Recombinant artemisia argyi pollen type III allergen as well as preparation method and application thereof

Abstract

The invention provides a recombinant artemisia argyi pollen type III allergen protein Art v3 and a coding gene and an expression and purification method thereof, the amino acid sequence of the protein is as shown in SEQ ID NO: 2, the coding nucleotide sequence is as shown in SEQ ID NO: 1, and the recombinant artemisia argyi pollen type III allergen protein Art v3 can be used for preparing drugs for treating artemisia pollen allergic diseases or diagnostic reagents for detecting artemisia pollen allergens.

Description

Recombinant Artemisia pollen type III allergen and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and relates to a recombinant Artemisia pollen type III allergen whose molecular characterization and immune activity are equivalent to those of natural allergens, as well as a preparation method and application thereof. Background Art

[0002] Pollen is a major cause of seasonal allergies. Unlike food allergies, pollen allergy is airborne and often difficult to avoid. It can trigger a range of allergic reactions, including rhinitis, dermatitis, and asthma, severely impacting patients' quality of life. In the United States, approximately 7% of adults and 9% of children suffer from pollen allergies (NIAID, National Institute of Allergy and Infectious Diseases), with the prevalence estimated as high as 40% in Europe (G. D'Amato, 2007). In recent years, with the continuous increase in the conversion of farmland to forest and green space in my country, the incidence of pollen allergies has been rising annually, reaching 5% in high-risk areas.

[0003] Artemisia pollen is one of the most important allergens (also known as allergens) causing hay fever in summer and autumn in my country. A domestic study of 215,210 allergen-specific IgE tests conducted between 2008 and 2010 showed that Artemisia pollen had the highest positive rate among inhaled allergens. A 2015 study by Yang Qiongliang et al. showed that Artemisia pollen is the most prevalent allergenic pollen in northern China.

[0004] Artemisia (Artemisia L.) is one of the largest genera in the Asteraceae family, with over 300 species worldwide, widely distributed across temperate, frigid, and subtropical regions of the Northern Hemisphere. Artemisia annua and mugwort are common pollen allergens. Artemisia annua (Artemisia annua), used to extract artemisinin, a potent antimalarial drug, is one of the most common Artemisia species in my country and the earliest studied plant for Artemisia allergy. Mugwort (Artemisia vulgaris), also known as northern mugwort, is widely distributed across China, Mongolia, Russia, Europe, the United States, and Canada, and is one of the most extensively studied allergenic pollens. Other major Artemisia allergens include Artemisia sieversiana, Artemisia capillaris, Artemisia lavabdykufikua, Artemisia desertorum, and Artemisia argyi. The major allergenic proteins in different Artemisia pollen are type I and type III allergens. Type III allergens belong to the non-specific lipid transfer protein (ns-LTP) family and have a molecular weight of approximately 9 kDa. Type III allergen proteins from different Artemisia species share a high degree of homology (sequence identity generally exceeding 80%).

[0005] The WHO proposes a "four-in-one" approach to allergic diseases: avoidance of allergens, symptomatic medication, specific immunotherapy, and physician and patient education. Avoidance of allergens involves identifying the allergen and implementing effective environmental controls to minimize exposure. Effectively avoiding allergens during the treatment of allergic diseases not only reduces the risk of allergic reactions but also improves medication efficacy and helps patients achieve faster desensitization. Specific immunotherapy, also known as desensitization therapy, is the only "cause-directed" treatment that can affect the natural course of allergic diseases and alter the immune response. By administering gradually increasing doses of the allergen, it aims to improve tolerance to the allergen, alleviate symptoms associated with exposure, and ultimately achieve tolerance and even immune tolerance.

