Recombinant artemisia annua 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 a3 and a coding gene and an expression and purification method thereof, the amino acid sequence of the protein is shown as SEQ ID NO: 2, the coding nucleotide sequence is shown as SEQ ID NO: 1, and the recombinant artemisia argyi pollen type III allergen protein Art a3 can be used for preparing drugs for treating artemisia pollen allergic diseases or diagnostic reagents for detecting artemisia pollen allergens.

Description

Recombinant Artemisia annua 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 annua pollen type III allergen having molecular characterization and immune activity equivalent to that of a natural allergen, 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 annua 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 annua pollen allergen.

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

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

[0012] The amino acid sequence, molecular weight, amino acid coverage, and disulfide bond pairing pattern of the recombinant Art a 3 protein of the present invention are completely consistent with those of the natural Art a 3 protein, and have similar biological activity to that of the natural Art a 3. Compared with natural Artemisia annua pollen extract, the recombinant Art a 3 protein avoids the differences between batches of major allergen content and activity of natural pollen from different sources, and has more stable and controllable process and quality. 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. It can be used for the treatment and diagnosis of Artemisia pollen allergic diseases such as allergic rhinitis and asthma, improves 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 a 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, which is more conducive to the expression of Art a 3 in Pichia pastoris.

[0014] Another object of the present invention is to provide a secretion signal peptide design that facilitates obtaining high expression of Art a 3 with the correct molecular weight in a Pichia pastoris expression system. The signal peptides are acid phosphatase signal peptide (Pho1), Art a 3 native protein signal peptide, and Saccharomyces cerevisiae α-factor signal peptide (α-factor). The α-factor signal peptide is shown in SEQ ID NO: 3, the Pho1 signal peptide amino acid sequence is shown in SEQ ID NO: 4, and the Art a 3 native protein signal peptide amino acid sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6. The inventors have discovered that using different secretion signal peptides to direct secretory expression of Art a 3 in Pichia pastoris significantly affects the uniformity and expression level of the final recombinant Art a 3 protein. Preferred signal peptides are more conducive to accurate and efficient expression of Art a 3 protein in the Pichia pastoris system. The resulting recombinant Art a 3 not only has increased protein expression, but also is completely consistent with the primary structure, molecular weight, amino acid coverage, and disulfide bond pairing pattern of native Art a 3.

[0015] Another object of the present invention is to provide a vector containing the codon-optimized Art a 3 gene described above. 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 inventors discovered that Art a 3 produced by recombinant expression strains constructed in Pichia pastoris using different secretion signal peptide and vector combinations exhibited 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 that produced a protein identical to native Art a 3 in terms of primary structure, molecular weight, amino acid coverage, and disulfide bond pairing.

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

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

[0020] When using the commonly used 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 a 3 expression cassette containing its own signal peptide or an artificially designed signal peptide such as Pho1. Specifically, the artificially synthesized Art a 3 sequence containing its own signal peptide (designed to contain a start codon and a stop codon) is cloned into the multiple cloning site of the corresponding vector (such as EcoRI and NotI), so that the open reading frame is located downstream of the promoter and upstream of the terminator, thereby constructing a recombinant expression vector.

[0021] When using the commonly used pPICZα series 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 a 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 a 3 gene sequence is located behind Kex2, the target gene can be cloned between the XhoI and NotI sites; if the Art a 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 a 3 using the α-factor signal peptide.

[0022] B. Linearize the vector from step A and transform it into a Pichia pastoris strain. Select clones with high expression levels using a stress resistance screening method, and culture them under appropriate conditions to further verify the expression level or perform downstream purification.

[0023] C. Recover and purify the protein.

[0024] The above-mentioned vector is preferably pPICZαA, pGAPZαA, 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 a 3 protein, wherein the purification method is as follows:

[0027] A. The Art a 3 fermentation broth was centrifuged at low temperature and high speed to collect the supernatant, concentrated by ultrafiltration using a 3KD membrane package, and then replaced with a 10 mM acetic acid-sodium acetate, pH 5.0 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 5.0.

[0029] C. In the second step, the Art a 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 a 3 protein solution was applied to a 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. In the third step, the target protein peak collected in C is ultrafiltered and replaced with a 10 mM citric acid-sodium citrate buffer, pH 5.0; after filtration and sterilization, the Art a 3 protein stock solution is obtained.

