CN111233995A

CN111233995A - New allergen NPC2 from cat

Info

Publication number: CN111233995A
Application number: CN202010098315.XA
Authority: CN
Inventors: 魏继福; 朱丹萱; 许志强; 张�成
Original assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Priority date: 2020-02-18
Filing date: 2020-02-18
Publication date: 2020-06-05
Anticipated expiration: 2040-02-18
Also published as: CN111233995B

Abstract

The invention discloses identification of a novel allergen protein NPC2 from cats (Felis domesticus). Mainly by cloning the allergen NPC2 gene and optimizing its codons. The optimized NPC2 gene is used for constructing a pET28a expression vector and converting an escherichia coli expression strain BL21(DE3), IPTG induces intracellular expression of His fusion protein, and the His fusion protein is purified by Ni-NTA affinity chromatography and then renatured to obtain the high-purity recombinant NPC2 protein. ELISA and Western Blot results showed a 25.5% sensitization rate of the cat NPC2 protein and cross-reactivity with the dog allergen Can f 7.

Description

New allergen NPC2 from cat

Technical Field

The invention belongs to the fields of molecular biology, immunology and bioinformatics, particularly relates to the field of allergic diseases, and particularly relates to identification of a novel allergen NPC2 from cats.

Background

Allergic diseases, also known as allergic diseases, are a common chronic inflammatory disease. With the development of the industrialized society and the change of the life style and the ecological environment of people, the incidence of the disease accounts for about 25 percent of the world population at present, is increased year by year, has seriously influenced the life quality of people, and is listed as one of three diseases which are mainly prevented and controlled by the World Health Organization (WHO) in the 21 st century. Allergic diseases mainly comprise pollinosis, allergic rhinitis, allergic bronchial asthma, allergic dermatitis, urticaria, angioneurotic edema and other allergic reactions, and the diseases are mainly caused by allergens in the air, such as pollen, house dust mites, animal dander, cockroaches, fungi and the like. In addition, some common foods such as peanuts, soybeans, milk, eggs, fish and shrimp, etc. also contain allergen components.

The conventional methods for detecting allergens include allergen skin prick test, serological detection and allergen elicitation test, but the treatment of allergic diseases mainly comprises allergen contact avoidance, drug treatment and specific immunotherapy (desensitization treatment), wherein the desensitization treatment is considered by WHO as the only treatment method for radically treating allergic diseases, and mainly induces immune tolerance by giving an immunogen preparation to a patient, the diagnosis and treatment of allergens mainly depend on crude extracts of allergens, but commercial crude extracts have the problems that the types of allergen components in ① crude extracts cannot be determined, the allergen components contain allergen components and a large amount of non-specific antigens, the allergen components in ② crude extracts are different in content to cause different effects, the sources and extraction methods of ③ crude extracts are different so that different batches have different differences, therefore, false positive or negative results are easy to occur in the detection process, local, systemic and fatal reactions are generated in the immunotherapy process, based on the above problems, the individual allergen components need to be separated and the main allergen components are clearly identified, the allergen and secondary allergen molecules are continuously cloned with the development of the allergen gene engineering, and the development of the allergen molecules is a single-based on the development and the clinical diagnosis and treatment of allergen.

