WO2023125958A1 - 重组艾蒿花粉i型变应原及其制备方法和应用 - Google Patents

重组艾蒿花粉i型变应原及其制备方法和应用 Download PDF

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WO2023125958A1
WO2023125958A1 PCT/CN2022/144040 CN2022144040W WO2023125958A1 WO 2023125958 A1 WO2023125958 A1 WO 2023125958A1 CN 2022144040 W CN2022144040 W CN 2022144040W WO 2023125958 A1 WO2023125958 A1 WO 2023125958A1
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protein
art
expression
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马永
赵百学
高慧
庄宇
王琴
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江苏众红生物工程创药研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biopharmaceuticals, and relates to a recombinant mugwort pollen type I allergen whose characterization and activity are consistent with natural allergens, its preparation method and application.
  • Pollen is one of the main causes of seasonal allergies. Unlike food allergies, pollen allergies are spread through the air and are often unavoidable. They can induce a series of allergic reactions such as rhinitis, dermatitis, and asthma, seriously affecting the quality of life of patients. About 7% of adults and 9% of children in the United States suffer from pollen allergy (NIAID, National Institute of Allergy and Infectious Diseases), and the prevalence in Europe is estimated to be as high as 40% (G.D'Amato, 2007). In recent years, with the continuous increase of the area of returning farmland to forests and green areas in my country, the incidence of pollen allergy has been increasing year by year, and the high incidence area can reach 5%.
  • NIAID National Institute of Allergy and Infectious Diseases
  • Artemisia (Genus Artemisia) plant pollen is one of the important allergens (also known as allergens) that cause hay fever in summer and autumn.
  • allergens also known as allergens
  • the results of 215,210 allergen-specific IgE tests in China from 2008 to 2010 showed that Artemisia pollen had the highest positive rate among inhaled allergens.
  • the research results of Yang Qiongliang et al. in 2015 showed that the most allergenic pollen in northern China was Artemisia pollen.
  • Artemisia L. is one of the large genera with the largest number of species in Compositae. There are more than 300 species of Artemisia plants in the world, which are widely distributed in temperate, temperate and subtropical regions of the northern hemisphere. Artemisia annua and Artemisia annua are common pollen-sensitizing plants, among which Artemisia annus (scientific name Artemisia annus) can be used to extract artemisinin, a special anti-malarial drug. Allergy to plants.
  • Artemisia vulgaris (scientific name Artemisia vulgaris), also known as northern mugwort, is widely distributed in China, Mongolia, Russia, Europe, the United States, Canada and other places, and is one of the most deeply studied allergenic pollen.
  • Other major Artemisia sensitizing plants include Artemisia sieversiana, Artemisia capillaris, Artemisia lavabdykufikua, Artemisia desertorum, Artemisia argyi )wait.
  • the main allergenic proteins of different Artemisia pollen are type I and type III allergens, among which the type I allergen belongs to the defensin-like protein (Defensin-like protein) family with a molecular weight of about 12kD, and the type I allergens of different Artemisia plants
  • the sequence of allergen protein is conserved; type III allergen belongs to non-specific lipid transport protein (nsLTP), and its variation degree is relatively high in pollen of different Artemisia plants.
  • WHO proposes a "four-in-one" therapy: avoiding contact with allergens, symptomatic drug treatment, specific immunotherapy, and education of doctors and patients.
  • avoiding contact with allergens refers to doing a good job in environmental prevention and control on the basis of determining the allergens, and avoiding contact with allergens as much as possible. Avoiding contact with allergens during the treatment of allergic diseases can not only reduce the chance of patients developing allergies, but also improve the efficacy of drugs and help patients desensitize faster.
  • Specific immunotherapy that is, desensitization therapy, is the only "causal" therapy that may affect the natural course of allergic diseases and change the immune response mechanism. Allergen tolerance can reduce the symptoms caused by exposure to the allergen, and finally achieve the purpose of tolerance and even immune tolerance.