[0006] In 2021, Zhejiang Wowu's Artemisia annua pollen allergen sublingual drops, a desensitization drug for the treatment of allergic rhinitis caused by Artemisia annua / Artemisia argyi pollen allergy, was approved for marketing. The main ingredient is Artemisia annua pollen allergen protein extract. Its patent CN101905022A states that "Artemisia pollen is used as raw material, and the pollen is defatted, extracted, and concentrated to prepare Artemisia pollen allergen extract." However, due to the limitations of raw material sources and production methods, natural allergen extracts inevitably have quality problems, such as the presence of undefined non-allergenic substances, pollutants, and high variability in allergen content and biological activity (Valenta R, et al. Allergen Extracts for in vivo diagnosis and treatment of allergy: is there a future[J]. Journal of allergy & Clinical immunology in practice, 2018.). The European Academy of Allergy and Clinical Immunology's 2018 Guidelines on Allergen Immunotherapy for Allergic Rhinitis (EAACI Guidelines on Allergen Immunotherapy: Allergic Rhinoconjunctivitis (2018)) also point out that mixed allergens present many potential drawbacks, including dilution effects, potential allergen degradation due to the enzymatic activity of certain allergens, and the difficulty in fully demonstrating the efficacy of allergen combinations. Standardized desensitization drugs approved for marketing by the EMA, HMA, and FDA generally limit their primary allergenic proteins. For example, ODACTRA, used to treat dust mite allergies, contains four primary allergenic proteins: Der p 1, Der p 2, Der f 1, and Der f 2; GRAZAX, used to treat timothy grass allergies, contains the primary allergenic protein Phl p 5; and RAGWIZAX, used to treat ragweed allergies, contains the primary allergenic protein Amb a 1. On the other hand, allergen diagnosis using natural extracts suffers from low sensitivity and specificity, and it is difficult to clearly determine the extent of a patient's response to the allergen components, which can easily lead to misdiagnosis.

[0007] Currently, no recombinantly expressed Artemisia pollen allergen protein drugs are on the market or undergoing clinical trials.

[0008] Summary of the Invention

[0009] The applicant hopes to provide a recombinant Artemisia pollen allergen with a clear main allergenic protein to improve the controllability of product quality, ensure the precision of desensitization immunotherapy drugs for Artemisia pollen allergic diseases and the accuracy of allergen diagnosis, and lay the foundation for the drugability of recombinant Artemisia pollen allergen.

[0010] One objective of the present invention is to provide a protein for treating Artemisia pollen allergy, comprising a recombinant Art v 3 protein. Art v 3 is a type III allergen protein of Artemisia pollen, the primary allergenic protein. It belongs to the non-specific lipid transfer protein (ns-LTP) family and has a molecular weight of approximately 9 kDa. Studies have shown that type III allergen proteins from different Artemisia plants share a high degree of homology (generally exceeding 80% sequence identity). The recombinant Art v 3 protein's amino acid sequence, disulfide bonds, and molecular weight are identical to those of the native protein, and exhibits similar biological activities.

[0011] Preferably, the amino acid sequence of the recombinant Art v 3 protein is shown in SEQ ID NO: 2.

[0012] The amino acid sequence, molecular weight, amino acid coverage and disulfide bond pairing mode of the recombinant Art v 3 protein of the present invention are completely consistent with those of the natural Art v 3 protein, and have similar biological activity to that of the natural Art v 3. Compared with the natural Artemisia pollen extract, it avoids the differences between batches of major allergens in natural pollen from different sources and activity, and the process and quality are more stable and controllable. It can also avoid the degradation of major allergens by interactions with other components in natural pollen and the generation of other allergic reactions, etc., meeting the safety, effectiveness and quality controllable requirements of modern biological products, and can be used for the treatment and diagnosis of Artemisia pollen allergic diseases such as allergic rhinitis and asthma, improving the precision of Artemisia pollen desensitization immunotherapy and the accuracy of Artemisia pollen allergen diagnosis, and has good medicinal prospects.

[0013] Another object of the present invention is to provide a DNA sequence encoding Art v 3 protein, the base sequence of which is shown in SEQ ID NO: 1. This sequence has been codon-optimized for the Pichia pastoris expression system, making it more conducive to the expression of Art v 3 in Pichia pastoris.

[0014] Another object of the present invention is to provide a secretory signal peptide design that facilitates the expression of Art v 3 protein in a Pichia pastoris expression system. This design not only increases protein expression, but also ensures that the resulting recombinant Art v 3 protein exhibits molecular characteristics identical to those of the native protein. The signal peptides are the acid phosphatase signal peptide Pho1 and the Art v 3 native protein signal peptide ns1 (Natural Signal 1). The amino acid sequence of the acid phosphatase signal peptide Pho1 is shown in SEQ ID NO:4, and the amino acid sequence of the Art v 3 native protein signal peptide ns1 is shown in SEQ ID NO:5. The inventors have discovered that using different secretory signal peptides to direct the secretory expression of Art v 3 in Pichia pastoris significantly affects the uniformity and expression level of the final recombinant Art v 3 protein. The Pho1 and ns1 signal peptides are more conducive to the correct and efficient expression of the Art v 3 protein in the Pichia pastoris system. The resulting recombinant Art v 3 not only exhibits increased protein expression but also is identical to the native Art v 3 in primary structure, molecular weight, amino acid coverage, and disulfide bond pairing.

