant expression plasmid, and transformed into BL21(DE3) Escherichia coli host to form BL21(DE3)-pET26b-Can f2 mutant expression strain;

[0121] Construction of eukaryotic expression strains: The DNA sequences of the above Can f2 mutants were cloned into the pGAPZαA vector to form the pGAPZαA-Can f2 mutant plasmid, and then electroporated into yeast host X33 to form the X33-pGAPZαA-Can f2 mutant expression strain.

[0122] Table 4 Summary of mutation information for each sample

[0123] Example 6: Recombinant expression of Can f2 and its mutants

[0124] The prokaryotic expression system uses the expression of Can f2 mutant 2 in the pET26b vector and BL21(DE3) host as an example, while the eukaryotic expression system uses the expression of Can f2 mutant 1 in the pAGPZαA vector and X33 host as an example. The recombination expression method for each mutant is the same as in Example 2.

[0125] Example 7: Extraction and purification of Can f2 and its mutants

[0126] The Can f2 and its mutants can be extracted and purified in prokaryotic or eukaryotic expression systems using the following process. This embodiment takes Can f2 mutant 2 expressed in a prokaryotic system and Can f2 mutant 1 expressed in a eukaryotic system as examples.

[0127] The main treatment method for Can f2 mutant 2 expressed in the prokaryotic expression system is as follows:

[0128] 1. The fermentation cells were resuspended in 10mM PB (pH 7.4) buffer to 100g / L and dispersed evenly; they were then homogenized in a high-pressure homogenizer at a pressure of 900bar±50bar for 3 cycles, with the material temperature controlled not to exceed 25℃ during the homogenization process; after homogenization, the supernatant was collected by centrifugation, and the supernatant was the crude extract of the target protein.

[0129] 2. The crude protein extract was salted out using ammonium sulfate at a ratio of 150g / 1000mL, and the supernatant was collected. The supernatant was then subjected to a second salting out using 100g / L ammonium sulfate, and the supernatant and precipitate were collected by centrifugation.

[0130] 3. Redissolution of precipitate: The salt-precipitate was redissolved using 10mM PB at a ratio of 500mL / g at pH 8.0. The salt-precipitate was dissolved by stirring at room temperature for 1 hour. After final dissolution, the sample was filtered through a 0.8μM membrane.

[0131] 4. First step purification: After changing the refolding buffer, the sample was purified using SepharoseQ Fast Flow. The sodium chloride concentration in the column bed was linearly reduced to 0.5M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM PB, pH 8.0; Purification elution buffer: 10mM PB + 1M sodium chloride, pH 8.0.

[0132] 5. Second purification step: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 1M ammonium sulfate, pH 7.4.

[0133] 6. Third step: Chromatographic purification. The sample purified in the second step was desalted using ultrafiltration until the conductivity was less than 5 mS / cm. The pH was adjusted to 6.0 with phosphate. Purification was then performed using a Sepharose SP High Performance chromatography column. Within 10 column volumes, the proportion of elution buffer passing through the column bed was linearly maintained at 20%. The target protein peak was collected. Purification binding buffer: 10 mM PB, pH 6.0; Purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 6.0.

[0134] 7. Replace the target protein peak collected by chromatography in step 3 with ultrafiltration buffer (20 mM PB, pH 7.0). After filtration and sterilization, the target protein stock solution is obtained.

[0135] 8. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0136] The main treatment method for Can f2 mutant 1 expressed in the eukaryotic expression system is as follows:

[0137] 1. Separate the fermentation cells by centrifugation, collect the fermentation supernatant, filter with 0.8 μM solution until clear, and then concentrate using an ultrafiltration system to replace the solution with 10 mM acetate-sodium acetate at pH 5.0.

[0138] 2. First step purification: After buffer replacement, the sample was purified using Sepharose SP High Performance. The sodium chloride concentration passing through the column bed was linearly reduced to 0.5 M within 10 column volumes, and the target protein peak was collected. The purification binding buffer was 10 mM acetate-sodium acetate, pH 5.0; the purification elution buffer was 10 mM acetate-sodium acetate and 1 M sodium chloride, pH 5.0.

[0139] 3. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 2.0M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 2.0M ammonium sulfate, pH 7.4.

[0140] 4. Third step: Chromatographic purification: The ultrafiltration buffer for the sample purified in the second step was changed to 10 mM PB, pH 8.0, until the conductivity was less than 5 mS / cm; after the buffer change, the sample was purified using a Sepharose Q Fast Flow chromatography column. Within 10 column volumes, the proportion of elution buffer passing through the column bed was linearly reached to 20%, 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;

[0141] 5. Replace the target protein peak collected by the chromatography purification in step 3 with ultrafiltration buffer of 20mM PB, pH 7.0; after filtration and sterilization, the target protein stock solution is obtained.

[0142] 6. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0143] Table 5 Summary of HPLC-SEC, HPLC-RP, and purity for each sample

[0144] The 27th amino acid of Can f2 is an N-glycosylation site. Since Pichia pastoris possesses a post-translational N-glycosylation modification system, wild-type Can f2 expressed in Pichia pastoris will undergo N-glycosylation modification. However, this N-glycosylation modification in Pichia pastoris is generally not uniform, resulting in the inability to integrate the HPLC-RP of wild-type Can f2 expressed in yeast systems due to the uneven degree of glycosylation. In contrast, the E. coli expression system does not possess post-translational glycosylation modification capabilities; therefore, wild-type Can f2 can be efficiently expressed and purified in E. coli systems to obtain highly purified and homogeneous products. In the Pichia pastoris system, when the 27th N-glycosylation site of Can f2 is mutated, the resulting mutants (e.g., mutant 1, mutant 2, mutant 4) no longer undergo N-glycosylation modification. Consequently, after expression in the Pichia pastoris system, a homogeneous product with high HPLC-SEC and HPLC-RP purity can be obtained, allowing for expression in both eukaryotic and prokaryotic systems. Based on the results of mutant 4, the substitution mutation at amino acid position 88 does not significantly alter the properties of the Can f2 molecule, thus resolving the issue of uneven Can f2 expression in Pichia pastoris and improving the applicability of Can f2 to both expression systems. Amino acid position 138 likely plays a crucial role in the stability of Can f2; when it is deleted, it cannot be expressed in the Pichia pastoris system, but in the E. coli system, it is converted to major inclusion body expression, and this inclusion body is difficult to convert to its active form through denaturation or renaturation.

[0145] Example 8: Primary structure analysis and molecular weight of Can f2 and its mutants

[0146] Using our Thermo Scientific TM A high-resolution mass spectrometry analysis system was used to perform LC-MS molecular weight and primary structure analysis on the purified wild-type Can f2 and its mutant recombinant protein. The mutants successfully expressed and purified in the above examples all showed uniform molecular weight, with the target molecule accounting for more than 90% and correct disulfide bond pairing, as shown in Figure 4 and the table below (using the E. coli system to express mutants as an example).

[0147] Table 6 Summary of molecular weight and disulfide bonds for each sample

[0148] Example 9: Design and Construction of Can f4 Mutant Expression Strains

[0149] The mature peptide sequence of Can f4 was obtained from the GenBank accession number, as shown in SEQ ID NO: 7. It was reverse-translated into a DNA sequence, and Nanjing GenScript Biotech Co., Ltd. was commissioned to optimize the codons and synthesize the artificially encoded DNA sequence according to the *E. coli* expression system. Mutations were performed on some sites using PCR (mutation information is shown in Table 7). The amino acid sequence of mutant 1 is shown in SEQ ID NO: 8, and the encoding DNA sequence is shown in SEQ ID NO: 9. During the PCR stage, when the mutant was expressed using *E. coli*, an NdeI restriction site was introduced at the 5' end and an XhoI restriction site was introduced at the 3' end of its encoding DNA sequence. When the mutant was expressed using *Pichia pastoris*, an XhoI restriction site was introduced at the 5' end and an XbaI restriction site was introduced at the 3' end of its encoding DNA sequence.