Cats, a common household pet, are one of the major sources of inhaled allergens in the house. Cat allergens can induce allergic reactions of varying severity, including allergic rhinitis, bronchial asthma, conjunctivitis, and the like. To date, 8 allergens have been named by WHO-IUIS in cats, Fel d 1, a 38kDa secretoglobin (Ulteroglobin); fel d 2, a 69kDa Serum albumin (Serum albumin); fel d 3, an 11kDa cysteine protease inhibitor (Cystatin); fel d4, a 22kDa lipid carrier protein (Lipocalin); fel d 5, a 400kDa immunoglobulin A (immunoglobulin A); fel d6, an 800-1000kDa immunoglobulin M (immunoglobulin M); fel d 7, a 17.5kDa lipocalin (von Edner glandprotein); fel d 8, a 24kDa Latherin-like protein (Latherin-like protein). These allergens are mainly distributed in the cat's dander, saliva, hair, serum and urine. Among them Fel d 1 is recognized by serum-specific IgE in 90% of cat allergic patients, and is currently considered to be the most major allergen in cats. However, the Fel domestica d 1-deficient cat fur extract still has 40% of allergenic activity, and therefore, other cat allergens and potential allergens still play a significant role in allergic patients. For cat-induced allergic diseases, the most effective way to avoid cat contact is at present, but because cat allergens have strong adhesion capability, are easy to adhere to clothes and furniture surfaces or adsorb on the surface of small particles (with the diameter less than 2 microns) and are suspended in the air for a long time, so that the cat allergen can be carried or spread to public environments without cats, and therefore, cat allergen contact prevention is difficult to realize in real life. In addition, the cat and dog allergen characteristics and biological functions are similar, and thus cross-allergic activity is present. Therefore, analysis of the IgE response of a single allergen component is advantageous for improving the accuracy and sensitivity of specific IgE diagnosis, and can also be used to distinguish between cross-reactive allergen sensitization and natural allergen sensitization. Desensitization therapy is currently considered the only therapy that can alter the course of allergic disease. However, the traditional desensitization treatment relying on crude extract has long period, is easy to generate adverse reaction and has poor patient compliance. With the intensive research of scientific technology and cat allergen components, the novel allergen vaccine with clear components and definite action mechanism is expected to become an alternative scheme for the treatment of cat crude extract, thereby efficiently preventing and treating cat allergic diseases.

Disclosure of Invention

The invention aims to identify an allergen Felis catus NPC intracellularcolesterol transporter 2(Cat-NPC2) from a Cat, provide a gene sequence of natural protein of Cat-NPC2, provide a gene optimized Cat-NPC2 sequence, provide a fusion expression recombinant Cat-NPC2 by taking His as a tag and a preparation method for purifying the recombinant Cat-NPC2, and prove the allergen activity of NPC 2.

In order to achieve the purpose, the invention adopts the following technical scheme: the amino acid sequence of the protein is SEQ ID No.1, or the protein has at least 90% homology with the amino acid sequence, wherein the amino acids from 1 st to 19 th are signal peptides.

The codon-optimized cat allergen NPC2 protein is a protein consisting of a nucleotide sequence shown in SEQ ID No. 2.

The recombinant cat allergen NPC2 protein is a protein consisting of a nucleotide sequence shown in SEQ ID No. 3.

The preparation method of the recombinant cat allergen NPC2 protein comprises the following steps:

extracting total RNA in Cat fur by using a Trizol method, performing reverse transcription to synthesize cDNA, designing a specific primer according to a sequence predicted by an NCBI Genbank gene bank, and synthesizing a Cat-NPC2 natural gene sequence SEQ ID No.2 by PCR;

carrying out codon optimization on a natural gene sequence of the allergen Cat-NPC2 to obtain an optimized Cat-NPC2 sequence SEQ ID No. 3;

cloning optimized genes of Cat-NPC2 to an expression vector of Escherichia coli pET28a, and transforming an Escherichia coli expression strain BL21 with the obtained recombinant plasmid pET28a-NPC 2;

inducing intracellular expression by IPTG, collecting thalli, performing ultrasonic disruption, purifying by Ni-NTA affinity chromatography, and performing dialysis renaturation to obtain high-purity recombinant Cat-NPC2 protein.

Use of the cat allergen NPC2 protein for the preparation of a pharmaceutical composition for the treatment or prevention of cat allergic diseases.

A pharmaceutical composition comprising the cat allergen NPC2 protein.

Further, the pharmaceutical composition is an allergen vaccine.

A diagnostic kit comprising the cat allergen NPC2 protein of SEQ ID No.1 or the composition of claim 6 for use in the in vitro diagnosis of cat allergic diseases.

The invention identifies and analyzes the NPC2 protein of the cat as the cat allergen by utilizing the technologies of molecular biology, cellular immunology, bioinformatics and the like. Codon optimization is carried out on the natural gene Cat-NPC2, recombinant escherichia coli capable of stably expressing allergen Cat-NPC2 is constructed, and a method for preparing recombinant Cat-NPC2 is provided. Meanwhile, immunological experiments prove that the Cat-NPC2 and the dog allergen Can f 7 have cross activity. The identification of new allergens is beneficial to improving the accuracy and sensitivity of diagnosis of allergic diseases on one hand, and can be used for desensitization treatment and prevention of allergic diseases by preparing allergen vaccines on the other hand. Therefore, the invention has important value for clinical diagnosis and treatment of cat allergy patients.