[0015] Another object of the present invention is to provide a vector containing the codon-optimized Art v 3 gene. These vectors provide strong promoter elements necessary for target gene transcription, such as the methanol oxidase promoter (AOX promoter) or the glyceraldehyde 3-phosphate dehydrogenase promoter (GAP promoter). The vectors are preferably pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ A, B, C, pGAPZ A, B, C, pPICZαA, B, C, or pGAPZαA, B, C.

[0016] Another object of the present invention is to provide a Pichia pastoris strain comprising the above-mentioned recombinant expression vector. Preferably, the Pichia pastoris strain is SMD1168, GS115, KM71, X33 or KM71H.

[0017] The recombinant Art v 3 protein-encoding gene of the present invention is more conducive to expression in Pichia pastoris. The inventors discovered that Art v 3 produced by recombinant expression strains constructed in Pichia pastoris using different secretion signal peptide and vector combinations showed significant differences in molecular weight uniformity, expression yield, and primary structural accuracy. The inventors compared multiple combinations and ultimately identified a vector and signal peptide design combination that can produce a primary structure, molecular weight, amino acid coverage, and disulfide bond pairing pattern that is completely consistent with native Art v 3. The cloned expression level can reach over 60 mg / L, and the amino acid sequence, disulfide bonds, and molecular weight are completely consistent with the native protein. The in vitro immune reactivity with specific antibodies in the serum of allergic patients is comparable to that of the native protein.

[0018] Another object of the present invention is to provide a method for expressing Art v 3 protein, the method comprising the following steps:

[0019] A. Construction of a vector containing the above-mentioned gene encoding Art v 3:

[0020] When using the pPICZ series or pGAPZ series as vectors, the exogenous gene expression cassette does not contain a signal peptide. Therefore, it can be used to construct an Art v 3 expression cassette containing its own signal peptide or an artificially designed signal peptide such as Pho1. Specifically, the artificially synthesized Art v 3 sequence containing its own signal peptide (designed to contain start and stop codons) is cloned into the multiple cloning site of the corresponding vector (such as EcoRI and NotI), with the open reading frame located downstream of the promoter and upstream of the terminator, to construct a recombinant expression vector.

[0021] When using the pPICZα or pGAPZα series vectors, the exogenous gene expression cassette contains the α-Factor signal peptide and signal peptide cleavage sites: Kex2 (amino acid sequence: KR) and Ste 13 (amino acid sequence: EAEA). The Art v 3 target gene can be cloned behind the Kex2 sequence (in this design, the α-Factor signal peptide is cleaved by the Kex2 protease) or behind the Ste 13 sequence (in this design, the α-Factor signal peptide is cleaved by both Kex2 and / or Ste 13 proteases). Taking the pPICZαA vector as an example, if the Art v 3 gene sequence is located behind Kex2, the target gene can be cloned between the XhoI and NotI sites; if the Art v 3 gene sequence is located behind Ste 13, the target gene can be cloned between the EcoRI and NotI sites. This example demonstrates the construction of a recombinant expression vector for secretory expression of Art v 3 using the α-Factor signal peptide.

[0022] B. Linearize the vector from step A and transform it into a Pichia pastoris strain, and culture it under appropriate conditions;

[0023] C. Recover and purify the protein.

[0024] The above-mentioned vector is preferably pPICZ A or pGAPZ A;

[0025] The Pichia pastoris strain is preferably KM71 or X33 strain.

[0026] Another object of the present invention is to provide a method for purifying recombinant Art v 3 protein, wherein the purification method is as follows:

[0027] A. The Art v 3 fermentation broth was centrifuged at low temperature and high speed to collect the supernatant, concentrated by ultrafiltration using a 3KD membrane, and then replaced with 10 mM PB, pH 7.0 buffer, and filtered through a 0.45 μm filter membrane.

[0028] B. In the first step of cationic chromatography, the column was equilibrated with an equilibration buffer. The fermentation broth obtained in step A was then passed through a separation medium using a purification system. The elution peak was then collected using a gradient elution buffer. The equilibration buffer was 10 mM acetic acid-sodium acetate, pH 5.0, and the elution buffer was 10 mM acetic acid-sodium acetate, 1.0 M NaCl, pH 7.0.

[0029] C. In the second step, the Art v 3 protein peak collected in step B was diluted with equilibration buffer, the equilibration buffer was used to equilibrate the chromatography column, the diluted Art v 3 protein solution was applied to the hydrophobic chromatography filler, and the eluted peak was collected; the equilibration buffer was 2.5 M (NH4)2SO4, 10 mM PB, pH 7.4, and the elution buffer was 10 mM PB, pH 7.4;

[0030] D. The third step is to ultrafiltration replace the target protein peak collected in C, the buffer is 10mM citric acid-sodium citrate, pH 5.0; after filtration and sterilization, the Art v 3 protein stock solution is obtained.