[0031] After optimizing the fermentation and purification process, the recombinant Art a 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., with SEC-HPLC purity >99%, expression level reaching 120 mg / L, and protein yield >0.15 g / L in scaled-up production. It also has an amino acid sequence, disulfide bonds, and molecular weight that are completely consistent with the natural protein. The in vitro immune reactivity with specific antibodies in the serum of allergic patients is comparable to that of the natural protein, showing good medicinal prospects. Compared with naturally extracted allergen products, the recombinant Art a 3 protein of the present 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 major 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 precision of desensitization immunotherapy drugs for Artemisia pollen allergic diseases and the accuracy of allergen diagnosis, laying the foundation for the drugability of recombinant Artemisia annua pollen allergen. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: Expression identification of Art a 3: Lanes 1-8: Electrophoresis analysis of the culture supernatant after shaker expression of 8 constructs, namely X33-pGAPZαA-Art a 3 (signal peptide is α-factor), X33-pGAPZ A-Art a 3 (signal peptide is Pho1), X33-pGAPZ A-Art a 3 (signal peptide is ns1), X33-pGAPZ A-Art a 3 (signal peptide is ns2), X33-pPICZαA-Art a 3 (signal peptide is α-factor), X33-pPICZ A-Art a 3 (signal peptide is Pho1), X33-pPICZ A-Art a 3 (signal peptide is ns1), and X33-pPICZ A-Art a 3 (signal peptide is ns2)

[0033] Figure 2-1: Disulfide bond ~C48, ~C50 / C3 / C87 fragment coverage map in Art a 3 sample

[0034] Figure 2-2: Disulfide bond ~C27, ~C28 / C13 / C73 fragment coverage map in Art a 3 sample

[0035] Figure 3: Molecular weight determination results of recombinant Art a 3

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

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

[0038] Figure 6: Electrophoretic analysis of fermentation supernatant from the recombinant Art a 3 fermentation process: Lane 1, SDS-PAGE analysis of fermentation supernatant (reducing electrophoresis)

[0039] Figure 7: Inhibition curves of recombinant Art a 3 protein and natural Art a 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 a 3 gene codon optimization

[0042] Based on the protein sequence of Art a 3 published in Genbank (Genbank Accession No. ANC85017.1), 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 a 3 recombinant expression strain

[0044] 1. Construction of different signal peptide forms of Art a 3

[0045] 1.1 α-Factor-Art a 3 Format Design: Primers were designed for the codon-optimized Art a 3 gene from Example 1, introducing appropriate restriction enzyme sites. The Art a 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 a 3 gene downstream of the α-Factor signal peptide (amino acid sequence shown in SEQ ID NO: 3) gene in the plasmid, achieving fusion expression.

[0046] 1.2 Pho1-Art a 3 format design: Primers were designed for the codon-optimized Art a 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 a 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 a 3 gene was 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 (the AOX1 terminator, the same below), forming an open reading frame.

[0047] 1.3 Design of natural signal peptide Art a 3 (nsArt a 3): The natural signal peptides explored in the present invention include two types, one denoted as ns1, with an amino acid sequence as shown in SEQ ID NO: 5, and the other denoted as ns2, with an amino acid sequence as shown in SEQ ID NO: 6. Primers were designed for the codon-optimized Art a 3 gene in Example 1, with overlapping sequences at both ends of the gene and at the vector insertion position, and a start codon and the corresponding natural signal peptide sequence were introduced at the 5' end. The ns1-Art a 3 and ns2-Art a 3 DNA sequences were obtained by PCR amplification, and cloned into predetermined positions in the pGAPZ A or pPICZ A plasmid using seamless cloning technology to form recombinant expression plasmids. In the resulting recombinant expression plasmid, the ns1-Art a 3 or ns2-Art a 3 gene was located downstream of the plasmid core promoter and upstream of the terminator, forming an open reading frame.

[0048] 2. Construction of recombinant expression strain

[0049] 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 X33, KM71H, GS115, SMD1168, or KM71, by electroporation and plate culture.

[0050] 3. Screening of expression strains

[0051] Pick a single colony from the transformation plate in "Step 2." and inoculate it onto a plate with a suitable antibiotic (taking the above-mentioned vector as an example, bleomycin antibiotic is used for culture). After culturing for a suitable time, use different antibiotic concentrations for pressure screening. Finally, take a single colony from the high-concentration antibiotic plate for culture, expression, and identification to screen for high-expression strains.

[0052] 4. Expression and Identification

[0053] Using the corresponding expression strains based on the GAP promoter constitutive expression vectors, such as pGAPZαA or pGAPZ A, they 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. Using the corresponding expression strains based on the AOX1 promoter inducible expression vectors, such as pPICZαA or pPICZ A, they were cultured in YPD liquid medium on a shaker. After 24 hours of culture, 0.5% methanol was added to the culture volume to induce expression (addition was made every 24 hours). After 72 hours of induction, equal amounts of expression supernatants were collected for SDA-PAGE or Dot blot analysis. The strains with the highest expression levels were finally selected for subsequent 3L fermentation studies. The fermentation expression yields were compared and summarized in Table 2 below. The results of SDS-PAGE analysis of the expression identification results are shown in Figure 1.