Drawings

FIG. 1 is a PCR electrophoretogram of Cat-NPC2 protein, and DNA2000 marker is in the leftmost M lane; lane 1 shows the PCR amplification product of the Cat-NPC2 native protein.

FIG. 2 shows the cDNA sequence before and after optimization of Cat-NPC2, and the amino acid sequence encoded by the cDNA sequence, wherein the amino acids from 1 st to 19 th are signal peptides.

FIG. 3A is an SDS-PAGE electrophoresis of induced expression of rCat-NPC2, the left-most lane M being the protein molecule marker; lane 1 is the E.coli liquid without IPTG induction; lane 2 is IPTG induced and recombined E.coli liquid; lane 3 is the supernatant of the disrupted biomass; lane 4 shows the pellet from the cell disruption solution.

FIG. 3B is an SDS-PAGE electrophoresis of rCat-NPC2 purified by Ni-NTA affinity chromatography, the leftmost M lane being the protein molecule Maker; lanes 1-4 are rCat-NPC2 eluted at 500mM imidazole.

FIG. 4A shows that the binding activity of rCat-NPC2 to specific IgE in the serum of cat allergic patients was measured by ELISA, and in the serum of 110 cat allergic patients, 28 of 110 cat allergic patients showed positive reaction of IgE to rCat-NPC 2.

FIG. 4B shows the Western Blot method to verify the binding activity of rCat-NPC2 to specific IgE in the serum of cat allergic patients.

FIG. 4C is a graph of the inhibitory efficiency of rCat-NPC2 on the binding of crude cat extract to serum IgE of cat allergic patients measured by a competitive ELISA method

FIG. 5 is a graph of inhibition of serum IgE binding to rCat-NPC2 by rCan f 7 in cat allergic patients, and rCat-NPC2 positive reaction sera were preincubated with 10 μ g/ml rCan f 7 (right) or PBS (left) prior to determining IgE binding to rCat-NPC 2.

FIG. 6 is the results of secondary structure analysis by circular dichroism chromatography of rCat-NPC2 and rCan f 7 after purification renaturation.

FIG. 7 is a diagram of the secondary structure of Cat-NPC2 protein analyzed by prediction software.

FIG. 8 is an alignment of the amino acid sequences of Cat-NPC2 and Can f 7, with consensus sequences on black and conserved sequences in yellow on white.

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of this application and the above-described drawings are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those expressly listed.

The invention provides a cat allergen NPC2 protein, the amino acid sequence is SEQ ID No.1, or at least 90% homology with the amino acid sequence, wherein, the amino acids at the 1 st to 19 th positions are signal peptides.

And provides the application of the cat allergen NPC2 protein in preparing a pharmaceutical composition for treating or preventing cat allergic diseases.

Pharmaceutical compositions comprising the allergen NPC2 protein are provided, including allergen vaccines, including DNA vaccines, recombinant live bacterial vaccines and recombinant protein vaccines.

A diagnostic kit comprising the allergen NPC2 protein is provided.

Total RNA in cat fur is extracted by a Trizol method and is subjected to reverse transcription to synthesize cDNA, a specific primer is designed according to a Sequence predicted by an NCBI Genbank gene bank (NCBI Reference Sequence: XM _003987833.5), an upstream primer is shown as SEQ ID No.4, and a downstream primer is shown as SEQ ID No. 5. PCR amplification was carried out in a 25uL system, the PCR amplification product was subjected to agarose gel electrophoresis, and then, after gel cutting, recovery and purification, the PCR amplification product was ligated to pCE2 TA/Blunt-zero plasmid vector and transformed into E. A positive colony containing a recombinant expression plasmid is selected from the clone bacteria of the Coli JM109 through anti-aminobenzyl screening, and the natural protein sequence SEQ ID No.2(Genbank accession number MN737596) of the Cat-NPC2 is obtained through colony PCR sequencing verification. The cloned Cat-NPC2 gene consists of 594 bases, wherein the open reading frame is 450 bases (containing a terminator), 150 amino acids are coded, and the 1 st to 19 th amino acids are signal peptides and have the theoretical molecular weight of 14.4 kDa.

Analyzing parameters such as codon preference and GC content of escherichia coli, and carrying out codon optimization on a natural gene sequence SEQ ID No.2 of the allergen Cat-NPC2 to obtain a gene capable of being efficiently expressed in the escherichia coli, wherein the gene sequence is SEQ ID No. 3.