  • the desensitization drug Artemisia annus pollen allergen sublingual drops which is used to treat allergic rhinitis caused by Artemisia annus pollen allergies, was approved for marketing in Wuwu, Zhejiang in 2021.
  • the main ingredient is Artemisia annus pollen Allergen protein extract, its patent CN101905022A points out that "Using Artemisia pollen as raw material, the pollen is degreased, extracted, and concentrated to obtain Artemisia pollen allergen extract".
  • Standardized desensitization drugs approved and marketed by EMA, HMA, and FDA basically limit the main allergenic proteins.
  • ODACTRA for the treatment of dust mite allergy contains four main allergenic proteins, Der P1, Der P2, Der F1, and Der F2.
  • GRAZAX for pasture allergy contains the main allergenic protein Phl P5, and RAGWIZAX for treating ragweed allergy contains the main allergenic protein Amb a1.
  • the use of natural extracts for allergen diagnosis has the problems of low sensitivity and specificity, and it is impossible to determine the degree of response of patients to allergen components, which easily leads to misdiagnosis.
  • the applicant hopes to provide a recombinant mugwort pollen allergen with a clear main allergenic protein to improve the controllability of product quality and ensure the accuracy of desensitization immunotherapy drugs for pollen allergic diseases of Artemisia genus and the accuracy of allergen diagnosis and lay the foundation for the druggability of the recombinant mugwort pollen allergen.
  • Art v 1 is the type I allergen protein of mugwort, which is the main allergenic protein. It is a glycoprotein composed of the N-terminal defensin domain and the C-terminal hydroxyproline-rich part. It belongs to the defensin-like protein (Defensin-like protein) )family. Studies have shown that the type I allergen proteins of different Artemisia plants belong to defensin proteins, and the sequences are highly conserved. The binding ability of each subtype to sIgE antibody is similar, and its immune activity is mainly determined by the N-terminal defensin domain. The amino acid sequence, disulfide bond and molecular weight of the recombinant Art v 1 protein are completely consistent with the natural protein, and have similar biological activity to the natural protein.
  • amino acid sequence of the Art v 1 protein is shown in SEQ ID NO:4.
  • the amino acid sequence, molecular weight, amino acid coverage and disulfide bond pairing mode of the recombinant Art v 1 protein of the present invention are completely consistent with the natural Art v 1 protein, and have similar biological activities to the natural Art v 1 protein. Extracts avoid differences in the content of major allergens and active batches of natural pollen from different sources, the process and quality are more stable and controllable, and it can also avoid the degradation of major allergens caused by the interaction of other components in natural pollen and other Sensitization, etc., meet the requirements of safe, effective and quality-controllable modern biological products, can be used for the treatment and diagnosis of allergic rhinitis, asthma and other Artemisia pollen allergic diseases, improve the accuracy of Artemisia pollen desensitization immunotherapy and improve the accuracy of Artemisia pollen desensitization immunotherapy. The accuracy of the diagnosis of pollen allergens has good medicinal prospects.
  • Another object of the present invention is to provide a DNA sequence encoding Art v 1 protein, the base sequence of which is shown in SEQ ID NO: 13.
  • the sequence is codon-optimized for the expression system of Pichia pastoris, which is more conducive to the expression of Art v 1 in Pichia pastoris.
  • Another object of the present invention is to provide a design form of secretion signal peptide that is conducive to the expression of Art v 1 protein in the expression system of Pichia pastoris, which not only improves the expression of Art v 1 protein, but also obtains recombinant Art v 1 protein and natural protein Compared with the molecular characterization, they are completely consistent.
  • the signal peptide is yeast ⁇ -factor signal peptide, aspergillus niger signal peptide, acid phosphatase signal peptide (PHO), Saccharomyces cerevisiae signal peptide (SUC2) and Art v 1 protein self signal peptide, preferably, the signal
  • the peptides are ⁇ -factor signal peptide (SEQ ID NO: 11) and self signal peptide (SEQ ID NO: 12).
