Engineering bacterium for efficiently expressing cat hair allergen component protein, construction method and application thereof

Abstract

The invention discloses an engineering bacterium for efficiently expressing cat hair allergen component protein as well as a construction method and application of the engineering bacterium. The nucleotide sequences of Fel d 2 and Fel d 4 after codon optimization are synthesized by a gene synthesis method, are recombined with a pPIC9K vector, and are transformed into pichia pastoris GS115 for fusion. Carrying out fermentation culture on the recombinant pichia pastoris for expressing the cat hair allergen protein to obtain a target allergen component protein; compared with the original gene, the expression yield of the gene sequence after codon optimization is remarkably increased, so that high-efficiency expression is realized, and the method can be used for industrial production of the cat hair allergen protein and meets the technical requirements of high-efficiency production of the cat hair allergen protein in China.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to an engineering bacterium for efficient expression of cat hair allergen component proteins, a construction method and an application thereof. Background Art

[0002] Allergic diseases are common non-infectious diseases, such as allergic asthma, allergic rhinitis, conjunctivitis and eczema, which seriously affect the health of the patient population. Allergic diseases are mainly related to genetic and environmental factors. When individuals with genetic predisposition are exposed to allergens in the environment, most allergens can directly activate innate immune cells such as alveolar macrophages and mast cells to release inflammatory factors and induce immune responses. The body produces specific IgE antibodies for different allergens. IgE antibodies bind to the surface of mast cells in the body, stimulating cells to produce and release inflammatory factors such as histamine and leukotrienes, thereby inducing corresponding allergic diseases.

[0003] Cats are one of the most important indoor allergens. Currently, more than 200 million people in the world are allergic to cats. Cat-sensitized patients suffer from severe respiratory symptoms such as severe chronic rhinitis and asthma. Cats are a complex allergen source donor. In addition to the main allergen Fel d 1, there are several different allergens: Fel d 2, Fel d 3, Fel d 4, Fel d 6, Fel d 7 and Fel d 8. Among them, Fel d 2, Fel d 4 and Fel d 7 are the main allergen components that cause allergic reactions in cats, corresponding to the highest level of specific IgE, and the allergen concentration required to induce basophil degranulation is a hundred times lower than other allergens. More than 65% of patients with respiratory allergies identify allergies to Fel d 1, Fel d 4 and Fel d 7, and 30% identify allergies to Fel d 2. Therefore, in addition to the major cat allergen Fel d 1, Fel d 2, Fel d 4 and Fel d 7 are also considered important allergens for cat allergy diagnosis and specific immunotherapy.

[0004] At present, the treatment methods for allergic diseases are mainly reflected in alleviating symptoms. For example, taking some antihistamines and steroid drugs can relieve symptoms. However, these drugs cannot inhibit the production of specific IgE antibodies secreted by immune cells caused by allergen inhalation, and the drugs also bring many side effects. In recent years, the role of specific immunotherapy (desensitization therapy) in allergic diseases has been confirmed and is considered to be the only causal treatment method for allergic diseases. This therapy is to give the patient low to high doses of allergens after determining the patient's allergens. Through repeated administration, tolerance to this allergen is induced, and when the patient is exposed to this allergen again, the allergic reaction will be reduced or not produced.

[0005] Allergen extracts used for treatment are crude mixtures of protein and non-protein components isolated from natural sources. Allergen extracts contain a large number of other components that are not related to allergens, and some crude allergen extracts do not even contain the allergen component that causes the patient's high sensitivity. Therefore, patients who are sensitive to a specific allergen component may receive treatment with a complex mixture containing all the extract components, which may lead to side effects. On the other hand, the use of the entire allergen extract for skin testing and serum testing in allergy diagnosis may affect the accurate detection of the specific allergen that causes the patient's sensitivity. In response to these shortcomings, many researchers have used biochemical separation and purification techniques to obtain single allergen components. However, although this method may be effective for the identification of allergens, it is not sufficient for the preparation of allergens on an industrial scale. At the same time, this method has many disadvantages such as low yield, time-consuming, labor-intensive, high cost and high pollution. For these reasons, recombinant DNA technology for synthesizing allergen component proteins has attracted people's attention. Summary of the invention

[0006] Based on this, the object of the present invention is to provide a vector, a recombinant engineered bacteria and a preparation method thereof for efficiently expressing cat hair allergen component proteins. The recombinant expression vector and the recombinant engineered bacteria of the present invention can achieve efficient expression of cat hair allergen component proteins in Pichia pastoris.