[0150] Construction of prokaryotic expression strains: The DNA sequences of the above Can f4 mutants were cloned into the pET26b vector to form the pET26b-Can f4 mutant expression plasmid, and transformed into the BL21(DE3) Escherichia coli host to form the BL21(DE3)-pET26b-Can f4 mutant expression strain.

[0151] Construction of eukaryotic expression strains: The DNA sequences of the above Can f4 mutants were cloned into the pGAPZαA vector to form the pGAPZαA-Can f4 mutant plasmid, which was then electroporated into the X33 yeast host to form the X33-pGAPZαA-Can f4 mutant expression strain.

[0152] Table 7 Summary of mutation information for each sample

[0153] Example 10: Recombinant expression of Can f4 and its mutants

[0154] The prokaryotic expression system uses the expression of Can f4 mutant 1 in the pET26b vector and BL21(DE3) host as an example, while the eukaryotic expression system uses the expression of Can f4 mutant 1 in the pAGPZαA vector and X33 host as an example. The recombination expression method for each mutant is the same as in Example 2.

[0155] Example 11: Extraction and purification of Can f4 and its mutants

[0156] The Can f4 and its mutants can be extracted and purified in prokaryotic or eukaryotic expression systems using the following process. This embodiment takes the Can f4 mutant 1 expressed in a prokaryotic system and the Can f4 mutant 1 expressed in a eukaryotic system as examples.

[0157] The main processing methods for prokaryotic expression systems are as follows:

[0158] 1. The fermentation cells were resuspended in 10mM PB 150mM sodium chloride (pH 7.4) buffer to 100g / L and dispersed evenly. The cells were then homogenized using a high-pressure homogenizer at a pressure of 900bar±50bar, and the homogenization was repeated 3 times. The material temperature was controlled not to exceed 25℃ during the homogenization process. After homogenization, the precipitate was collected by centrifugation, and the precipitate was the crude extract of the target protein inclusion bodies.

[0159] 2. The obtained inclusion bodies were resuspended in 10mM PB, 2M urea, 0.5% Triton X-100, and 150mM sodium chloride at pH 7.4 at a ratio of 1 / 100 (mass / volume), dispersed evenly, stirred and mixed at 4℃ for 2h, and centrifuged to collect the inclusion body precipitate. The above operation was repeated twice to obtain the crude purified inclusion bodies.

[0160] 3. After crude purification, the inclusion bodies were resuspended in denaturing solution at a ratio of 2g / 100mL and stirred at room temperature for more than 1 hour until basically clear; denaturing solution: 10mM PB, 6M guanidine hydrochloride, 10mM DTT, pH 8.0; after denaturation, the supernatant was collected by centrifugation.

[0161] 4. Refolding: Slowly add the denaturing solution to the refolding solution at a ratio of 1 / 5 to 1 / 20, stirring rapidly to disperse the protein and prevent excessively high local protein concentration. Control the addition time of the denaturing solution to 3 hours. Control the temperature of the refolding solution to 8°C during the addition process. After the denaturing solution is added, maintain the refolding process for 48 hours. Refolding solution: 20mM Tris, 0.5M Urea, 3mM Reduced Glutathione, 1mM Oxidized Glutathione, pH 8.0.

[0162] 5. After refolding, centrifuge or filter the refolded solution to remove insoluble matter, then use an ultrafiltration membrane with a pore size of 3-10 kDa to concentrate the solution to 10 mM PB at pH 8.0, filter and set aside.

[0163] 6. First step purification: After changing the refolding buffer, the sample was purified using Sepharose Q Fast Flow. The sodium chloride concentration passing through the column bed was linearly reduced to 0.3M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM PB, pH 8.0; Purification elution buffer: 10mM PB + 1M sodium chloride, pH 8.0.

[0164] 7. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1.5M, and the pH was adjusted to 7.4-8.0. A Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 1.5M ammonium sulfate, pH 7.4.

[0165] 8. Third step: Chromatographic purification: The sample purified in the second step was desalted using ultrafiltration until the conductivity was less than 5 mS / cm. The pH was adjusted to 6.0 using phosphate. The sample was then purified using a Sepharose SP High Performance chromatography column. Within 10 column volumes, the proportion of elution buffer passing through the column bed was linearly achieved to 50%. The target protein peak was collected. Purification binding buffer: 10 mM PB, pH 6.0; Purification elution buffer: 10 mM PB + 1 M sodium chloride, pH 6.0.

[0166] 9. Replace the target protein peak collected by the chromatography purification in step 3 with ultrafiltration buffer of 20mM PB, pH 7.0; after filtration and sterilization, the target protein stock solution is obtained.

[0167] 10. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0168] The main processing methods for eukaryotic expression systems are:

[0169] 1. Separate fermentation cells by centrifugation, collect the fermentation supernatant, filter with 0.8 μM solution until clear, and then concentrate using an ultrafiltration system to replace the solution with 10 mM acetate-sodium acetate at pH 5.0;

[0170] 2. First step purification: After changing the buffer, the sample was purified using Sepharose SP High Performance. The sodium chloride concentration passing through the column bed was linearly reduced to 0.5M within 10 column volumes, and the target protein peak was collected. The purification binding buffer was 10mM acetate-sodium acetate, pH 5.0; the purification elution buffer was 10mM acetate-sodium acetate and 1M sodium chloride, pH 5.0.

[0171] 3. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1.0 M, and the pH was adjusted to 7.4-8.0. A Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10 mM PB, pH 7.4; Purification binding buffer: 10 mM PB + 1 M ammonium sulfate, pH 7.4.

[0172] 4. Replace the target protein peak collected by the second step of chromatography with ultrafiltration buffer (20 mM PB, pH 7.0); after filtration and sterilization, the target protein stock solution is obtained.

[0173] 5. The final samples were analyzed using HPLC-SEC and HPLC-RP.

[0174] Table 8 Summary of HPLC-SEC and HPLC-RP purity of each sample

[0175] Can f4 is an apolipoprotein with a typical "barrel" structure formed by β-sheets in its higher-order structure. This barrel structure can bind lipid molecules such as sterols and retinol, and participate in lipid transport in the body.

[0176] Can f4 or its mutants 1, 4, and 5, whether expressed in an E. coli system or a Pichia pastoris system, can all be purified to a high purity of over 95% by the corresponding purification methods described in the above examples.

[0177] In HPLC-RP analysis, wild-type Can f4 or its mutant expressed in the Pichia pastoris system exhibited a multi-peak pattern with an indistinct main peak. Analysis revealed the cause to be similar to that of Can f1.

[0178] The HPLC-RP purity of Can f4 mutants 1, 4, and 5 expressed in *E. coli* reached over 95%. One possible explanation is that the lipid metabolism profiles of the *Pichia pastoris* and *E. coli* systems are different. These mutants could bind different types and numbers of lipid molecules in the two systems. Overall, in the *E. coli* system, the HPLC-RP and HPLC-SEC purities of mutants 1, 4, and 5 all reached over 95%, indicating that each mutant is more advantageous than wild-type Can f4 in obtaining recombinant Can f4 with uniform molecular weight in the *E. coli* system. In the *Pichia pastoris* system, the HPLC-RP purity of both wild-type Can f4 and the mutants was poor. Based on the above analysis, the Can f4 mutants of this application solve the problems of poor HPLC-RP purity and non-uniform molecular weight of expression products in the *E. coli* system, which may impair the lipid-binding ability of Can f4 in the expression system, thus making it more beneficial for the uniformity control of expression products and the scale-up of recombinant expression production in the *E. coli* system. On the other hand, different mutants are significantly more suitable for Escherichia coli than the Pichia pastoris system. This may be because Escherichia coli is more primitive than Pichia pastoris and its lipid metabolism pathway is simpler.