  • Another object of the present invention is to provide a carrier containing the above-mentioned optimized Art v 1 gene, preferably, the carrier is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ ⁇ A, B, C or pGAPZ ⁇ A, B, C, more preferably pPICZA or pGAPZA.
  • Another object of the present invention is to provide a Pichia strain comprising the above-mentioned vector, preferably, the Pichia strain is SMD1168, GS115, KM71, X33 or KM71H, more preferably KM71 or X33 strain.
  • the recombinant Art v 1 protein coding gene of the present invention is more conducive to the expression of Pichia pastoris, and the inventors found that the engineering strain Art v 1 protein expression amount obtained by the combination of the above different secretion signal peptides, expression vectors and bacterial strains has a large difference, preferably The highest expression level of Art v 1 clone obtained by the combination method can reach 210mg/L.
  • the purified recombinant Art v 1 protein has the same amino acid sequence, disulfide bond and molecular weight as the natural protein, and is specific in vitro and in the serum of allergic patients. The immunological reactivity of the sex antibody is comparable to that of the native protein.
  • Another object of the present invention is to provide a kind of expression method of Art v 1 protein, described method comprises the following steps:
  • the exogenous gene expression cassette does not contain a signal peptide, so it can be used to construct an Art v 1 expression cassette containing its own signal peptide.
  • the Art v 1 artificially synthesized sequence (designed to contain a start codon and a stop codon) containing its own signal peptide is cloned into the multiple cloning site of the corresponding vector (such as EcoRI and NotI), so that the open reading frame is located at Downstream of the promoter and upstream of the terminator, a recombinant expression vector is constructed.
  • the foreign gene expression frame contains ⁇ -factor signal peptide and signal peptide cleavage site: Kex2 (amino acid sequence is KR), Ste 13 (amino acid sequence is EAEA).
  • Kex2 amino acid sequence is KR
  • Ste 13 amino acid sequence is EAEA
  • the Art v 1 target gene can be cloned behind the Kex2 sequence (in this design method, the ⁇ -factor signal peptide is excised by Kex2 protease), or it can be cloned into the Ste 13 sequence (in this design method, the ⁇ -factor signal peptide can be combined with Kex2 and/or) Ste 13 protease acts simultaneously to excise).
  • the Art v 1 gene sequence is positioned behind Kex2, it can be achieved by cloning the target gene between the XhoI and NotI sites; if the Art v 1 gene sequence is positioned behind Ste 13, it can be achieved by Cloning of the target gene is achieved between the EcoRI and NotI sites.
  • a recombinant expression vector for secreting and expressing Art v 1 using the ⁇ -factor signal peptide was constructed by way of example operation.
  • step B Linearize the vector in step A and transfer it into Pichia pastoris strains, and obtain clones with high expression levels through resistance pressure screening method, and culture under appropriate conditions to further verify the expression level or carry out downstream purification;
  • the aforementioned vector is preferably pPICZ ⁇ A or pGAPZ ⁇ A.
  • the above-mentioned Pichia strain is preferably KM71 or X33 strain.
  • Another object of the present invention is to provide a kind of recombinant Art v 1 protein purification method, described purification method is as follows:
  • the first step of cation chromatography equilibrate the chromatographic column with an equilibrating buffer, then use the purification system to pass the fermentation broth obtained in step A through the separation filler, and then use the gradient elution of the elution buffer to collect the elution peak; equilibrate
  • the buffer is 25mM PB, pH7.0
  • the elution buffer is 25mM PB, 1.0M NaCl, pH7.0.
  • the Art v 1 protein peak collected in B is diluted with an equilibrating buffer, and the equilibrating buffer equilibrates the chromatographic column, and the diluted Art v 1 protein solution is applied to a hydrophobic chromatography filler to collect the elution peak;
  • the equilibration buffer is 1.0M (NH 4 ) 2 SO 4 , 25 mM PB, pH 7.0, and the elution buffer is 25 mM PB, pH 7.0.