[0007] The first object of the present invention can be achieved by adopting the following technical solutions:

[0008] The first aspect of the present invention provides a cat hair allergen component protein, wherein the cat hair allergen component protein is FeI d 2 or FeI d 4; the amino acid sequence of FeI d 2 is shown in SEQ ID No: 3; the amino acid sequence of FeI d 4 is shown in SEQ ID No: 4; or a sequence identical to part of the amino acids of SEQ ID No: 3 and SEQ ID No: 4.

[0009] The second aspect of the present invention provides the coding gene of the cat hair allergen component protein, the nucleotide sequence of the coding gene of Fel d 2 is shown as SEQ ID No: 1, or a sequence complementary to the sequence of SEQ ID No: 1, or a nucleotide sequence that is partially identical or complementary to the sequence of SEQ ID No: 1; the nucleotide sequence of the coding gene of Fel d 4 is shown as SEQ ID No: 2, or a sequence complementary to the sequence of SEQ ID No: 2, or a nucleotide sequence that is partially identical or complementary to the sequence of SEQ ID No: 2.

[0010] The third aspect of the present invention provides a recombinant expression vector into which the coding gene of the above-mentioned cat hair allergen component protein is inserted.

[0011] As one embodiment, pPIC9K plasmid is used as a template plasmid.

[0012] The fourth aspect of the present invention provides a recombinant engineered bacterium that expresses the above-mentioned recombinant expression vector.

[0013] The fifth aspect of the present invention provides a method for preparing the above-mentioned recombinant engineered bacteria, comprising the following steps:

[0014] The recombinant vector was linearized and purified using Sal I endonuclease;

[0015] The recovered linearized recombinant vector is transformed into Pichia pastoris cells, and positive transformants are screened to obtain the recombinant engineered bacteria.

[0016] The sixth aspect of the present invention provides a method for efficiently expressing the component proteins of cat hair allergens, wherein the above-mentioned recombinant engineered bacteria are shaken in BMGY medium to obtain a seed solution, and then transferred to BMMY medium for continued cultivation; a carbon source is supplemented and the induction culture is continued; at the end of the cultivation, the culture solution is centrifuged and the supernatant is collected for purification of the target protein.

[0017] As one example, the recombinant engineered bacteria were cultured in BMGY medium until OD 600 12-15 Get the seed solution;

[0018] The seed solution was washed 1-4 times, OD 600 Dilute to 1 and transfer to BMMY medium for further culture;

[0019] The carbon source was supplemented every 24 h, and the carbon source was 1-2% methanol.

[0020] As one embodiment, FeI d 2 and FeI d 4 proteins are purified by affinity chromatography; preferably, the affinity chromatography is nickel column affinity chromatography.

[0021] The seventh aspect of the present invention provides an application of a cat hair allergen component protein as an anti-cat hair allergen pharmaceutical reagent, an application in preparing an anti-cat hair allergen drug, or an application as a cat hair allergen component detection reagent.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention synthesizes the codon-optimized Fel d 2 and Fel d 4 gene sequences by gene synthesis, recombines with the pPIC9K vector, and transforms into Pichia pastoris GS115 for fusion expression. Compared with the original gene, the expression yield of the codon-optimized gene sequence is significantly increased. The method of the present invention can be used for the industrial production of cat hair allergen component proteins, meeting the technical requirements of my country for the efficient production of cat hair allergen component proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The SDS-PAGE image shows the expression of two components, FeI d 2 and FeI d 4;

[0025] Lane 6 is a protein molecular weight standard; Lanes 2 and 4 are the fermentation supernatants of the original gene sequences of Fel d 2 and Fel d 4 after being expressed in yeast; Lanes 3 and 5 are the fermentation supernatants of the gene sequences of Fel d 2 and Fel d 4 after being expressed in yeast after codon optimization;

[0026] Figure 2 The SDS-PAGE image of FeI d 2 and FeI d 4 after being purified by nickel column;