[0031] After optimizing the fermentation culture process and purification method, the recombinant Art v 3 finally prepared by the present invention meets the requirements of human recombinant DNA products in terms of purity, process impurity residues, molecular characterization, etc. The HPLC-SEC and HPLC-RP purity reaches more than 99%, the expression level reaches more than 60 mg / L, the protein yield of scaled production is>0.2 g / L, the amino acid sequence, disulfide bonds and molecular weight are completely consistent with the natural protein, and the immune reactivity of specific antibodies in the serum of allergic patients in vitro is equivalent to that of the natural protein, and has good medicinal prospects. Compared with naturally extracted allergen products, the recombinant Art v 3 protein of this application has many advantages, such as avoiding the differences in the content and activity of major allergens in natural pollen from different sources, more stable and controllable allergen processing and quality, avoiding the interaction of other components in natural pollen to degrade the main allergens and produce other allergic reactions, etc., meeting the safety, effectiveness and quality control requirements of modern biological products. It can be used for diagnosis to identify the allergen molecules that actually cause allergic reactions and reveal cross-reactivity. It has good tolerance, safety and effectiveness for desensitization immunotherapy, ensuring the accuracy of desensitization immunotherapy drugs for Artemisia pollen allergic diseases and the accuracy of allergen diagnosis, laying the foundation for the drugability of recombinant Artemisia pollen allergens. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: Lanes 1-4: Electrophoresis analysis of culture supernatants after shaker expression of four constructs: X33-pGAPZαA-Art v 3 (signal peptide is α-factor), X33-pGAPZ A-Art v 3 (signal peptide is ns1), X33-pGAPZ A-Art v 3 (signal peptide is ns2), and X33-pGAPZ A-Art v 3 (signal peptide is Pho1).

[0033] Figure 2-1: Disulfide bond ~C27, ~C28 / C13 / C73 fragment coverage map in Art v 3 sample

[0034] Figure 2-2: Disulfide bond ~~C48,~C50 / C3 / C87 fragment coverage map in Art v 3 sample

[0035] Figure 3: Art v 3 molecular weight determination results

[0036] Figure 4: HPLC-SEC purity analysis chromatogram of recombinant Art v 3 pure product

[0037] Figure 5: HPLC-RP purity analysis chromatogram of recombinant Art v 3 pure product

[0038] Figure 6: Electrophoresis analysis of fermentation supernatant from recombinant Art v 3 fermentation process: Lane 1 is SDS-PAGE analysis of fermentation supernatant (reducing electrophoresis)

[0039] Figure 7: Inhibition curves of recombinant Art v 3 protein and natural Art v 3 protein on IgE antibodies in positive serum DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to specific examples. It should be understood that the examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] Example 1: Art v 3 gene codon optimization

[0042] Based on the protein sequence of Art v 3 published in Genbank (Genbank Accession No.: ACE07186), as shown in SEQ ID NO: 2, the inventors reverse-translated the protein sequence to obtain a DNA sequence and codon-optimized the coding DNA sequence, ultimately obtaining the optimized gene sequence shown in SEQ ID NO: 1. The codon-optimized DNA sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and delivered as a subcloned plasmid dry powder.

[0043] Example 2: Construction of Art v 3 recombinant expression strain

[0044] In this example, plasmids pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ A, B, C, pGAPZ A, B, C, pPICZαA, B, C or pGAPZαA, B, C, host bacteria SMD1168, GS115, KM71, X33 or KM71H and various signal peptides were used to construct Art v 3 recombinant expression strains. The plasmids pGAPZαA, pPICZαA, pGAPZ A, pPICZ A and signal peptides α-Factor, Pho1 or natural signal peptides ns1, ns2 are used as examples to illustrate the construction process of the Art v 3 recombinant expression strain. The processes of other construction forms are not listed one by one.

[0045] 1. Construction of different signal peptide forms of Art v 3

[0046] 1.1 α-Factor-Art v 3 Format Design: Primers were designed for the codon-optimized Art v 3 gene from Example 1, introducing appropriate restriction enzyme sites. The Art v 3 DNA sequence (including the stop codon) was amplified by PCR and cloned into the pGAPZαA or pPICZαA plasmid between the XhoI and NotI multiple cloning sites via the XhoI and NotI restriction enzyme sites to form a recombinant expression plasmid. This positioned the Art v 3 gene downstream of the α-Factor signal peptide (amino acid sequence shown in SEQ ID NO: 3) gene within the plasmid, achieving fusion expression.