[0179] Compared to wild-type Can f4 and mutants 1, 4, and 5, mutants 2 and 3 lack amino acids at positions 62 and 154. Amino acids at positions 62 and 154 are cysteine ​​residues, which, according to the higher-order structure predicted by ALPHA FOLD, pair to form a disulfide bond. The deletion or mutation of these two amino acids significantly disrupts the stability of Can f4, resulting in: expression failure in the Pichia pastoris system, significantly reduced refolding and purification yields in the E. coli system, and inability to effectively purify the protein.

[0180] Example 12: Primary structure analysis and molecular weight of Can f4 and its variants

[0181] Using our Thermo Scientific TM A high-resolution mass spectrometry analysis system was used to perform LC-MS molecular weight and primary structure analysis on the purified wild-type Can f4 and its variant recombinant proteins. The mutants successfully expressed and purified in the above examples all showed uniform molecular weight, with the target molecule accounting for more than 90% and correct disulfide bond pairing, as shown in Figures 5-7 and the table below.

[0182] Table 9 Summary of molecular weight and disulfide bond count for each sample

[0183] Example 13: Design and Construction of Can f5 Mutant Expression Strains

[0184] The mature peptide sequence of Can f5 was obtained from the Genbank accession number, as shown in SEQ ID NO: 10. It was reverse-translated into a DNA sequence, and codon optimization was performed by Nanjing GenScript Biotech Co., Ltd. according to the *E. coli* expression system. The coding DNA sequence of Can f5 was then artificially synthesized. Mutations were performed at certain sites using PCR (mutation information is shown in Table 10). The amino acid sequence of mutant 1 is shown in SEQ ID NO: 11, and the coding DNA sequence is shown in SEQ ID NO: 12. During the PCR stage, when the mutant was expressed using *E. coli*, an NdeI restriction site was introduced at the 5' end and an XhoI restriction site was introduced at the 3' end of its coding DNA sequence. When the mutant was expressed using *Pichia pastoris*, an XhoI restriction site was introduced at the 5' end and an XbaI restriction site was introduced at the 3' end of its coding DNA sequence.

[0185] Construction of prokaryotic expression strains: The DNA sequences of the above Can f 5 mutants were cloned into the pET26b vector to form the pET26b-Can f 5 mutant expression plasmid, and transformed into the BL21(DE3) Escherichia coli host to form the BL21(DE3)-pET26b-Can f 5 mutant expression strain.

[0186] Construction of eukaryotic expression strains: The DNA sequences of the above Can f5 mutants were cloned into the pGAPZαA vector to form the pGAPZαA-Can f5 mutant plasmid, which was then electroporated into the X33 yeast host to form the X33-pGAPZαA-Can f5 mutant expression strain.

[0187] Table 10 Summary of mutation information for each sample

[0188] Example 14: Recombinant expression of Can f5 and its mutants

[0189] The prokaryotic expression system uses the expression of Can f 5 mutant 1 in the pET26b vector and BL21(DE3) host as an example, while the eukaryotic expression system uses the expression of Can f 5 mutant 1 in the pAGPZαA vector and X33 host as an example. The recombination expression method for each mutant is the same as in Example 2.

[0190] Example 15: Extraction and purification of Can f5 and its mutants

[0191] In this application, Can f5 and its mutants can be extracted and purified in prokaryotic and eukaryotic expression systems using the following processes. This embodiment uses Can f5 mutant 1 expressed in a prokaryotic system and Can f5 mutant 1 expressed in a eukaryotic system as examples. The processing techniques for mutant 2 and other mutants are completely consistent with those for mutant 1.

[0192] The main treatment method for Can f5 mutant 1 expressed in the prokaryotic expression system is as follows:

[0193] 1. The fermentation cells were resuspended in 10mM PB 150mM sodium chloride (pH 7.4) buffer to a concentration of 80-120 g / L and dispersed evenly. The mixture was then homogenized using a high-pressure homogenizer at a pressure of 900 bar ± 50 bar, with three cycles of homogenization. The material temperature was controlled to not exceed 25℃ during the homogenization process. After homogenization, the precipitate was collected by centrifugation; the precipitate is the crude extract of the target protein inclusion bodies.

[0194] 2. The obtained inclusion bodies were resuspended in 10mM PB, 2M urea, 0.5% Triton X-100, and 150mM sodium chloride at pH 7.4 at a ratio of 1 / 100 (mass / volume), dispersed evenly, stirred and mixed at 4℃ for 2h, and centrifuged to collect the inclusion body precipitate. The above operation was repeated twice to obtain the crude purified inclusion bodies.

[0195] 3. After crude purification, the inclusion bodies were resuspended in denaturing solution at a ratio of 2g / 100mL and stirred at room temperature for more than 1 hour until basically clear; denaturing solution: 10mM PB, 8M urea, 10mM DTT, pH 8.0; after denaturation, the supernatant was collected by centrifugation.

[0196] 4. Refolding: Slowly add the denaturing solution to the refolding solution at a ratio of 1 / 20, stirring rapidly to disperse the protein and prevent excessively high local protein concentration. Control the addition time of the denaturing solution to 10 hours. Control the temperature of the refolding solution to 8°C during the addition process. After the denaturing solution is added, maintain the refolding process for 48 hours. Refolding solution: 20mM Tris, 0.5M Urea, 3mM Cysteine, 1mM Cystine, pH 8.0.

[0197] 5. After refolding, centrifuge or filter the refolded solution to remove insoluble matter, then use a 10kDa ultrafiltration membrane to concentrate and replace the solution with 10mM PB at pH 8.0. Filter the solution and set it aside for later use.

[0198] 6. First step purification: After changing the refolding buffer, the sample was purified using Sepharose SP High Performance. The sodium chloride concentration passing through the column bed was linearly reduced to 0.5M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM PB, pH 6.0; Purification elution buffer: 10mM PB + 1M sodium chloride, pH 6.0.

[0199] 7. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1.5M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 1.5M ammonium sulfate, pH 7.4.

[0200] 8. Replace the target protein peak collected by the second step of chromatography with ultrafiltration buffer (20 mM PB, pH 7.0); after filtration and sterilization, the target protein stock solution is obtained.

[0201] 10. The final samples were analyzed using HPLC-RP and non-reducing SDS-PAGE.

[0202] The main treatment method for Can f5 mutant 1 expressed in the eukaryotic expression system is as follows:

[0203] 1. The fermentation supernatant was filtered with 0.8 μM solution and then concentrated using an ultrafiltration system to obtain 10 mM PB solution at pH 7.4. After the solution was changed, the pH of the sample was adjusted to 7.4 using sodium hydroxide.

[0204] 2. First step purification: After changing the buffer, the sample was purified using Sepharose SP High Performance. The sodium chloride concentration passing through the column bed was linearly reduced to 0.5M within 10 column volumes, and the target protein peak was collected. Purification binding buffer: 10mM PB, pH 7.4; Purification elution buffer: 10mM PB + 1M sodium chloride, pH 7.4.

[0205] 3. Second step purification: Ammonium sulfate was added to the sample purified in the first step to a final concentration of 1.5M, and the pH was adjusted to 7.4-8.0. A Sepharose Phenyl High Performance 6 (HS) chromatography column was used for purification. Within 10 column volumes, the elution buffer ratio passing through the column bed was linearly achieved to 100%, and the target protein peak was collected. Purification elution buffer: 10mM PB, pH 7.4; Purification binding buffer: 10mM PB + 1M ammonium sulfate, pH 7.4.

[0206] 4. Replace the target protein peak collected by the second step of chromatography with ultrafiltration buffer of 20mM PB, pH 7.0; after filtration and sterilization, the target protein stock solution is obtained.