The optimized Cat-NPC2 gene (shown as SEQ ID No. 3) is cloned between Sac I and Xho I sites of an expression vector (fused His tag) of Escherichia coli pET28a to obtain a recombinant plasmid pET28a-NPC 2.

The recombinant plasmid pET28a-npc2 is transformed into an escherichia coli expression strain BL21(DE3), a monoclonal colony is selected and inoculated into LB liquid culture medium containing 50ug/ml kanamycin for overnight culture, the next day is inoculated into a fresh culture solution according to the inoculation amount of 1 percent, the bacterial is shaken at 37 ℃ until the OD600 is 0.5, isopropyl thiogalactoside (IPTG) with the final concentration of 1mM is added, the induction expression is carried out for 8 hours at 37 ℃ at 160 rpm, the bacterial is collected at 4 ℃, then the bacterial cell lysis equilibrium liquid LE buffer (50mM NaH2PO4, 300mM NaCl, pH 8.0) is used for heavy suspension, and after ultrasonic disruption, the supernatant and the precipitate are centrifugally collected. The theoretical molecular weight of the His fusion protein Cat-NPC2 is about 18.7 kDa. SDS-PAGE electrophoresis verifies that the molecular weight of the His fusion protein Cat-NPC2 is consistent with the theoretical value and is expressed in inclusion bodies.

The inclusion bodies were solubilized with a Urea-containing LE buffer and the supernatant was collected by centrifugation and applied to a Ni-NTA sepharose column equilibrated with Urea-containing buffer, washed for 2 column volumes with washing buffer (100mM NaH2PO4, 10mM Tris, 8Murea, 10mM Imidazole, pH 8.0) to remove non-specifically bound contaminating proteins, and finally competitively eluted with elution buffer (100mM NaH2PO4, 10mM Tris, 500mM Imidazole, 8M Urea, pH 8.0) to remove the fusion protein His-Cat-NPC 2. The biologically active recombinant Cat-NPC2 protein (rCat-NPC2) was renatured using a urea gradient and a redox renaturation solution (0.1M Tris, 0.5M L-argine, 1mM Moxidized glutamthione, 1mM EDTA, 5mM reduced glutamthione, pH 8.5). The purity of SDS-PAGE analysis is about 90-95%.

Serum IgE reaction of rCat-NPC2 with cat allergic patients was determined using ELISA method. Polystyrene plates were coated with 10. mu.g/mL rCat-NPC2 at 4 ℃ overnight at 100. mu.L per well. After the next day of washing, BSA blocking was performed for 2 hours. A10-fold dilution of the feline allergen patient serum was added and incubated at 37 ℃ for 2 hours. After washing, 100. mu.L of anti-human IgE peroxidase was added and incubated at 37 ℃ for 1 hour, after washing, the solution was added with a developing solution for 30 minutes and then stopped, and then the detection was carried out by an enzyme-linked immunosorbent assay (OD 450). Serum from 110 cat allergic patients, 28 showed a positive IgE response to rCat-NPC 2.

Western blot further demonstrated the binding activity of rCat-NPC2 to specific IgE in the serum of cat allergic patients. rCat-NPC2 protein was separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with milk for 2 hours, and incubated overnight at 4 ℃ with 10-fold diluted serum from cat allergic patients. After the next day of washing, anti-human IgE peroxidase was added and incubated at room temperature for 1 hour, and after washing, specificity detection was performed.

Competitive ELISA method to determine the inhibition efficiency of rCat-NPC2 protein on the binding of crude cat extract to serum IgE of cat allergic patients. Polystyrene plates were coated with 10. mu.g/mL cat crude extract, 100. mu.L per well, overnight at 4 ℃. The next day BSA blocking was 2 hours. The cat crude extract and rCat-NPC2 were diluted in several dilutions and preincubated with 10-fold diluted serum (No. 80 & No. 92) at 37 ℃ for 1 hour. The serum was then transferred to a well plate and incubated at 37 ℃ for an additional 1 hour. After washing, 100. mu.L of anti-human IgE peroxidase was added and incubated at 37 ℃ for 1 hour, after washing, the solution was added with a developing solution for 30 minutes and then stopped, and then the detection was carried out by an enzyme-linked immunosorbent assay (OD 450). The maximum inhibition rate of rCat-NPC2 protein on the combination of the crude extract of the cat and serum IgE of a cat allergic patient is 14-17% as shown by a formula calculation result.