[0047] 1.2 Pho1-Art v 3 format design: Primers were designed for the codon-optimized Art v 3 gene described in Example 1. Sequences overlapping the vector insertion site were designed at both ends of the gene, and a start codon and Pho1 signal peptide (amino acid sequence such as SEQ ID NO: 4) DNA sequence were introduced at the 5' end. The Pho1-Art v 3 DNA sequence was obtained by PCR amplification and cloned into a predetermined position in the pGAPZ A or pPICZ A plasmid using seamless cloning technology to form a recombinant expression plasmid. In the recombinant expression plasmid, the Pho1-Art v 3 gene is located downstream of the plasmid core promoter (e.g., the pGAPZ A core promoter is the GAP promoter, and the pPICZ A core promoter is the AOX1 promoter, the same below) and upstream of the terminator (AOX1 terminator, the same below), forming an open reading frame.

[0048] 1.3 Design of natural signal peptide form Art v 3 (nsArt v 3): The natural signal peptides explored in the present invention include two types, one is denoted as ns1, and the amino acid sequence is shown in SEQ ID NO: 5, and the other is denoted as ns2, and the amino acid sequence is shown in SEQ ID NO: 6. Primers were designed for the Art v 3 gene after codon optimization in Example 1, and overlapping sequences with the vector insertion position were designed at both ends of the gene, and a start codon and the corresponding natural signal peptide sequence were introduced at the 5' end. The ns1-Art v 3 and ns2-Art v 3 DNA sequences were obtained by PCR amplification, and cloned into the predetermined positions in the pGAPZ A or pPICZ A plasmid by seamless cloning technology to form recombinant expression plasmids. In the formed recombinant expression plasmid, the ns1-Art v 3 or ns2-Art v 3 gene is located downstream of the plasmid core promoter and upstream of the terminator, forming an open reading frame.

[0049] 2. Construction of recombinant expression strain

[0050] Transform the recombinant expression plasmid generated in "Step 1" above into an E. coli cloning host, such as DH5α or Top10. After culture, extract the plasmid and confirm the correct construction of the recombinant plasmid using sequencing to generate a subcloning strain. Cultivate the subcloning strain and extract the plasmid to obtain a high-concentration recombinant expression plasmid. Linearize the recombinant expression plasmid using an appropriate restriction endonuclease. Purify and recover the linearized fragment, then transform it into a suitable Pichia pastoris competent cell line, such as SMD1168, GS115, KM71, X33, or KM71H, by electroporation and plate culture.

[0051] 3. Screening of expression strains

[0052] A single colony was picked from the transformation plate in step 2 above and inoculated onto a suitable antibiotic plate (using the above vector as an example, bleomycin antibiotic was used for culture). After culturing for a suitable period of time, pressure screening was performed using different antibiotic concentrations. Finally, a single colony was cultured from a high-concentration antibiotic plate for expression and identification to screen for highly expressing strains.

[0053] 4. Expression and Identification

[0054] Strains expressing the corresponding GAP promoter-based constitutive expression vectors, such as pGAPZαA or pGAPZ A, were cultured in YPD liquid medium on a shaker. After 72 hours of culture, equal amounts of expression supernatants were collected for SDA-PAGE or Dot blot analysis. Strains expressing the corresponding AOX1 promoter-based inducible expression vectors, such as pPICZαA or pPICZ A, were cultured in YPD liquid medium on a shaker. After 24 hours of culture, expression was induced by adding 0.5% methanol to the culture volume (addition every 24 hours). After 72 hours of induction, equal amounts of expression supernatants were collected for SDA-PAGE or Dot blot analysis. Strains expressing the highest expression levels were subsequently selected for subsequent 3L fermentation studies. The fermentation expression yields were compared and summarized in Table 1. The results of SDS-PAGE analysis of the expression identification results are shown in Figure 1.

[0055] Example 3: Purification of recombinant Art v 3 protein

[0056] A. The Art v 3 fermentation broth was centrifuged at low temperature and high speed to collect the supernatant, concentrated by ultrafiltration using a 3KD membrane package, replaced with 10mM PB, pH 7.0 buffer, and filtered through a 0.45μm filter membrane.

[0057] B. In the first step of cationic chromatography, the column was equilibrated with an equilibration buffer. The fermentation broth obtained in step A was then passed through a separation medium using a purification system. The broth was then gradient eluted using an elution buffer, and the elution peak was collected. The equilibration buffer was 10 mM acetic acid-sodium acetate, pH 5.0, and the elution buffer was 10 mM acetic acid-sodium acetate, 1.0 M NaCl, pH 7.0.