[0207] Figure 8 shows the results of non-reducing electrophoresis analysis. The mutant 2 sample contained two components, A and B, in the second purification step. With increasing purification elution gradient, the proportion of A gradually decreased, while the proportion of B gradually increased. At least one of components A and B existed in a form where they were paired via intermolecular disulfide bonds (① and ②). The non-reducing molecular weights of samples lane 1 and lane 8 in Figure 8 were determined using LC-MS, and the results are shown in Figures 9 and 10. The component corresponding to lane 8 was mainly band B in Figure 8, and the determination showed the correct molecular weight, 25941.935 Da. The component corresponding to lane 1 in Figure 8 was mainly band A, whose molecular weight was slightly larger than band B. LC-MS determination showed a significant signal at 25959.997 Da, which, according to inference analysis, represents a break at a specific site in the Can f 5 mutant 2. It can be inferred that the molecules marked ① and ② in Figure 8 are Can f 5 mutant 2 that have been cleaved at a specific site. ① and ② are connected by disulfide bonds to form the complete Can f 5 mutant 2. The mutant 2 formed by this cleavage and subsequent connection by disulfide bonds is different in structure or shape from the mutant 2 formed by non-cleavage, which is reflected in the inconsistent migration rate on the non-reducing electrophoresis gel.

[0208] The band marked ① in lane 9 of Figure 8 was cut out, and then amino acid coverage analysis was performed. The results are shown in Figure 11. The amino acid coverage results confirmed that no signal was detected in the first 83 amino acids of band ② in Figure 8. Therefore, it can be inferred that ① and ② were characteristically cleaved at position 83, and after cleavage, the two were linked together by intermolecular disulfide bonds to form a complete molecule.

[0209] The above phenomenon was not observed in mutants 1 and 3, which were mutated at position 83 of the wild type. Their non-reducing electrophoresis (as shown in Figure 12) showed a single band, and the HPLC-RP purity showed a single peak with a purity of over 95%, which further confirmed the above hypothesis.

[0210] Further HPLC-RP purity analysis of the mutants expressed in the eukaryotic and prokaryotic expression systems (results shown in Table 11, using the *E. coli* system as an example) revealed that under the same process conditions, except for mutant 2, the purity of all mutants reached over 95%. The goal of the mutation targeting the 83rd amino acid was to prevent molecular fragmentation by protease after expression. Based on the expression and analysis studies of the mutants of this invention, it was confirmed that after mutating the 83rd amino acid of natural Can f5, the Can f5 mutant could be expressed in both eukaryotic and prokaryotic systems, yielding recombinant Can f5 with correct molecular weight, good purity, and uniformity. The mutation at position 47 primarily aims to reduce intermolecular disulfide bond mismatches and prevent dimer formation, as the cysteine ​​at this position does not participate in disulfide bond formation and is a free cysteine. The presence of this free cysteine ​​may lead to non-specific mismatch aggregation during expression, process operation, and storage. HPLC-SEC analysis of the mutant samples of this invention showed that the purity of each mutant remained above 95%. In summary, the Can f5 mutant of the present invention has advantages over wild-type Can f5 in terms of purity, molecular weight and uniformity of recombinant protein, which is more conducive to the scale-up of recombinant expression production.

[0211] Table 11 Summary of HPLC-RP purity for each sample (E. coli system)

[0212] Example 16: Primary structure analysis and molecular weight of Can f5 and its mutants

[0213] Using our Thermo Scientific TM A high-resolution mass spectrometry analysis system was used to perform LC-MS molecular weight and primary structure analysis on the purified wild-type Can f5 and its mutant recombinant protein. The mutants successfully expressed and purified in the above examples all showed uniform molecular weight, with the target molecule accounting for more than 90% and correct disulfide bond pairing, as shown in Figure 13 and the table below (using the E. coli system to express mutants as an example).

[0214] Table 12 Molecular weights and disulfide bond pairings of each sample (E. coli system)

[0215] Example 17: Activity detection of Can f1, Can f2, Can f4, and Can f5 mutants

[0216] In this embodiment, reactivity was tested using clinical canine allergy serum (a mixed serum from multiple hospitals) and the canine recombinant allergen mutant prepared in this application. The detection method is as follows:

[0217] 1. Coating: Dilute the recombinant protein to 1 μg / mL with 0.05M carbonate coating buffer (pH 9.6), add 100 μL / well to the microplate, and incubate overnight at 2-8℃;

[0218] 2. Blocking: The next day, remove the microplate, wash once with PBST, and add 200 μL of PBST containing 2% BSA to the microplate at 37°C for 2 hours.

[0219] 3. Sample preparation: Dilute the clinical canine allergy positive and negative sera separately with PBST containing 2% BSA (dilution factor is shown in the table below). Use PBST containing 2% BSA as blank background. Discard the liquid in the ELISA plate. Add 100uL of each dilution sample to the ELISA plate at 37℃ and 300rpm for 2h.

[0220] 4. Detection: Wash the microplate three times with PBST, add 100 μL of mouse anti-human IgE-HRP secondary antibody diluted 1:1500 per well, and incubate at 37℃ and 300 rpm for 1 h.

[0221] 5. Color development, termination, and reading: Wash the ELISA plate three times with PBST, add 100 μL of TMB VII color development solution per well, react at 37°C for 15 min, then add 50 μL of 2M sulfuric acid per well, and immediately place it under a wavelength of 450 nm for reading (ELISA reader: Thermo Multi SKANGO). The ELISA reader reading reflects the binding level of recombinant allergens to specific IgE in serum.

[0222] The table below shows the results (ELISA reader readings) of the canine recombinant allergen mutants (expressed in E. coli system) prepared in this application and different clinical canine allergy sera. The results show that the positive rate of Can f1 mutant 1 with clinical positive sera is the highest, and the correlation with clinical reported values ​​is also high (such as clinical serum samples SZ22, 8629, 7485, 8603, serum 3, etc.). Can f4 and Can f5 recombinant allergen mutants also showed positive reactions with some clinical positive sera (such as O35, CZ461, 8603, serum 3, etc.). Some serum samples with high clinical positive values ​​only showed high reactivity with Can f5 mutant (such as CZ461), while some serum samples with high clinical positive values ​​showed high reactivity with Can f1, Can f4 and Can f5 mutants at the same time (such as 8603, serum 3). Furthermore, the clinical serum samples collected in this study showed weak reactivity to the Can f2 mutant overall, which may be related to the low Can f2 positivity rate among canine allergy patients. Further expansion of the serum sample size is needed to screen for Can f2-sensitive patients. In addition, the reactivity of Can f1, Can f2, Can f4, and Can f5 mutants expressed and purified in the *E. coli* or *Pichia pastoris* systems to canine positive serum was similar to the detection results of the serum exemplified in this embodiment.

[0223] Table 13

[0224] Example 18: Evaluation of in vivo sensitization effects of Can f1, Can f2, Can f4, Can f5 mutants and their combinations

[0225] 1. Experimental Materials

[0226] BALB / c mice, female, 6-7 weeks old (at delivery week), provided by Shanghai Slack Laboratory Animal Co., Ltd.

[0227] Aluminum hydroxide adjuvant, batch number: 230017, provided by SERVA.

[0228] 2. Allergen preparation

[0229] 2.1 Sensitized Samples

[0230] Using normal saline (NS) for injection, prepare solutions of Can f1, Can f2, Can f4, and Can f5 with a concentration of 0.0067 mg / mL (the allergens used were the samples prepared in the examples above, taking Can f1 mutant 1, Can f2 mutant 2, Can f4 mutant 1, and Can f1 mutant as examples, the same applies below). Separately, mix the above four allergen solutions in equal volumes to prepare an allergen compound solution. Add aluminum hydroxide adjuvant to each solution, with a volume ratio of allergen solution to aluminum hydroxide adjuvant of 3:1, to prepare sensitized samples. Shake thoroughly before injection.

[0231] 2.2 Excitation of Samples

[0232] Using normal saline (NS) for injection, Can f1, Can f2, Can f4, and Can f5 were prepared into Can f1 solution, Can f2 solution, Can f4 solution, and Can f5 solution with a concentration of 0.5 mg / mL. In addition, the above four allergen solutions were mixed in equal volumes to prepare a compound solution, which was used as the stimulation sample.