[0027] Lanes 1 and 4 are the supernatant samples of the fermentation broth after 120 hours of fermentation of Fel d 2 and Fel d 4, respectively; Lanes 2 and 5 are the flow-throughs after the fermentation broth of Fel d 2 and Fel d 4 is bound to the nickel column, respectively; Lanes 3 and 6 are the target proteins of Fel d 2 and Fel d 4 eluted from the nickel column with 20 mM Tris-HCl buffer (containing 250 mM NaCl, 250 mM imidazole, pH 8.0), respectively;

[0028] Figure 3 The two allergen components, Fel d 2 and Fel d 4, were spotted on the silica matrix membrane, and the color development results after immunofluorescence detection;

[0029] The first row in the figure is the Fel d 2 allergen point, the second row is the Fel d 4 allergen point, position 1 corresponds to the immunofluorescence experiment results of commercially purchased Fel d 2 and Fel d 4 standards, and positions 2 and 3 are the experimental results of the recombinant Fel d2 and Fel d 4 proteins of this application. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] Recombinant allergens can be obtained on a large scale using microbial expression systems that can be grown in large fermenters, allowing the recombinant allergens to be produced in large quantities with consistent purity.

[0032] Conventional protein recombinant expression systems include prokaryotic expression and eukaryotic expression. Among them, the most common prokaryotic expression is Escherichia coli recombinant expression and Bacillus subtilis secretory expression, and the eukaryotic expression system includes yeast expression system and mammalian expression system. Among them, the Escherichia coli expression system in the prokaryotic expression system has extremely high gene expression efficiency. It has fast growth rate, simple culture conditions and low cost, so it is suitable for large-scale production of recombinant proteins. In addition, the engineering technology of Escherichia coli expression system is very mature, with simple gene operation, high conversion efficiency, and easy cloning and expression of target proteins. However, the Escherichia coli expression system lacks the post-translational modification mechanism of eukaryotic cells, especially the inability to perform correct glycosylation and disulfide bond formation, which may cause the expressed complex proteins to misfold and form inclusion bodies without biological activity. At the same time, Escherichia coli itself contains endotoxins (Lipopolysaccharides, LPS), which poses a safety hazard to biological products used by humans. Therefore, Escherichia coli expressing large allergen components is not suitable for skin prick experiments on patients, and additional removal steps are required, which increases the complexity of the experiment and production costs. For Bacillus subtilis, the chassis cells have a strong secretion capacity and can secrete recombinant proteins directly into the culture medium, simplifying the downstream protein purification process. Bacillus subtilis does not produce endotoxins, so the expressed proteins are safer and are particularly suitable for the production of medical-grade proteins and enzyme preparations. However, the proteins secreted by Bacillus subtilis are easily degraded by the proteases secreted by itself, which is a problem that cannot be ignored when producing highly active proteins. Compared with Escherichia coli, the expression level of Bacillus subtilis is relatively low and it is not suitable for expressing highly complex proteins. Although Bacillus subtilis is closer to the eukaryotic system than Escherichia coli, its post-translational modification is still limited and cannot perform complex glycosylation modifications. Compared with the prokaryotic expression system, the Pichia pastoris expression system has natural advantages, such as: (1) it can achieve high-density fermentation and significantly increase protein expression yield; (2) it can efficiently secrete recombinant proteins and simplify subsequent purification treatment; (3) it has a relatively complete post-translational modification ability, which is conducive to the correct folding of proteins, disulfide bond formation, and glycosylation modification mechanisms, and is suitable for expressing complex eukaryotic proteins, which is crucial for expressing functional eukaryotic proteins. In addition, compared with mammalian cell expression, the cost of the Pichia pastoris expression system is lower, and the culture conditions are relatively simple, which is suitable for large-scale industrial production. Based on the above factors, Pichia pastoris is the preferred expression system for expressing cat hair allergen components.

[0033] In some embodiments of the present invention, two cat hair allergen component proteins are disclosed, wherein the cat hair allergen component proteins are FeI d 2 or FeI d 4; the amino acid sequence of FeI d 2 is shown in SEQ ID No: 3; the amino acid sequence of FeI d 4 is shown in SEQ ID No: 4.

[0034] SEQ.ID No:3

[0035] *.

[0036] SEQ.ID NO:4

[0037] HEEENVVRSNIDISKISGEWYSILLASDVKEKIEENGSMRVFVEHIKALDNSSLSFVFHTKENGKCTEIFLVADKTKDGVYTVVYDGYNVFSIVETVYDEYILLHLLNFDKTRPFQLVEFYAREPDVSQKLKEKFVKYCQEHGIVNILDLTEVDRCLQARGSEVAQDSSVEHHHHHH*.