  • the third step is to replace the target protein peak collected in C by ultrafiltration, and the buffer solution is 25mM PB, pH7.0; after filtration and sterilization, the Art v 1 protein stock solution is obtained.
  • the recombinant Art v 1 finally prepared by the present invention has reached the requirements of human recombinant DNA products in terms of purity, residual process impurities, molecular characterization, etc., the SEC-HPLC purity> 99%, and the expression level It can reach 210mg/L, and has the same amino acid sequence, disulfide bond and molecular weight as the natural protein.
  • the immunoreactivity of the specific antibody in the serum of allergic patients is equivalent to that of the natural protein, and has a good medicinal prospect.
  • recombinantly expressed allergen molecules have more advantages, such as avoiding differences in the main allergen content and active batches of natural pollen from different sources, more stable and controllable allergen process and quality, Avoid the degradation of major allergens and other sensitization reactions caused by the interaction of other components in natural pollen, and meet the needs of safe, effective and quality-controlled modern biological products; in addition, recombinant allergen proteins such as those used in allergy diagnostic kits To develop the allergen proteins that can accurately identify the allergens that trigger the body's allergies.
  • Figure 1 shows the sequence comparison of Art v 1 gene before and after optimization.
  • the unoptimized sequence corresponds to the natural Art v 1 gene nucleotide sequence; Art v 1-01 is the first optimized nucleotide sequence, and Art v 1-02 is the second optimized nucleotide sequence.
  • Figure 2-a, 2-b, and 2-c are the distribution area diagrams of the average GC base content of the Art v 1 gene in the Pichia pastoris expression system before and after codon optimization.
  • Figure 2-a shows that the average GC base content of Art v 1 natural gene nucleotide sequence in Pichia pastoris expression system is 50.71%;
  • Figure 2-b shows Art v 1-01 codon in Pichia pastoris expression system The average GC base content in Pichia pastoris expression system is 55.48%;
  • Figure 2-c shows that the average GC base content of Art v 1-02 codon in Pichia pastoris expression system is 56.31%.
  • Figure 3 is an agarose gel electrophoresis image of the PCR products of Art v 1-01 and Art v 1-02 genes (including their own signal peptide) after codon optimization.
  • lane 1 is 200bp DNA Ladder
  • lane 2 is the PCR product of Art v 1-01 gene
  • lane 3 is the PCR product of Art v 1-02 gene.
  • Figure 4 is an agarose gel electrophoresis image of the PCR products of Art v 1-01 and Art v 1-02 genes (without self-signal peptide) after codon optimization.
  • lane 1 is the PCR product of Art v 1-01 gene
  • lane 2 is the 200bp DNA Ladder
  • lane 3 is the PCR product of Art v 1-02 gene.
  • Fig. 5 is the expression identification map of Art v 1-01, 02 gene in 4 kinds of engineering bacteria in pPIC system.
  • Figure 5-a is the SDS-PAGE gel electrophoresis image of the supernatant of the pPICZ-Art v 1-01 engineering strain after 72 hours of expression.
  • lane 1 is the non-prestained protein marker in the range of 10-94KD;
  • lane 2-9 is the culture supernatant of each positive monoclonal host engineering strain of Art v 1-01 gene screened by Zeocin.
  • Figure 5-b is the SDS-PAGE gel electrophoresis image of the supernatant of the pPICZ ⁇ -Art v 1-02 engineering strain 72 hours after expression.
  • lane 1 is the non-prestained protein marker in the range of 10-94KD
  • lane 2-10 is the culture supernatant of each positive monoclonal host engineering strain of Art v 1-02 gene screened by Zeocin.
  • Fig. 6 is the expression identification figure of Art v 1-01,02 gene in 4 kinds of engineering bacteria in pGAP system.