[0233] 3. Drug delivery system

[0234] 3.1 Sensitization Stage

[0235] Seventy-two mice were randomly divided into six groups of 12 mice each, based on their body weight. Detailed group information is shown in the table below. Groups G2-G6 received an intraperitoneal injection of 4 μg / mouse / injection of the corresponding allergen-sensitized sample plus aluminum adjuvant. Group G1 received an equal volume of blank sample (physiological saline + aluminum adjuvant). The injections were administered once a week for a total of three times.

[0236] Table 14 Grouping and Dosage

[0237] 3.2 Stimulation Phase

[0238] One week after the last sensitization, groups G2-G6 were challenged with nasal drops of the corresponding allergen, 40 μL / animal / time, once a day for 7 consecutive days. The negative group was given physiological saline under the same conditions.

[0239] 4. Evaluation Indicators

[0240] 4.1 Penh:

[0241] Twenty-four hours after the last challenge, all mice had their Penh values ​​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.

[0242] 4.2 Serum antibodies:

[0243] Forty-eight hours after the last challenge, whole blood was collected by enucleation. After standing at room temperature for 2-3 hours, serum was obtained by centrifugation at 3500 rpm for 10 minutes. Serum antibodies were detected by ELISA.

[0244] 4.3 White blood cell (WBC) count in BALF;

[0245] Bronchoalveolar lavage fluid (BALF) was collected, and the three BALF samples were combined and centrifuged at 800 rpm for 10 min. The supernatant was discarded, and the precipitated cells were resuspended in 0.5 mL of pre-cooled sterile 1×PBS buffer containing 1% BSA and mixed well.

[0246] The remaining BALF suspension was diluted 10 times with 1×PBS buffer containing 1% BSA, centrifuged at 800 rpm for 5 min, and stained according to the "Wright-Giemsa Staining Solution Instructions". Three fields of view were randomly selected under a 200x microscope, and the average value was obtained by counting the stained cells using ImageJ software.

[0247] The antibody level was detected by ELISA using BALF supernatant.

[0248] 4.4 In vitro stimulation of spleen cells with supernatant cytokines

[0249] Except for the compound treatment group, spleen cells were isolated from mice in each group under sterile conditions and stimulated with the corresponding allergens. After co-culturing the spleen cells with the allergens for 144 h, the supernatant was collected. The levels of cytokines IL-4, IL-5, IL-13, and IFN-γ in the in vitro stimulation supernatant of spleen cells were detected by the CBA method.

[0250] 5. Experimental Results

[0251] 5.1 Penh:

[0252] Under stimulation with Mch at a concentration of 6.25 mg / mL, the Penh values ​​of mice in the Can f 2, Can f 4, Can f 5 and combination groups were higher than those in the negative group, with the Can f 5 group showing the highest value.

[0253] Under stimulation with Mch at a concentration of 12.5 mg / mL, the Penh values ​​of mice in all groups were higher than those in the negative control group, with the highest value observed in the combination therapy group.

[0254] Under stimulation with Mch at a concentration of 25 mg / ml, the Penh values ​​of mice in the Can f 1, Can f 4, Can f 5 and combination groups were higher than those in the negative group, with the highest value in the Can f 5 group.

[0255] Under stimulation with Mch at a concentration of 50 mg / mL, the Penh values ​​of mice in the Can f 1, Can f 5 and combination groups were higher than those in the negative group, with the combination group showing the highest value.

[0256] Based on the results of Mch stimulation at various concentrations (Table 15, Figure 14), the Penh values ​​of mice in each component were higher than those in the negative group under Mch stimulation, with the Can f 5 and combination groups showing relatively higher values ​​at their respective Mch concentrations.

[0257] Table 15. Penh values ​​in mice (Mean ± SEM) Note: * indicates P < 0.05 compared to the negative group, ** indicates P < 0.01 compared to the negative group.

[0258] 5.2 Serum resistance:

[0259] IgE: The levels of specific IgE in all groups of mice were higher than those in the negative group, with Can f2 showing the highest level.

[0260] IgG1: The levels of specific IgG1 in all groups of mice were higher than those in the negative control group, with Can f4 and Can f5 being the highest.

[0261] IgG2a: The levels of specific IgG2a in all groups of mice were higher than those in the negative control group, with Can f4 and Can f5 being the highest.

[0262] IgA: The level of specific IgGA in all groups of mice was higher than that in the negative group, with the highest levels observed in the Can f4, Can f5, and combination therapy groups.

[0263] The combined results of serum antibody levels (Table 16, Figure 15) show that each component can induce different forms of immune response in mice, thereby inducing the symptoms of an allergic airway inflammation model.

[0264] Table 16 Serum antibody values ​​(Mean ± SEM) Note: * indicates P < 0.05 compared to the negative group, ** indicates P < 0.01 compared to the negative group.

[0265] 5.3 BALF antibody:

[0266] The test results are shown in Table 17 and Figure 16. The levels of specific IgE, IgG1, IgG2a and IgGA in the BALF of mice in each group were higher than those in the negative group, with the highest levels in Can f4, Can f5 and the combination therapy group.

[0267] Table 17 Serum antibody values ​​(Mean ± SEM) Note: * indicates P < 0.05 compared to the negative group, ** indicates P < 0.01 compared to the negative group.

[0268] 5.4 WBC count in BALF:

[0269] The results are shown in Table 18 and Figure 17. Except for the Can f 2 group, the number of WBCs in the BALF of mice in all groups was higher than that in the negative group, with the highest values ​​in the Can f 5 and the combination therapy group. The Can f 1 group and the combination therapy group showed significant differences compared with the negative group.

[0270] Table 18 Number of WBCs in BALF (Mean ± SEM) Note: * indicates P < 0.05 compared to the negative group.

[0271] 5.5 In vitro stimulation of spleen cells with supernatant cytokines:

[0272] The test results are shown in Table 19 and Figure 18. The results of TH2 cytokines IL-4, IL-5 and IL-13 showed that, compared with the negative group, except for the Can f 1 group which had a lower IL-5 level, all other indicators in the other groups were increased to varying degrees.

[0273] Results of TH1-type cytokine IFN-γ showed that the levels in all groups were lower than those in the negative group.

[0274] The levels of TH1-type cytokines (such as IFN-γ) in each group were significantly lower than those in the negative group, while the levels of TH2-type cytokines (such as IL-4, IL-5, and IL-13) were generally upregulated. This indicates that the various allergen proteins can induce an immune imbalance of TH1 / TH2 in mice, causing the immune response to shift towards TH2 and thus inducing an allergic immune response.

[0275] Table 19. Cytokines in the supernatant of spleen cells stimulated in vitro (Mean ± SEM) Note: * indicates P < 0.05 compared to the negative group; ** indicates P < 0.01 compared to the negative group.

[0276] 6. Experimental Conclusions

[0277] The results of this study show that after mice were sensitized by injection and challenged by nasal drops with the Can f1 mutant, Can f2 mutant, Can f4 mutant, and Can f5 mutant of this invention, different mutants showed varying degrees of elevation or downregulation in different detection indicators, including symptoms, cellular levels, and immune levels. Overall, this indicates that each mutant can induce an immune response in mice, leading to T-cell inflammation and enhanced airway hyperresponsiveness, with significant effects on mice. The combination group containing the above four mutants showed significant differences compared to the negative group in several detection indicators, such as Penh value (stimulated at Mch concentrations of 12.5 mg / mL, 25 mg / mL, and 50 mg / mL), serum resistance (IgE, IgG2a, IgA, IgG1), BALF antibodies (IgE, IgA, IgG1), and WBC count in BALF. It also exhibited a sensitizing effect that induced an allergic immune response, and the overall sensitizing effect was more reliable than that of a single mutant component.