[0054] Example 3: Purification of recombinant Art a 3 protein

[0055] A. The Art a 3 fermentation broth was centrifuged at low temperature and high speed to collect the supernatant, concentrated by ultrafiltration using a 3KD membrane package, and then replaced with a 10 mM acetic acid-sodium acetate, pH 5.0 buffer, and filtered through a 0.45 μm filter membrane.

[0056] 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 5.0.

[0057] C. In the second step, the Art a 3 protein peak collected in B was diluted with equilibration buffer, the equilibration buffer was used to equilibrate the chromatography column, the diluted Art a 3 protein solution was applied to a 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.

[0058] D. In the third step, the target protein peak collected in C is ultrafiltered and replaced with a 10 mM citric acid-sodium citrate buffer, pH 5.0; after filtration and sterilization, the Art a 3 protein stock solution is obtained.

[0059] Example 4: Evaluation of the effects of different signal peptide and vector combinations on Art a 3 expression

[0060] 1. The purified product prepared in Example 3 was analyzed for molecular weight using LC-MS. The molecular weight results were used to determine whether the molecular weight of the purified Art a 3 was correct. For designs with incorrect molecular weights, the amino acid sequences were further analyzed. The results are shown in the table below.

[0061] Table 2 Expression levels and trait evaluation of Art a 3 expressed in different signal peptide and vector combinations

[0062] As can be seen from the table above, the inventors explored the effects of different signal peptide and vector combination selections on the final Art a3 protein expression. We found that the target proteins produced by recombinant expression strains constructed with various signal peptide combinations and different vectors had significant differences in molecular accuracy and expression levels. The target protein yields of expression strains formed by the signal peptide combination pGAP vector (mainly based on the GAP promoter) were significantly higher than those of the corresponding combination pPIC vector (mainly based on the AOX1 promoter). As shown in Table 2, the expression levels of various pGAP vector constructs were generally higher than those of the pPIC vector. The inventors also found that the choice of different signal peptides affected the molecular weight accuracy and yield of the target protein. Insufficient cleavage of some signal peptides during secretion resulted in residual signal peptide amino acids. Residual signal peptide amino acids may lead to negative effects such as decreased target protein activity and increased immunogenicity. At the same time, different signal peptides have different abilities to guide the secretion and expression of target proteins, which also reflects differences in expression levels. For example, in Table 2, the expression level of the pGAPZ A-Pho1 construct was 90 mg / L, while the expression level of the pGAPZ A-ns1 construct was 20 mg / L.

[0063] Example 5: Molecular weight and disulfide bond detection of recombinant Art a 3 protein

[0064] Correct disulfide bond pairing is crucial for maintaining the higher-order structure and activity of biomacromolecules such as proteins. Using our company's Thermo Scientific Q Exactive LC-MS system, the molecular weight and disulfide bond analysis of the recombinant Art a 3 protein, obtained after purification of the correctly designed construct, were performed. The four theoretical disulfide bond pairs identified for the recombinant Art a 3 protein, C3-50, C13-27, C28-73, and C48-87, were consistent with those reported in the database, and the molecular weight was completely consistent with the theoretical molecular weight. The results are shown in Figures 2 and 3, using the recombinant Art a 3 expressed in the recombinant expression strain constructed with X33-pGAPZαA and an α-factor signal peptide as an example.

[0065] Example 6: Purity Analysis of Recombinant Art a 3

[0066] Take the recombinant Art a 3 expressed by the recombinant expression strain constructed with X33-pGAPZαA and the signal peptide as α-factor as an example:

[0067] 1. The highly purified Art a 3 (in the form of the signal peptide α-Factor) from Example 3 was analyzed using HPLC-SEC and HPLC-RP. The analytical chromatograms are shown in Figures 4 and 5 , respectively. After integration of the main peak, the purities by SEC and RP were 99.90% and 98.10%, respectively, meeting pharmaceutical-grade purity requirements.

[0068] Example 7: Purification of native Art a 3 protein

[0069] 1. Preparation of crude extract: Weigh defatted Artemisia annua 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.

[0070] 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.

[0071] 3. Ultrafiltration replacement: Combine the target fractions in "Step 2." and use ultrafiltration replacement buffer. The buffer is replaced with pH 7.4 PBS solution and frozen below -20°C until use.