Purified rCat-NPC2 protein was adsorbed to PVDF solid phase support using Western blot and binding of IgE was determined using serum from cat allergic patients incubated with rCanf 7 (10. mu.g/mL) or PBS overnight at 4 ℃. The results showed that rCan f 7 in 9/10 sera almost completely inhibited IgE binding to rCat-NPC2 protein, indicating that there was cross-sensitizing activity in rCat-NPC2 and rCan f 7.

The secondary structure of the recombinant Cat-NPC2 Protein is analyzed by PSIPRED, Predict Protein and NetSurfP v1.1 online software, and the comprehensive result shows that 9 β folds exist in Cat-NPC2, which are respectively the 22 th to 26 th, 34 th to 40 th, 48 th to 50 th, 54 th to 63 th, 70 th to 78 th, 81 th to 85 th, 106 th to 114 th, 122 th to 132 th and 137 th to 148 th amino acid sequences of the Cat-NPC2 amino acid sequence SEQ ID No. 1.

Although the embodiments of the present invention have been described in detail with reference to the examples, it should be noted that the scope of the present invention is not limited by the embodiments, but is defined by the claims. Those skilled in the art can appropriately modify the embodiments without departing from the technical spirit and scope of the present invention, and the modified embodiments are also clearly included in the scope of the present invention.

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> a novel allergen NPC2 from cats

<160>5

<210>SEQ ID N0.1

<211>149

<212>PRT

<213> Cat (Felis family)

<400>1

Met Arg Ser Leu Ala Val AlaPhe Val Leu Leu Ala Leu Ser Ala Ser

1 5 10 15

Gly Leu Ala Glu Pro Val Ile Phe Lys Asp Cys Gly Ser Gly Phe Gly

20 25 30

Val Ile Lys Glu Leu Asn Val Ser Pro Cys Pro Thr Gln Pro Cys Lys

35 40 45

Leu His Lys Gly Gln Ser Tyr Ser Val Asn Val Thr Phe Thr Ser Asn

50 55 60

Val Ser Ser Gln Gly Ser Lys Ala Leu Val Tyr Gly Ile Leu MET Gly

65 70 75 80

Val Ala Val Pro Phe Pro Ile Pro Glu Ala Asp Gly Cys Lys Ser Gly

85 90 95

Ile Asn Cys Pro Ile Gln Gln Gly Lys Thr Tyr Ser Tyr Leu Asn Lys

100 105 110

Leu Pro Val Lys Asn Glu Tyr Pro Ser Ile Lys Val MET Val Lys Trp

115 120 125

Gln Leu Leu Gly Asp Lys Glu Gln Asn Leu Phe Cys Trp Glu Ile Pro

130 135 140

Val Gln Ile Glu Gly

145

<210>SEQ ID N0.2

<211>839

<212>DNA

<221>CDS

<222>(120)...(566)