[0278] Example 19: Evaluation of the sensitization and desensitization effects of canine recombinant allergen protein mixture in vivo

[0279] 1. Experimental materials:

[0280] BALB / c mice, female, 8-9 weeks old (use age), were provided by Shanghai Slack Laboratory Animal Co., Ltd.

[0281] Dog dander extract (hereinafter referred to as DD), with a total protein content of 1 mg / mL, batch number: O2723A, was provided by Wokawi (Beijing) Biotechnology Co., Ltd.

[0282] Aluminum hydroxide adjuvant, batch number: 230017, provided by SERVA.

[0283] 2. Sample preparation:

[0284] 2.1 Sensitized Samples:

[0285] Recombinant Can f1, Can f2, Can f4, and Can f5 were prepared into a 0.4 mg / mL solution using normal saline (NS) (the recombinant allergen protein used was the sample prepared in the above examples, taking Can f1 mutant 1, Can f2 mutant 2, Can f4 mutant 1, and Can f1 mutant as examples, the same applies below). Equal volumes of the four solutions were thoroughly mixed to obtain a recombinant protein solution. Separately, a 0.4 mg / mL dilution of the DD solution was prepared using NS. Equal volumes of the above recombinant protein solution and DD solution were thoroughly mixed. Aluminum hydroxide adjuvant was added at 1 / 3 volume of the allergen solution, and the mixture was thoroughly mixed using a 3D mixer for 30 min. The solution was prepared fresh and used immediately, shaken well before use, and injected immediately after extraction to prevent precipitation.

[0286] 2.2 Excitation of the sample:

[0287] Recombinant Can f1, Can f2, Can f4, and Can f5 were prepared into a 0.25 mg / mL solution using neutralizing agents (NS). Equal volumes of these solutions were then thoroughly mixed to prepare a recombinant protein solution. Separately, a 0.25 mg / mL solution of diethyldi ...

[0288] 2.3 Recombinant protein compound:

[0289] High-dose recombinant protein group: Weigh 11.37 mg of recombinant protein mixed powder (0.7803 mg based on the content of the major allergen; recombinant Can f 1, recombinant Can f 2, recombinant Can f 4 and recombinant Can f 5 are mixed in a mass ratio of 1:1:1:1), add 650 μL of ultrapure water, and mix thoroughly with a pipette to prepare a recombinant protein solution with a concentration of 1.2 mg / mL (calculated based on the content of the major allergen), which is used for administration to the high-dose recombinant protein group.

[0290] Medium-dose recombinant protein group: Take 325 μL of the high-dose recombinant protein solution with a concentration of 1.2 mg / mL and add 325 μL of ultrapure water to prepare a recombinant protein solution with a concentration of 0.6 mg / mL (calculated according to the content of the main allergen), which is used for administration of the medium-dose recombinant protein group.

[0291] Low-dose recombinant protein group: Take 325 μL of the medium-dose recombinant protein solution with a concentration of 0.6 mg / mL, add 325 μL of ultrapure water to prepare a recombinant protein solution with a concentration of 0.3 mg / mL (calculated according to the content of the major allergen), which is used for administration of the low-dose recombinant protein group.

[0292] 3. Drug delivery system:

[0293] 3.1 Modeling Stage:

[0294] Seventy-two female BALB / c mice were randomly divided into two groups according to their body weight: a negative control group and a model group.

[0295] Sensitization: After grouping, the model group was intraperitoneally injected with 0.2 mL / dog / time of canine allergen compound sensitization sample solution, once a week for 3 consecutive weeks; the negative group was given NS solution in the same way.

[0296] Challenge: Five days after the last sensitization, mice in the model group were given 40 μL of dog allergen compound challenge sample solution via nasal drops once a day for 5 consecutive days; mice in the negative group were given NS in the same way.

[0297] For grouping and dosing information, please refer to Table 20.

[0298] Table 20. Grouping and Drug Administration Information for Mice During the Modeling Period Note: Animals were tested for serum-specific antibodies, and animals were selected for inclusion in the group based on the test results.

[0299] 3.2 Treatment Phase:

[0300] Sixty mice were used in the model group. Based on the serum levels of specific IgE and IgG1, 48 mice with high levels and good general condition were selected and randomly divided into four groups: model group, low-dose recombinant treatment group, medium-dose recombinant treatment group, and high-dose recombinant treatment group, with 12 mice in each group. The negative control group consisted of 12 mice, which were also the negative control group during the modeling stage. See Table 21 for details.

[0301] Table 21 Grouping and Dosing Information

[0302] Each group was administered the corresponding test solution according to the information in Table 21 (the blank powder did not contain allergens and mainly contained buffer salts). Sublingual administration procedure: Hold the mouse and use a pipette to accurately pipette 20 μL of the corresponding test solution from Table 21 and drip it evenly under the mouse's tongue. Continue to hold the mouse for 30 seconds to prevent immediate swallowing of the test solution. Duration and frequency of administration: 5 times / week, for 8 consecutive weeks. Within 3 days of the last sublingual administration, each group of mice was given 40 μL / mouse / time of canine allergen compound challenge sample solution via nasal drops, once daily for 5 consecutive days; the negative control group and model group were given the blank powder solution in the same manner.

[0303] 4. Evaluation indicators:

[0304] 4.1 Penh:

[0305] Twenty-four hours after the last challenge, all mice were tested for Penh values ​​using a non-invasive whole-body volume plethysmography (WBP) system at methacholine (Mch) concentrations of 0, 10, 20, 40, and 80 mg / mL.

[0306] 4.2 Serum IgE and IgG1 levels

[0307] Forty-eight hours after the last challenge, whole blood was collected by enucleation. After standing at room temperature for 2–3 hours, serum was obtained by centrifugation at 3500 rpm for 10 minutes. Serum IgE and IgG1 levels were detected by indirect ELISA.

[0308] 4.3 White blood cells in BALF:

[0309] Half of the animals in the group underwent bronchoalveolar lavage fluid (BALF) collection. The BALF samples from three separate collections were then combined and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the precipitated cells were resuspended in 0.5 mL of pre-chilled sterile 1×PBS buffer and mixed thoroughly. The EOS% in the BALF suspension was measured using a Siemens ADVIA 2120i hematology analyzer. The remaining BALF suspension was diluted 10-fold with 1×PBS buffer, centrifuged at 800 rpm for 10 min, and smeared according to the Wright-Giemsa staining instructions. Three fields of view were randomly selected under a 200x microscope, and the average number of stained cells was calculated using ImageJ software.

[0310] 4.4 In vitro stimulation of spleen cells with supernatant cytokines:

[0311] In each group, spleen cells were isolated from half of the animals under aseptic conditions and stimulated with the corresponding allergens. After co-culturing the spleen cells with the allergens for 168 hours, the supernatant was collected. The levels of cytokines IL-4, IL-5, and IL-13 in the in vitro stimulation supernatant of spleen cells were detected using the CBA method.

[0312] 5. Experimental Results:

[0313] 5.1 Penh:

[0314] The results are shown in Table 22 and Figure 19. After sublingual administration, the Penh values ​​of the model group mice were higher than those of the negative group at all Mch concentrations (P<0.05). The Penh values ​​of the low, medium, and high dose groups were lower than those of the model group at all Mch concentrations. Specifically, the Penh values ​​of the high-dose groups (Mch 10 mg / mL and 20 mg / mL) were significantly lower than those of the model group (P<0.05, P<0.01), and the Penh value of the medium-dose group (Mch 80 mg / mL) was significantly lower than that of the model group (P<0.01). These results indicate that sublingual administration of the canine recombinant allergen compound to the mouse allergic airway inflammation model can improve airway hyperresponsiveness symptoms.

[0315] Table 22. Penh values ​​in mice (Mean ± SEM) Note: * indicates P < 0.05, ** indicates P < 0.01, compared with the model group.