[0038] In some embodiments of the present invention, the amino acid sequence of the cat hair allergen component protein is a sequence identical to a portion of the amino acids in SEQ ID No: 3 or SEQ ID No: 4, and the sequence includes a 6His-tag at the C-terminus.

[0039] In some embodiments of the present invention, genes encoding cat hair allergen component proteins are disclosed, and the nucleotide sequence of the gene encoding Fel d 2 is shown in SEQ ID No: 1; the nucleotide sequence of the gene encoding Fel d 4 is shown in SEQ ID No: 2. The nucleotide sequences of the genes encoding Fel d 2 and Fel d 4 are recombinantly fused with a 6His-tag tag at their C-termini.

[0040] SEQ.ID No:1

[0041]

[0042] SEQ.ID No:2

[0043] .

[0044] In some embodiments of the present invention, the nucleotide sequence of the gene encoding Feld 2 is a sequence complementary to SEQ ID No: 1, or a nucleotide sequence that is partially identical to or complementary to SEQ ID No: 1.

[0045] In some embodiments of the present invention, the nucleotide sequence of the gene encoding FeI d 4 is a sequence complementary to the sequence of SEQ ID No: 2, or a nucleotide sequence that is partially identical to or complementary to the sequence of SEQ ID No: 2.

[0046] In some embodiments of the present invention, a recombinant expression vector having inserted therein the gene encoding the above-mentioned cat hair allergen component protein is disclosed.

[0047] In this embodiment, the gene encoding the cat hair allergen component protein is the nucleotide sequence of the gene encoding Fel d 2, as shown in SEQ ID No: 1, or the nucleotide sequence of the gene encoding Fel d 4, as shown in SEQ ID No: 2.

[0048] As one embodiment, pPIC9K plasmid is used as a template plasmid.

[0049] In some embodiments of the present invention, a recombinant engineered bacterium expressing the above-mentioned recombinant expression vector is disclosed.

[0050] In some embodiments of the present invention, a method for preparing the above-mentioned recombinant engineered bacteria is disclosed, comprising the following steps:

[0051] The recombinant vector was linearized and purified using Sal I endonuclease;

[0052] The recovered linearized recombinant vector is transformed into Pichia pastoris cells, and positive transformants are screened to obtain the recombinant engineered bacteria.

[0053] In some embodiments of the present invention, a method for efficiently expressing cat hair allergen component proteins is disclosed, wherein the above-mentioned recombinant engineered bacteria are shaken in BMGY medium to obtain seed liquid, and then transferred to BMMY medium for further cultivation; carbon source is supplemented and induction culture is continued; after the cultivation is completed, the culture liquid is centrifuged and the supernatant is collected for purification.

[0054] As one example, the recombinant engineered bacteria were cultured in BMGY medium until OD 600 12-15 Get the seed solution;

[0055] The seed solution was washed 1-4 times, OD 600 Dilute to 1 and transfer to BMMY medium for further culture;

[0056] The carbon source was supplemented every 24 h, and the carbon source was 1-2% methanol.

[0057] As one embodiment, FeI d 2 and FeI d 4 proteins are purified by affinity chromatography; preferably, the affinity chromatography is nickel column affinity chromatography.

[0058] In some embodiments of the present invention, an anti-cat hair allergen pharmaceutical agent is disclosed, comprising at least the above-mentioned cat hair allergen component protein Fel d 2.

[0059] The following is a further description with reference to specific embodiments.

[0060] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be purchased from conventional commercial channels or obtained by existing known methods.

[0061] The molecular biology experimental techniques used in the following examples include plasmid extraction, recombinant vector digestion, gel electrophoresis, SDS-PAGE, immunofluorescence detection, etc., which are specifically referred to in the Molecular Cloning Experiment Guide (3rd Edition).

[0062] Example 1 Construction of a high-efficiency secretory expression system of Pichia pastoris and detection of protein secretory expression levels

[0063] (1) pPIC9K-Fel d 2-1 and pPIC9K-Fel d 2-2 as well as pPIC9K-Fel d 4-1 and pPIC9K-Feld 4-2 vectors were constructed, where -1 represents the original gene base sequence and -2 represents the base sequence after codon optimization; the above vectors were synthesized and delivered by Guangzhou Aiji Biotechnology Co., Ltd., and the Sal I restriction site was avoided in the synthesized target gene.