  • Figure 6-a is the SDS-PAGE gel electrophoresis picture of the supernatant of the pGAPZ-Art v 1-01 engineering strain 48 hours after expression.
  • lane 1 is the non-prestained protein marker in the range of 10-94KD
  • lane 2-10 is the culture supernatant of Art v 1-01 gene monoclonal engineering strain screened by Zeocin.
  • Figure 6-b is the SDS-PAGE gel electrophoresis image of the supernatant of the bacterial liquid after the pGAPZ ⁇ -Art v 1-02 engineering strain was expressed for 48 hours.
  • lane 1 is the non-prestained protein marker in the range of 10-94KD
  • lane 2-10 is the culture supernatant of Art v 1-02 gene monoclonal engineering strain screened by Zeocin.
  • Figure 7 is the chromatogram and electrophoresis identification diagram of the first step of cationic chromatography purification of the recombinant Art v 1 fermentation broth.
  • Fig. 7-a is the first step cationic chromatography purification chromatogram of recombinant Art v 1 fermented liquid supernatant; There are three elution peaks.
  • Figure 7-b is the positive ion chromatography electrophoresis image of the first step of the supernatant of the recombinant Art v 1 fermentation broth; lane 1 is the 10-94KD non-prestained protein Marker; lane 2 is the sample before purification of the fermentation broth of Art v 1; lane 3 is the sample of Art v 1 fermentation broth v 1 fermentation broth first step cation chromatography breakthrough sample; lane 4 is the elution peak 1 of the first step cation chromatography purification of Art v 1 fermentation broth; swimming lane 5 is the first step cation chromatography purification wash of Art v 1 fermentation broth Off-peak 2; lane 6 is peak 3 eluted in the first step of cation chromatography purification of Art v 1 fermentation broth.
  • Fig. 8 is the second-step hydrophobic chromatography purification chromatogram and electrophoresis identification diagram of the recombinant Art v 1 protein;
  • Figure 8-a is the second-step hydrophobic chromatography purification chromatogram of the recombinant Art v 1 protein, with only one elution peak.
  • Figure 8-b is the second-step hydrophobic chromatography electrophoresis identification diagram of the recombinant Art v 1 protein; where lane 1 is the 10-94KD non-prestained protein Marker; lane 2 is the second-step hydrophobic chromatography purification and elution of the recombinant Art v 1 protein Peak 1.
  • Fig. 9 is the natural Art v 1 protein cation chromatography chromatogram and electrophoresis identification result
  • Fig. 9-a is the natural Art v 1 protein cation chromatography chromatogram, there are 3 elution peaks.
  • Figure 9-b is the cation chromatography electrophoresis image of natural Art v 1 protein; lane 1 is the 10-94KD non-prestained protein marker; lane 2 is the elution peak 1; lane 3 is the elution peak 2; and lane 4 is the elution peak 3.
  • Figure 10 shows the detection results of recombinant Art v 1 protein peptide coverage.
  • Figure 11 is the identification result of the disulfide bond of the Art v 1 protein;
  • Figure 11-a is the identification result of the disulfide bond of the recombinant Art v 1 protein, and
  • Figure 11-b is the identification result of the disulfide bond of the natural Art v 1 protein.
  • Figure 12 is the SEC-HPLC purity detection result of recombinant Art v 1 protein.
  • Figure 13 is the inhibition curve of recombinant Art v 1 protein and natural Art v 1 protein to IgE antibody in positive serum
  • Embodiment 1 Art v 1 gene codon optimization
  • GC content will affect the expression level of genes.
  • the ideal distribution area of GC content is 30%-70%. Any peak outside this area will affect transcription and translation efficiency to varying degrees. From the comparison of the average GC base content distribution area map of Art v 1 gene in Figure 2-a, Figure 2-b and Figure 2-c, it can be seen that the average GC base content of Art v 1 gene in Figure 2-a is 50.71%, The average GC base content of Art v 1-01 after optimization in Figure 2-b is 55.48%, and the average GC base content of Art v 1-02 after optimization in Figure 2-c is 56.31%; the average GC content after optimization It has been improved, Art v 1-01 and Art v 1-02 are not much different.