[0316] 5.2 Serum IgE and IgG1 levels

[0317] The test results are shown in Table 23 and Figure 20. After treatment with the recombinant protein compound, the serum IgE levels in mice were lower than those in the model group, with the high-dose group showing significantly lower serum IgE levels (p<0.05). The trend of serum IgG1 levels in mice was consistent with that of IgE, and all dose groups showed significantly lower levels than the model group (p<0.01). These results indicate that the systemic allergen immune response decreased after treatment with the recombinant protein compound.

[0318] Table 23 Antibody values ​​(Mean ± SEM) Note: * indicates P < 0.05, ** indicates P < 0.01, compared with the model group.

[0319] 5.3 EOS% and WBC count in BALF

[0320] The results are shown in Table 24 and Figure 21. After sublingual administration, the EOS% in the BALF of the model group mice was significantly higher than that in the negative control group (P<0.01), while the EOS% in all treatment groups of the recombinant protein compound was lower than that in the model group. Among them, the EOS% in the medium and high dose groups of the recombinant protein compound was significantly lower than that in the model group (P<0.01). The WBC count in the BALF of the model group mice was significantly higher than that in the negative control group (P<0.01), while the WBC count in all treatment groups of the recombinant protein compound was significantly lower than that in the model group (P<0.01). These results indicate that the recombinant protein compound treatment significantly improved lung inflammation in mice.

[0321] Table 24 Inflammatory cells in BALF (Mean ± SEM) Note: ** indicates p < 0.01 compared to the model group.

[0322] 5.4 In vitro stimulation of spleen cells with supernatant cytokines

[0323] The results are shown in Table 25 and Figure 22. After sublingual administration of the recombinant protein compound, IL-4 levels in all treatment groups were significantly lower than those in the model group (P<0.05, P<0.01). Except for the low-dose group, IL-5 levels in all treatment groups were lower than those in the model group, with significant differences between the medium- and high-dose groups (P<0.05). Except for the low-dose group, IL-13 levels in all treatment groups were lower than those in the model group, with significant differences between the medium- and high-dose groups (P<0.05, P<0.01). Low-dose recombinant protein may enhance the immune system's recognition and response to allergens, leading to elevated IL-5 and IL-13 levels. Overall, these results indicate that treatment with the recombinant protein compound downregulates Th2 cytokines in the model animals, which is closely related to the improvement of allergic reactions.

[0324] Table 25. TH2-type cytokines in the supernatant of spleen cells stimulated in vitro (Mean ± SEM) Note: * indicates p < 0.05 compared to the model group; ** indicates p < 0.01 compared to the model group.

[0325] 6. Experimental Conclusion:

[0326] The results of this study demonstrate that the recombinant protein compound has a clear sublingual immunotherapy effect in a mouse model of allergic airway inflammation induced by canine allergens. It effectively alleviates allergic airway inflammation by downregulating Th2 cytokine production and IgE, and inhibiting the infiltration and activation of inflammatory cells. In conclusion, the recombinant protein compound significantly improves the symptoms of allergic rhinitis and allergic asthma by regulating the immune system, reducing allergic reactions, and enhancing protective immunity, exhibiting good specific immunotherapeutic effects.

[0327] Example 20: In vivo safety evaluation of canine recombinant allergen protein mixture

[0328] This embodiment compares the effects of sublingual administration of canine recombinant allergen protein mixtures (Can f1, Can f2, Can f4, and Can f5) to dogs once daily for 28 consecutive days. The potential toxicity of these four canine recombinant allergen protein mixtures was assessed through a 28-day recovery period, evaluating the reversibility, persistence, or delayed effects of their toxicity. Simultaneously, the immunogenicity and immunotoxicity of the canine recombinant allergen proteins in vivo were also evaluated.

[0329] 1. Preparation of canine recombinant allergen protein compound tablets

[0330] The recombinant canine allergen protein compound sublingual tablets were prepared according to PCT / CN2023 / 108667 patent. Recombinant Can f 1, recombinant Can f 2, recombinant Can f 4, and recombinant Can f 5 (using samples prepared in the above examples, taking Can f 1 mutant 1, Can f 2 mutant 2, Can f 4 mutant 1, and Can f 5 mutant as examples) were freeze-dried separately using a vacuum freeze-drying oven to prepare lyophilized powders. The lyophilized powders of Can f 1, Can f 2, and Can f 5 contained citric acid, sodium citrate, mannitol, and capsule gelatin; the lyophilized powder of Can f 4 contained tromethamine, mannitol, and capsule gelatin. The blank lyophilized powder contained citric acid, sodium citrate, tromethamine, mannitol, and capsule gelatin. The obtained lyophilized powders were pulverized, passed through a 30-mesh sieve, and the protein content was determined. Following the additive method, lactose was used as the layer-by-layer additive. The following steps were performed: 20% lactose, low-substituted hydroxypropyl cellulose, 20% lactose, canine recombinant allergen protein Can f1, Can f2, Can f4, and Can f5 lyophilized powders (for low-dose sublingual tablets, blank lyophilized powder was mixed in), 20% lactose, microcrystalline cellulose, mannitol, and the remaining lactose were added and mixed separately. Finally, magnesium stearate was added and mixed. A rotary tableting machine was used to compress the tablets, controlling the core hardness to 10–70 N and the tablet weight to 75 mg, to prepare high-dose and low-dose canine allergen protein compound sublingual tablets. Blank tablets were prepared by replacing the allergen lyophilized powder with blank lyophilized powder. The formulation amounts for each tablet are shown in Tables 26-28.

[0331] Table 26 High-dose canine allergen protein mixture sublingual tablets

[0332] 2. Grouping and Experimental Design

[0333] A total of 15 female and 15 male beagle dogs were randomly divided into three groups and administered medications sublingually once daily for 28 consecutive days. The administered medications were a blank tablet, a low-dose canine recombinant allergen protein mixture sublingual tablet, and a high-dose canine recombinant allergen protein mixture sublingual tablet, respectively. Each administration consisted of 3 tablets per animal. At the start of administration, the animals were 6-7 months old and weighed between 7.51 and 10.28 kg (males) and 6.19 and 8.27 kg (females), respectively. Day 1 of the experiment was the first day of administration. After 28 days of administration, 3 animals / sex / groups were necropsized on day 29. After the administration period, 2 animals / sex / groups were assigned to a 28-day recovery period for observation, and necropsies were performed at the end of the recovery period on day 57. Evaluation criteria included survival rate (dying / death), clinical observation, body weight, food intake, ophthalmological examination, safety pharmacology parameters (electrocardiogram, blood pressure, respiratory and nervous system), body temperature, immunogenicity (ADA), lymphocyte subset analysis, cytokine analysis (IL-6, IL-8, IL-10, MCP-1, TNF-α, IFN-γ and TGF-β1), clinicopathology (hematology, serum biochemistry, coagulation and urine analysis), gross (necropsy) observation, organ weight and histopathology.

[0334] 3. Experimental Conclusions

[0335] All animals survived to planned euthanasia. No clinical observations, body weight, food intake, ophthalmological examinations, safety pharmacology (ECG, blood pressure, respiratory and nervous system), body temperature, immunogenicity (ADA), lymphocyte subset analysis (IPT), cytokines (CK), body temperature, clinicopathology (hematology, serum biochemistry, hemagglutination and urinalysis), gross (necropsy) observations, organ weight, or histopathological changes were observed in any dose group.

[0336] In summary, beagles were well-tolerated after sublingual administration of low-dose or high-dose canine recombinant allergen protein mixture once daily for 28 consecutive days, with a 28-day recovery period. No adverse events related to the test product were observed, indicating that the canine recombinant allergen protein and its mixture of the present invention have good safety in animals.