<400>2

cctggttgct ggtgagagtc gcctgacctg gctcctcccc aggcccgccc gggcaggttt 60

atcttgtgac tgaggcggtc gcttcttcct tcttcgtgct tggaacctgt tcagctgcg 119

atg cgt tcc ctg gcc gtc gcg ttc gtg ctc ctg gcg ctc agc gcc tcc 167

Met Arg Ser Leu Ala Val Ala Phe Val Leu Leu Ala Leu Ser Ala Ser

5 10 15

ggc ctc gcc gag cca gtg att ttc aag gac tgc ggt tct ggg ttt gga 215

Gly Leu Ala Glu Pro Val Ile Phe Lys Asp Cys Gly Ser Gly Phe Gly

20 25 30

gtt ata aag gag ctg aat gtg agc cca tgc ccc acc cag ccc tgc aaa 263

Val Ile Lys Glu Leu Asn Val Ser Pro Cys Pro Thr Gln Pro Cys Lys

35 40 45

ttg cac aaa ggc cag tct tac agt gtc aat gtc acc ttc acc agt aat 311

Leu His Lys Gly Gln Ser Tyr Ser Val Asn Val Thr Phe Thr Ser Asn

50 55 60

gtt tca tct cag ggt agc aaa gct ttg gtg tat ggc atc ctg atg ggc 359

Val Ser Ser Gln Gly Ser Lys Ala Leu Val Tyr Gly Ile Leu Met Gly

65 70 75 80

gta gca gtt ccc ttt ccc att cct gag gct gatggt tgt aag agt gga 407

Val Ala Val Pro Phe Pro Ile Pro Glu Ala Asp Gly Cys Lys Ser Gly

85 90 95

atc aac tgc ccc atc cag caa ggc aag acc tat agc tac ctg aat aaa 455

Ile Asn Cys Pro Ile Gln Gln Gly Lys Thr Tyr Ser Tyr Leu Asn Lys

100 105 110

cta cca gtg aag aat gaa tac ccc tct ata aaa gtg atg gtg aag tgg 503

Leu Pro Val Lys Asn Glu Tyr Pro Ser Ile Lys Val MET Val Lys Trp

115 120 125

cag ctt ctg ggc gac aag gaa cag aat ctc ttc tgc tgg gag atc cca 551

Gln Leu Leu Gly Asp Lys Glu Gln Asn Leu Phe Cys Trp Glu Ile Pro

130 135 140

gtg cag att gaa ggc tagaggtgct aatgccagca tgttgcgtgg gggtgagaga 606

Val Gln Ile Glu Gly

145

ggaacgggtg gagggagggc gggagaaatc gagtctaacc taaaggaaag gaattttgac 666

acagctgttt ggtgcctctc agtctccaga aggttccaga ccctgtttcc tgagagctca 726

gaactattgc ccttgtagta tctttggtga agggttggag ggaagaagag agggggagag 786

aggcacggat ttaatttgtc ttcagtattt tttgcgttgg aaaaaggaag gtc 839

<210>SEQ ID N0.3

<211>450

<212>CDS

<400>3

atgcgttctc tggctgttgc tttcgttctg ctggctctgt ctgcttctgg tctggctgaa 60

ccggttatct tcaaagactg cggttctggt ttcggtgtta tcaaagaact gaacgtttct 120

ccgtgcccga cccagccgtg caaactgcac aaaggtcagt cttactctgt taacgttacc 180

ttcacctcta acgtttcttc tcagggttct aaagctctgg tttacggtat cctgatgggt 240

gttgctgttc cgttcccgat cccggaagct gacggttgca aatctggtat caactgcccg 300

atccagcagg gtaaaaccta ctcttacctg aacaaactgc cggttaaaaa cgaatacccg 360

tctatcaaag ttatggttaa atggcagctg ctgggtgaca aagaacagaa cctgttctgc 420

tgggaaatcc cggttcagat cgaaggttag 450

<210>SEQ ID N0.4

<211>23

<212>DNA

<213> Artificial sequence

<400>4

gcaggtttat cttgtgactg agg 23

<210>SEQ ID N0.5

<211>22

<212>DNA

<213> Artificial sequence

<400>5

aggttagact cgatttctcc cg 22

Claims

1. A cat allergen NPC2 protein characterized by: the amino acid sequence of the protein is SEQ ID No.1, or has at least 90% homology with the amino acid sequence, wherein the amino acids from the 1 st to the 19 th are signal peptides.

2. Codon-optimized cat allergen NPC2 protein, characterized in that: the protein is composed of a nucleotide sequence shown in SEQ ID No. 2.

3. Recombinant cat allergen NPC2 protein characterized by: the recombinant protein is a protein consisting of a nucleotide sequence shown in SEQ ID No. 3.

4. The preparation method of the recombinant cat allergen NPC2 protein is characterized by comprising the following steps:

carrying out codon optimization on a natural gene sequence of the allergen Cat-NPC2 to obtain an optimized Cat-NPC2 sequence SEQID No. 3;

5. Use of the cat allergen NPC2 protein of claim 1 for the preparation of a pharmaceutical composition for the treatment or prevention of cat allergic diseases.

6. A pharmaceutical composition characterized by: comprising the cat allergen NPC2 protein of claim 1.

7. The pharmaceutical composition of claim 6, wherein: the pharmaceutical composition is an allergen vaccine.

8. A diagnostic kit characterized in that: the cat allergen NPC2 protein comprising SEQ ID No.1 or the composition of claim 6 for use in the in vitro diagnosis of cat allergic diseases.