[0064] (2) Use Sal I endonuclease to linearize the recombinant vector and purify and recover it. The specific experimental method is as follows: take 10 μg of recombinant plasmid, 5 μl of Sal I endonuclease, 10×Sal I reaction buffer, a total volume of 50 μl, and react in a 37°C water bath for 3-4 hours. The reaction product is purified and recovered using Tiangen's PCR product recovery kit;

[0065] (3) The recovered linearized recombinant vector was used in a yeast transformation kit purchased from Coollab Biotech Co., Ltd., and the Pichia pastoris was efficiently transformed according to the instructions. The transformed yeast cells were plated on MD plates for culture, and after 3-4 days, the monoclonal colonies with good growth were transferred to YPD plates containing 6 mg / mL G418 to screen for high-copy strains; PCR was used to verify the positive transformants;

[0066] (4) The positive monoclonal strain was cultured in BMGY medium at 30°C and 220 rpm. After 24 h, the OD 600 When the cell count reaches 12-15, wash the cells and switch to methanol induction medium BMMY for further culture. Methanol with a final concentration of 1% is added every 24 hours. After 96-120 hours of induction culture, the expression of the target protein is detected.

[0067] (5) Take 30 μL of the supernatant of the Pichia yeast fermentation broth after 96-120 h of induction culture and perform SDS-PAGE electrophoresis detection. The results are as follows: Figure 1 shown. Figure 1Lanes 2 and 4 in the middle are the results of expression of the original gene sequences of Fel d 2 and Fel d 4 in Pichia pastoris, respectively; lanes 3 and 5 are the results of expression of the codon-optimized Fel d 2 and Fel d 4 gene sequences in Pichia pastoris, respectively.

[0068] Depend on Figure 1 It can be seen that the protein yields of Fel d 2 and Fel d 4 after codon optimization increased to varying degrees, among which the yield of Fel d 2 increased most significantly, by about 4-5 times, and that of Fel d 4 increased by about 3-4 times. This indicates that the selected codon-optimized Fel d 2 and Fel d 4 gene sequences have a significant high expression effect.

[0069] (6) The codon-optimized Pichia fermentation broth was centrifuged to separate the bacterial cells and the fermentation broth supernatant, and the fermentation broth supernatant was fully mixed and combined with the nickel column filler. After loading, the flow-through was removed, and non-specific binding proteins were washed away with 20mM Tris-HCl, 250-500mM NaCl, 10-20mM imidazole, pH 8.0, and then replaced with 20mM Tris-HCl, 250-500mM NaCl, 200-250mM imidazole, pH 8.0 for elution. The samples generated in each step of the purification process were subjected to SDS-PAGE detection. The results are shown in FIG. Figure 2 As shown, lanes 1 and 4 are the supernatant samples of the fermentation broth after 120 hours of fermentation of Fel d 2 and Fel d 4, respectively; lanes 2 and 5 are the flow-through after the fermentation broth of Fel d 2 and Fel d 4 is bound to the nickel column, respectively; lanes 3 and 6 are the target proteins eluted from the nickel column by 20mM Tris-HCl buffer (containing 250mM NaCl, 250mM imidazole, pH 8.0) of Fel d 2 and Fel d 4 proteins, respectively.

[0070] like Figure 2 As shown in the figure, the target bands were clearly contained in the fermentation broth supernatant. After binding with the nickel column filler, the target protein in the flow-through was significantly reduced, indicating that the target protein was effectively bound by the nickel column. At the same time, after elution with high-concentration imidazole buffer, the target proteins in lanes 3 and 6 were fully eluted, and the protein purity reached more than 95%.

[0071] Example 2 Application of immunofluorescence method to detect recombinant cat hair allergen components

[0072] (1) Construction of immunofluorescence detection chip: The purified cat hair allergen components Fel d 2 and Fel d 4 proteins were spotted on the protein-specific adsorption silica matrix membrane (protein chip membrane) developed and provided by Shanghai Saga Biotechnology Co., Ltd. The membrane has a strong protein adsorption and binding capacity. 2 mg / ml of allergen components and commercially purchased standards were spotted on the silica matrix membrane using a protein micro-spotter. Each sample was spotted with 50 nl, for a total of 3 groups, one group of standard products, and one group of recombinantly expressed Fel d 2 and Fel d 4. The spotted protein chip matrix membrane was sealed and dried to ensure that the spotted allergen proteins were fully and firmly bound to the silica matrix membrane.