[0072] Example 8: Scale-up of the fermentation and purification process of the recombinant Art a 3 strain

[0073] In this example, the recombinant expression strain X33-pGAPZαA, constructed with an α-factor signal peptide, was used as an example to scale up the fermentation and purification process of recombinant Art a 3, further improving the expression level and purification yield. Other preferred constructions of the present invention can also be well scaled up for production.

[0074] 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.

[0075] 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.

[0076] 3. Inoculate the primary seed solution in "Step 2" into the YPD secondary 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.

[0077] 4. Inoculate the secondary seed solution in "Step 3" at 1% (v / v) into the seed tank 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 until OD 600 The culture is terminated between 8 and 15 minutes, and the fermentation volume of the seed tank is 30L.

[0078] 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. The fermentation can be terminated by continuing the fed-batch culture for 24-36h. The initial fermentation volume after inoculation of the working tank is 300L.

[0079] 6. After the fermentation is completed, the fermentation supernatant is collected by centrifugation using a continuous flow centrifuge. The fermentation supernatant is filtered using a filter device with a pore size of 0.45 μm to remove impurities. The fermentation supernatant is analyzed by electrophoresis, and the results are shown in Figure 6. The target protein yield in the fermentation supernatant reaches more than 0.15 g / L. The fermentation supernatant is purified according to Example 3, and the final pure product HPLC-SEC and HPLC-RP can reach more than 99%, meeting pharmaceutical standards. Art a 3 protein can obtain more than 17 g of pure product in a single batch, which has good process scale-up feasibility and lays the foundation for future production.

[0080] Example 9: Detection of Art a 3 protein activity

[0081] This example uses X33-pGAPZαA and an α-factor combination of recombinantly expressed Art a 3 as an example to illustrate that the recombinant Art a 3 protein of the present application has equivalent activity to the natural Art a 3 protein. The recombinantly expressed Art a 3 proteins of other preferred constructions of the present application also have equivalent activity to the natural Art a 3 protein.

[0082] 1. The highly purified Art a 3 (signal peptide in the form of α-factor) in Example 3 and the natural Art a 3 protein purified and prepared in Example 7 were diluted to 0.5 μg / ml with 50 mM NaH 2 PO 4 buffer, pH 7.2, 100 μl per well, and coated at 4°C overnight.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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 450nm Get tested now.

[0088] 7. Analysis of Results: As shown in Figure 7 , the inhibition curves of the recombinant Art a 3 protein of the present invention against IgE antibodies in positive serum were essentially identical to those of the native Art a 3 protein. Using the native Art a 3 protein as the standard, the relative activity of the recombinant Art a 3 protein was 102%, indicating that the in vitro immunoreactivity of the recombinant protein with IgE-specific antibodies in the serum of allergic patients was comparable to that of the native protein.

Claims

1. A protein for treating Artemisia pollen allergy, which is a recombinant Artemisia annua pollen type III allergen protein. The amino acid sequence, disulfide bonds and molecular weight of the recombinant Artemisia annua pollen type III allergen protein are consistent with those of the natural protein, and the immunoreactivity of the recombinant Artemisia annua pollen type III allergen protein in vitro with specific antibodies in the serum of allergic patients 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, pPICZαA, B, C, pGAPZαA, B, C, pPICZ A, B, C or pGAPZ A, 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, the method comprising the following steps: A. constructing a vector containing the gene encoding Art a 3 according to claim 4; B. linearizing the vector in step A and transferring it into a Pichia yeast strain, and culturing it under appropriate conditions; C. recovering and purifying the protein.

7. A method for purifying a protein for treating artemisia pollen allergy according to claim 1 or 2, the purification method being as follows: A. collecting the supernatant by low-temperature high-speed centrifugation of Art a 3 fermentation broth, ultrafiltration and concentration by 3KD membrane package, 10mM acetic acid-sodium acetate, pH5.0 buffer exchange, and 0.45μm filter membrane filtration; B. a first step of cationic chromatography, balancing the chromatography column with an equilibrium buffer, then using a purification system to pass the fermentation broth obtained in step A through a separation filler, and then using an elution buffer gradient elution to collect the elution peak; the equilibrium buffer is 10mM acetic acid-sodium acetate, pH5.0, and the elution buffer is 10mM acetic acid-sodium acetate, 1.0M NaCl, pH5.0; C. a second step of diluting the Art a 3 protein peak collected in B with an equilibrium buffer, balancing the chromatography column with the equilibrium buffer, applying the diluted Art a 3 protein solution to a hydrophobic chromatography filler, and collecting the elution peak; the equilibrium buffer is 2.5M (NH4)2SO4, 10mM PB, pH7.4, and the elution buffer is 10mM 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, pH5.0; after filtration and sterilization, Art a 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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