[0337] In addition to the safety evaluation in beagle dogs described above, the canine recombinant allergen protein mixture of the present invention also demonstrated good safety in rat toxicity tests. For example, in SD rats (6 to 7 weeks old), low-dose or high-dose canine recombinant allergen protein mixture sublingual tablets were administered once daily for 28 consecutive days, one tablet per animal, with a 28-day recovery period. The rats tolerated the treatment well, and no changes were observed in clinical symptoms, body weight, food intake, clinicopathological findings (hematology, serum biochemistry, hemagglutination, urine), ophthalmic examination, histopathology, organ weight, or gross observation related to the test product. The results of drug resistance antibody and bone marrow micronucleus test were negative. In young rats (4 weeks old), low-dose or high-dose canine recombinant allergen protein mixture sublingual tablets were administered once daily for 36 consecutive days, 1 tablet per animal, with a 28-day recovery period. The rats tolerated the test product well. No adverse changes were observed in animal deaths, clinical observations, administration site irritation, body weight, food intake, ophthalmological findings, vaginal opening / prepuce separation, hematology, hemagglutination, serum biochemical parameters and urinalysis, FOB, gross observation, organ weight and histopathology, femur length and femur mineral density. All test product samples were ADA negative.

Claims

A dog recombinant allergen vaccine comprising one or more of a Can f 1 mutant or a derivative thereof, a Can f 2 mutant or a derivative thereof, a Can f 4 mutant or a derivative thereof, a Can f 5 mutant or a derivative thereof. The dog recombinant allergen vaccine of claim 1, wherein the Can f 1 mutant or a derivative thereof, is mutated from a native Can f 1 or an allogenic allergen or a variant of the native Can f 1, the amino acid sequence of the native Can f 1 is shown as SEQ ID NO: 1, the mutation of the native Can f 1 is deletion or substitution of C at position 100, and / or N at position 62, and / or R at position 83, and / or K at position 113 of the native Can f 1 into other amino acids; the mutation of the allogenic allergen or the variant of the native Can f 1 is deletion or substitution of the amino acid corresponding to position 100, and / or position 62, and / or position 83, and / or position 113 of the native Can f 1 into other amino acids. The dog recombinant allergen vaccine of claim 2, wherein the mutation site of the Can f 1 mutant is replaced by any one or several 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. The dog recombinant allergen vaccine of claim 1, wherein the Can f 2 mutant or a derivative thereof, is mutated from a native Can f 2 or an allogenic allergen or a variant of the native Can f 2, the amino acid sequence of the native Can f 2 is shown as SEQ ID NO: 4, the mutation of the native Can f 2 is deletion or substitution of the amino acid at position 27, and / or position 88 of the native Can f 2 into other amino acids; the mutation of the allogenic allergen or the variant of the Can f 2 is deletion or substitution of the amino acid corresponding to position 27, and / or position 88 of the native Can f 2 into other amino acid. The dog recombinant allergen vaccine of claim 4, wherein the mutation site of the Can f 2 mutant is replaced by any one or several of Q or G or A or V or L or I or Y or S or K or R or H of F or W or M or T. The dog recombinant allergen vaccine of claim 5, wherein the Can f 4 mutant or a derivative thereof, is mutated from a native Can f 4 or an allogenic allergen or a variant of the native Can f 4, the amino acid sequence of the native Can f 4 is shown as SEQ ID NO: 7, the mutation of the native Can f 4 is deletion or substitution of the amino acid at position 85 of the native Can f 4 into other amino acids; the mutation of the allogenic allergen or the variant of the natural Can f 4 is deletion or substitution of the amino acid corresponding to position 85 of the native Can f 4 into other amino acid. The dog recombinant allergen vaccine of claim 6, wherein the mutation site of the Can f 4 mutant is replaced by any one or several of Q or 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. The dog recombinant allergen vaccine of claim 1, wherein the Can f 5 mutant or its derivative, which is mutated to the native Can f 5 or to the native Can f 5's alloallergen or variant, has an amino acid sequence as shown in SEQ ID NO: 10, The mutation of the native Can f 5 is to replace the amino acid at position 83, and / or position 47, and / or position 55 of the native Can f 5 with other amino acid; The mutation of the native Can f 5's alloallergen or variant is to replace the amino acid corresponding to position 83, and / or position 47, and / or position 55 of the native Canf 5 with other amino acid. The dog recombinant allergen vaccine of claim 8, wherein the mutation site of the Can f 5 mutant is replaced by any one or several of G or Q or A or V or L or I or Y or S or H or F or W or M or T. The nucleotide encoding the mutant or its derivative of any one of claims 2, 4, 6, 8. The mutant or its derivative of any one of claims 2, 4, 6, 7, which has one more methionine or formylmethionine at the N-terminus. The vector containing the nucleotide of claim 10, which is a T7 promoter-based expression vector for prokaryotic expression: pET32a, pET26b, pET28a, pDEST14; or a temperature- controlled promoter PL-PR-based expression vector for prokaryotic expression: pBV220; or a secretory expression vector for eukaryotic expression: pPIC9K, pPIC9, pGAPZαA, pPICZαA, pH1L-S1, pYAM75P; or an intracellular expression vector for eukaryotic expression: pH2L-D2, pAO815, pPIC3K, pHWO10, pPIC3.5K. The strain containing the vector of claim 12, which is E. coli with PL-PR promoter-based expression vector: BL21 (DE3), BL21 AI (DE3), Top10, DH5α, JM109, Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE3) plys; or E. coli with T7 promoter-based expression vector: BL21 (DE3), BL21 AI (DE3), Rosetta (DE3), Rosetta gamiB (DE3), BL21 (DE7) plys; or Pichia pastoris: GS115, X33, KM71H, SMD1168, Y11430, MG1003. A method for expressing a Can f 1 mutant or a derivative thereof, a Can f 2 mutant or a derivative thereof, a Can f 4 mutant or a derivative thereof, a Can f 5 mutant or a derivative thereof, The method is a prokaryotic expression system expression method, comprising the following steps: A. Constructing a vector containing the vector of claim 13; B. Transforming the recombinant plasmid of step A into an E. coli strain and culturing under suitable conditions; C. Recovering and purifying the protein; Or the method is a eukaryotic expression system expression method, comprising the following steps: A. Constructing a vector containing a vector of claim 13; B. Transforming the recombinant plasmid of step B into an E. coli strain by heat shock transformation method and culturing under suitable conditions; C. Performing plasmid large extraction on the recombinant strain of step B to obtain a recombinant plasmid; linearizing the recombinant plasmid using a suitable restriction enzyme and recovering the linearization product; D. Electrically transforming the linearization product of the recombinant plasmid of step C into a Pichia pastoris host, culturing under suitable conditions and obtaining a recombinant under pressure screening; E. Recovering and purifying the protein. A method for purifying a Can f 1 mutant or a derivative thereof, a Can f 2 mutant or a derivatives thereof, a Can f 4 mutant or a derivative thereof, a Can f 5 mutant, or a derivative thereof, The purification method is a purification method for prokaryotic system expression products, comprising the following steps: A. Resuspending and crushing the E. coli strain obtained by culturing according to claim 14, collecting the precipitate or supernatant, and the precipitate is the crude extract of the target protein inclusion body; B. If the inclusion body is collected, it is subjected to rough purification, denaturation and renaturation treatment to obtain a renaturation liquid; If the supernatant is collected, it is filtered and concentrated by ultrafiltration membrane to obtain a filtrate; C. Collecting the renaturation liquid or the filtrate, and performing three-step purification with Sepharose Q Fast Flow, Sepharose SP High Performance, and Sepharose Phenyl High Performance 6(HS) to obtain the target protein stock solution. Or the purification method is a purification method for eukaryotic system expression products, comprising the following steps: A. Collecting the target protein secreted product expressed by the Pichia pastoris strain in claim 14, and obtaining a crude extract by desalting; B. The crude extract of step A is subjected to three-step purification with Sepharose Q Fast Flow, Sepharose SP High Performance, and Use of the dog recombinant allergen vaccine of claim 1 in the preparation of a diagnostic reagent for detecting dog allergy or in the preparation of a drug for treating dog allergic diseases.

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