[0058] C. In the second step, the Art v 3 protein peak collected in step B was diluted with equilibration buffer, the equilibration buffer was used to equilibrate the chromatography column, the diluted Art v 3 protein solution was applied to the hydrophobic chromatography filler, and the eluted peak was collected; the equilibration buffer was 2.5 M (NH4)2SO4, 10 mM PB, pH 7.4, and the elution buffer was 10 mM PB, pH 7.4;

[0059] D. The third step is to ultrafiltration replace the target protein peak collected in C, the buffer is 10mM citric acid-sodium citrate, pH 5.0; after filtration and sterilization, the Art v 3 protein stock solution is obtained.

[0060] Example 4: Evaluation of the effects of different signal peptide forms on Art v 3 expression

[0061] 1. The molecular weight of the purified product prepared in Example 3 was analyzed by LC-MS. The molecular weight result was used to determine whether the molecular weight of Art v 3 after purification was correct.

[0062] 2. Further analyze the amino acid sequences of the designs with correct and incorrect molecular weights.

[0063] Table 1 Expression levels and character evaluation of Art v 3 expressed in different signal peptide and vector combinations

[0064] This example compares the recombinant expression effects of various signal peptide forms of Art v 3 and the effects of different signal peptide selections on the correct molecular identity of the final Art v 3 protein product. The acid phosphatase signal peptide Pho1 and the native protein signal peptide ns1 can guide the correct and efficient expression of Art v 3 in Pichia pastoris, resulting in high expression levels of the recombinant Art v 3 protein that is completely consistent with the molecular characteristics of the native Art v 3. Signal peptide and vector combinations that fail to express correctly or express at low levels are shown in Table 1, for example, pGAPZαA-α-Factor and pGAPZ A-ns2.

[0065] The acid phosphatase signal peptides Pho1 and ns1 are completely removed after secretion. The recombinant Art v 3 proteins produced with these two signal peptide forms exhibited the correct molecular weight and amino acid sequence identical to that of the native Art v 3. Furthermore, the resulting expression strains expressed high levels of the target protein. As shown in Examples 5 and 8, the recombinant Art v 3 proteins produced with these two signal peptide forms exhibited correct disulfide bond pairing compared to the native Art v 3 protein and possessed similar in vitro biological activity to the native protein. As shown in Table 1, which exemplifies the expression results of different signal peptide forms of pGAPZ A-Art v 3, Art v 3 recombinant expression strains constructed with the signal peptides Pho1 and ns1 produced target proteins with correct molecular characterization and high expression levels. However, signal peptides such as α-Factor and the native signal peptide ns2 are not completely removed after secretion of the target protein, resulting in recombinant Art v 3 proteins with molecular weights and amino acid sequences inconsistent with those of the native protein. Residual signal peptide amino acids may negatively impact target protein activity, such as decreased activity and increased immunogenicity.

[0066] Example 5: Detection of Art v 3 protein molecular weight and disulfide bonds

[0067] Correct disulfide bond pairing is crucial to maintaining the higher-order structure and activity of biomacromolecules such as proteins. Our Thermo Scientific Q Exactive LC-MS system was used to determine the disulfide bonds of the recombinant Art v 3 proteins obtained from the Pho1-Art v 3 and ns1-Art v 3 construction designs with the correct molecular weights. Four pairs of theoretical disulfide bonds were identified for the recombinant Art v 3 protein: C3-50, C13-27, C28-73, and C48-87. These were consistent with those reported in the database, and the molecular weight was completely consistent with the theoretical molecular weight. Taking the recombinant Art v 3 expressed in the recombinant expression strain constructed with X33-pGAPZ A and the signal peptide ns1 as an example, the results are shown in Figures 2 and 3, respectively.

[0068] Example 6: Art v 3 purity analysis

[0069] Taking the recombinant Art v 3 expressed in Example 3 using the recombinant expression strain constructed with X33-pGAPZ A and a signal peptide in the form of ns1 as an example, the highly purified Art v 3 was analyzed using HPLC-SEC and HPLC-RP. The results showed purities exceeding 99-100%, respectively, meeting pharmaceutical-grade purity requirements. Figures 4 and 5 show the analytical chromatograms of recombinant Art v 3 with the signal peptide in the form of ns1. The main peak integration results in purities of 100% and 99.80%, respectively.