[0073] (2) The matrix membrane after spotting was fully sealed with milk powder, and the protein chip membrane was incubated with serum from patients with cat hair allergy level 3-4, incubated at 37°C with shaking for 20 minutes, washed 3 times with PBST, incubated with biotinylated IgE secondary antibody, incubated at 37°C with shaking for 20 minutes, washed 3 times with PBST, and then combined with streptavidin-conjugated fluorescent substance for 5 minutes, washed 3 times with PBST, and finally detected by fluorescence detection analyzer. The results are shown in Figure 3 shown. Figure 3 The first row is the Feld 2 allergen spot, the second row is the Feld 4 allergen spot, position 1 corresponds to the immunofluorescence experiment results of commercially purchased Feld 2 and Feld 4 standards, and positions 2 and 3 are the experimental results of the recombinant Feld 2 and Feld 4 proteins of the present application.

[0074] from Figure 3 It can be seen that the immunofluorescence effect is significant, and the two recombinant cat hair allergen components Feld 2 and Feld 4 can better detect specific IgE in the serum of allergic patients.

[0075] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A cat hair allergen component protein, characterized in that: The cat hair allergen component protein is FeI d 2 or FeI d 4; the amino acid sequence of FeI d 2 is shown in SEQ ID No: 3; the amino acid sequence of FeI d 4 is shown in SEQ ID No: 4, or a sequence identical to part of the amino acids in SEQ ID No: 3 and SEQ ID No:

4.

2. The gene encoding the cat hair allergen component protein according to claim 1, characterized in that: The nucleotide sequence of the gene encoding Fel d 2 is shown as SEQ ID No: 1, or a sequence complementary to SEQ ID No: 1, or a nucleotide sequence partially identical to or complementary to SEQ ID No: 1; the nucleotide sequence of the gene encoding Fel d 4 is shown as SEQ ID No: 2, or a sequence complementary to SEQ ID No: 2, or a nucleotide sequence partially identical to or complementary to SEQ ID No:

2.

3. A recombinant expression vector into which is inserted the gene encoding the cat hair allergen component protein according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The pPIC9K plasmid was used as the template plasmid.

5. A recombinant engineered bacterium expressing the recombinant expression vector according to claim 3 or 4.

6. The recombinant engineered bacterium according to claim 5, characterized in that The chassis cells of the recombinant engineered bacteria are Pichia pastoris; preferably, the chassis cells of the recombinant engineered bacteria are Pichia pastoris GS115 strain.

7. The method for preparing the recombinant engineered bacteria according to claim 5 or 6, characterized in that: The following steps are involved: The recombinant vector was linearized using Sal I endonuclease and purified and recovered; The recovered linearized recombinant vector is transformed into Pichia pastoris cells, and positive transformants are screened to obtain the recombinant engineered bacteria.

8. A method for efficiently expressing cat hair allergen component proteins, characterized in that: The recombinant engineered bacteria according to claim 5 or 6 are shaken and cultured in BMGY medium to obtain a seed solution, OD 600 When the value reaches the expected value, the cells are transferred to BMMY medium for continued cultivation; carbon source is supplemented and induction culture is continued; at the end of the cultivation, the culture medium is centrifuged and the supernatant is collected for protein purification.

9. The method for efficiently expressing cat hair allergen component proteins according to claim 8, characterized in that: The cells were cultured in BMGY medium until OD 600 When the value reaches 12-15, you will get the seed solution; The seed solution was washed 1-4 times and diluted to OD 600 =1, transfer to BMMY medium and continue culture; Supplementing a carbon source every 24 hours, wherein the carbon source is 1-2% methanol; The FeI d 2 and FeI d 4 proteins are purified by affinity chromatography; preferably, the affinity chromatography is nickel column affinity chromatography.

10. An application of cat hair allergen component protein, characterized in that: The invention can be used for preparing anti-cat hair allergen drugs or as a reagent for detecting cat hair allergen components.

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