[0070] Example 7: Purification of native Art v 3 protein

[0071] 1. Preparation of crude extract: Weigh defatted Artemisia argyi pollen (purchased from Stallergenes Greer) and prepare a pH 7.0, 50 mM PB solution. Add the PB solution at a w / v ratio of 1:10 and extract at 2-8°C for 48-72 hours. Centrifuge at 4000 rpm and collect the supernatant to obtain the crude extract.

[0072] 2. Chromatographic purification: The crude extract in "Step 1" was purified according to the purification steps in Example 3. After purification, electrophoresis analysis was performed and the components with good purity were subjected to subsequent operations.

[0073] 3. Ultrafiltration replacement: The target component in "Step 2" is replaced with a buffer solution by ultrafiltration. The buffer solution is replaced with a pH 7.4 PBS solution and stored at -20°C or below until use.

[0074] Example 8: Scale-up of the fermentation and purification process of the recombinant Art v 3 strain

[0075] In this example, the recombinant expression strain constructed with X33-pGAPZ A and signal peptide ns1 was used as an example to scale up the fermentation and purification process of recombinant Art v 3, and the expression level and purification yield were further improved. Other preferred construction forms of the present invention can also be well scaled up for production.

[0076] 1. The genetically engineered strain constructed in Example 2 was streaked onto YPD agar solid medium and cultured at 30°C for 48-72 hours until a single colony grew, completing the strain activation.

[0077] 2. Pick a single colony from "Step 1." and inoculate it into YPD primary seed medium. Incubate it in a shaker at 30℃ and 220rpm for 12-16h. The final OD 600 It should be between 1-4, which is the first-level seed liquid.

[0078] 3. Inoculate the primary seed solution in "Step 2" into the YPD secondary culture medium at 1% (v / v), and culture on a shaker at 30°C and 220 rpm for 18-24 hours. The final OD 600 It should be between 8-15, this is the secondary seed liquid.

[0079] 4. Inoculate the secondary seed solution in step 3 into the seed tank at 1% (v / v) for cultivation. The seed tank culture medium is YPD medium. Seed tank culture conditions: tank pressure 0.2-0.5 bar, temperature 28-30 ° C, dissolved oxygen DO controlled between 15-35%, which is achieved by linking the fermentation tank speed and ventilation volume. Culture to OD 600 The culture is terminated between 8 and 15 minutes, and the fermentation volume of the seed tank is 30L.

[0080] 5. Transfer the culture solution in the seed tank in "Step 4." to the working tank and start culturing. Working tank culture conditions: tank pressure is 0.2-0.5 bar, temperature is 28-30°C, dissolved oxygen DO is controlled between 15-35%, which is achieved by associating the fermentation tank speed and ventilation volume. The working tank culture medium is BSM inorganic culture medium, and the culture is carried out using a batch-fed culture strategy. After the carbon source (glycerol) in the initial culture medium is exhausted in the working tank, the second stage of fed-batch culture begins. The feeding carbon source is 50% glycerol or glucose, and the feeding rate is 5-15ml / L / h. Continue the fed-batch culture for 24-36h to end the fermentation. The initial fermentation volume after inoculation of the working tank is 300L.

[0081] 6. After fermentation, the fermentation supernatant was collected by centrifugation in a continuous flow centrifuge. The fermentation supernatant was filtered using a filter device with a pore size of 0.45 μm to remove impurities. The fermentation supernatant was analyzed by electrophoresis, and the results are shown in Figure 6. The target protein yield in the fermentation supernatant reached more than 0.2 g / L. The fermentation supernatant was purified according to Example 3, and the final pure product HPLC-SEC and HPLC-RP both reached more than 99%, meeting pharmaceutical standards. Art v 3 protein can obtain more than 20 g of pure product in a single batch, which has good process scale-up feasibility and lays the foundation for future production.

[0082] Example 9: Detection of Art v 3 protein activity

[0083] Taking Art v 3 expressed by the recombinant expression strain constructed with X33-pGAPZ A and signal peptide ns1 as an example, it is shown that the activity of the recombinant Art v 3 protein of this application is equivalent to that of the natural Art v 3 protein. The recombinantly expressed Art v 3 proteins of other preferred construction forms of this application also have equivalent activity to that of the natural Art v 3 protein.

[0084] 1. The highly purified rArt v 3 in Example 3 and the natural Art v 3 protein purified and prepared in Example 7 were diluted to 0.5 μg / ml with 50 mM NaH2PO4 buffer pH 7.2, 100 μl per well, and coated at 4°C overnight.

[0085] 2. Sample preparation: Use blocking solution (2% BSA / PBST) to dilute the recombinant protein or native protein to a starting concentration of 500ug / ml (S1), then dilute it in a 4-fold gradient, for a total of 10 gradients (S2-S11). Mix each dilution sample with positive serum (diluted 15-fold) in a 1:1 ratio. After mixing, incubate the samples at 4°C overnight.

[0086] 3. The next day, remove the ELISA plate, wash it 4 times with PBST, add 200 μl of 2% BSA / PBST solution to each well, and block it at 37°C for 2 h.

[0087] 4. After blocking, discard the blocking solution and add 100ul of the mixed incubated sample to each well, incubate at 300rpm and 37℃ for 1.5h.

[0088] 5. Wash three times with PBST, add 1:1500 diluted mouse anti-human IgE-HRP secondary antibody to each well, 100 μl per well, incubate at 300 rpm and 37°C for 1 h.

[0089] 6. Wash 3 times with PBST, add 100 μl TMB VII colorimetric solution to each well, react at 37°C for 10 min, then add 50 μl stop solution (2M H2SO4) to each well, and OD 450nmGet tested now.

[0090] 7. Analysis of results: As shown in Figure 7, the inhibition curves of the recombinant Art v 3 protein of the present invention against IgE antibodies in positive serum are basically the same as those of the natural Art v 3 protein. Taking the natural Art v 3 protein as the standard, the recombinant Art v 3 protein is comparable to it, indicating that the immunoreactivity of the recombinant protein with IgE-specific antibodies in the serum of allergic patients in vitro is comparable to that of the natural protein.

Claims

1. A protein for treating Artemisia pollen allergy, which is a recombinant Artemisia pollen type III allergen protein, the amino acid sequence, disulfide bonds and molecular weight of the recombinant Artemisia pollen type III allergen protein are consistent with those of the natural protein, and the immunoreactivity of the recombinant Artemisia pollen type III allergen protein with specific antibodies in the serum of allergic patients in vitro is equivalent to that of the natural protein.

2. The protein for treating Artemisia pollen allergy according to claim 1, wherein the amino acid sequence is as shown in SEQ ID NO:

2.

3. A nucleotide encoding the protein for treating artemisia pollen allergy according to claim 2, wherein the base sequence is shown in SEQ ID NO:

1.

4. A vector containing the nucleotide of the protein for treating Artemisia pollen allergy according to claim 3, wherein the vector is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ A, B, C, pGAPZ A, B, C, pPICZαA, B, C or pGAPZαA, B, C.

5. A Pichia strain comprising the vector of claim 4, wherein the Pichia strain is SMD1168, GS115, KM71, X33 or KM71H.

6. The method for expressing a protein for treating Artemisia pollen allergy according to claim 1 or 2, comprising the following steps: A. constructing a vector containing the gene encoding Art v 3 as claimed in claim 4; B. linearizing the vector in step A and transferring it into a Pichia yeast strain, and culturing it under appropriate conditions; C. Recover the purified protein.

7. The method for purifying the protein for treating Artemisia pollen allergy according to claim 1 or 2, wherein the purification method is as follows: A. Collect the supernatant of Art v 3 fermentation broth by low-temperature high-speed centrifugation, concentrate it by ultrafiltration with 3KD membrane package, replace it with 10mM PB, pH7.0 buffer, and filter it with 0.45μm filter membrane; B. The first step is cationic chromatography, wherein the chromatographic column is equilibrated with an equilibration buffer, and then the fermentation broth obtained in step A is passed through a separation filler using a purification system, and then gradient eluted with an elution buffer to collect the elution peak; the equilibration buffer is 10 mM acetic acid-sodium acetate, pH 5.0, and the elution buffer is 10 mM acetic acid-sodium acetate, 1.0 M NaCl, pH 7.0; C. In the second step, the Art v 3 protein peak collected in B is diluted with an equilibrium buffer, the equilibrium buffer is used to equilibrate the chromatography column, the diluted Art v 3 protein solution is applied to a hydrophobic chromatography filler, and the elution peak is collected; the equilibrium buffer is 2.5 M (NH4)2SO4, 10 mM PB, pH7.4, and the elution buffer is 10 mM PB, pH7.4; D. In the third step, the target protein peak collected in C is ultrafiltered and replaced with a buffer of 10 mM citric acid-sodium citrate, pH 5.0; after filtration and sterilization, the Art v 3 protein stock solution is obtained.

8. Use of the protein of claim 1 or 2 in the preparation of a medicament for treating allergic diseases caused by Artemisia pollen.

9. Use of the protein of claim 1 or 2 in the preparation of a diagnostic reagent for detecting Artemisia pollen allergens.

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