  • Embodiment 2 Art v 1 gene expression plasmid construction containing self signal peptide
  • the Art v 1-01, 02 gene after codon optimization in Example 1 was introduced into the EcoR I restriction site sequence at the 5' end, and the XhoI restriction site sequence was introduced at the 3' end, and the whole gene synthesis was carried out.
  • the synthetic gene fragments were constructed into the pPICZ plasmid (provided by Nanjing KingScript Technology Co., Ltd.) to obtain a long-term storage plasmid, which was respectively recorded as pPICZ-Art v 1-01 and pPICZ-Art v 1-01 according to different optimization methods. 02 plasmid.
  • the pPICZ-Art v 1-01 and pPICZ-Art v 1-02 plasmids were respectively used as templates for PCR amplification, and the primer sequences used were as follows:
  • the sequence of the upstream primer 5'AOX primer is shown in SEQ ID NO:5; the sequence of the downstream primer 3'AOX primer is shown in SEQ ID NO:6.
  • the total reaction volume was 50 ⁇ L, in which 2.5 ⁇ L was added to each primer with a concentration of 10 ⁇ mol/L, and 1 ⁇ L was added to dNTP with a concentration of 10 mmol/L.
  • the DNA polymerase used was Q5 (purchased from New England Biolabs), 2 U/ ⁇ L, and 0.5 ⁇ L was added.
  • the reaction conditions were 98°C for 5 seconds, 55°C for 45 seconds, and 72°C for 30 seconds. After 25 cycles, the product was analyzed by 1.0% agarose gel electrophoresis, and the results showed that the size of the product was consistent with the expected size (400bp) (the result is shown in Figure 3 shown).
  • T4 ligase (M0202S, purchased from New England Biolabs) was connected to the pGAPZ A plasmid (purchased from Invitrogen), and transformed into DH5 ⁇ competent cells (CB101, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), Mycin (purchased from Invitrogen) in LB solid medium and cultured overnight at 37°C. The next day, the positive clones were picked for sequencing and compared, and they were completely consistent with the expected sequence, that is, the expression plasmids after codon optimization of Art v 1 were obtained, which were denoted as pGAPZ-Art v 1-01 and pGAPZ-Art v 1-02.
  • Embodiment 3 Art v 1 gene expression plasmid construction containing yeast ⁇ -factor signal peptide
  • the upstream primer is SEQ ID NO: 7
  • the downstream primer is SEQ ID NO: 8.
  • the upstream primer is SEQ ID NO: 9
  • the downstream primer is SEQ ID NO: 10.
  • the total reaction volume was 50 ⁇ L, in which 2.5 ⁇ L was added to each primer with a concentration of 10 ⁇ mol/L, and 1 ⁇ L was added to dNTP with a concentration of 10 mmol/L.
  • the DNA polymerase used was Q5 (purchased from New England Biolabs), 2 U/ ⁇ L, and 0.5 ⁇ L was added.
  • the reaction conditions were 98°C for 5 seconds, 55°C for 45 seconds, and 72°C for 30 seconds. After 25 cycles, the product was analyzed by 1.0% agarose gel electrophoresis, and the results showed that the size of the product was consistent with the expected size (400bp) (the result is shown in Figure 4 shown).
  • T4 ligase (M0202S, purchased from New England Biolabs) was connected to the pPICZ ⁇ A plasmid (purchased from Invitrogen), and transformed into DH5 ⁇ competent cells (CB101, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), in the presence of Bleomyces Incubate overnight at 37°C in LB solid medium (purchased from Invitrogen). The next day, the positive clones were picked for sequencing and compared, and they were completely consistent with the expected sequence, that is, the expression plasmids after Art v 1 codon optimization were obtained, which were designated as pPICZ ⁇ -Art v 1-01 and pPICZ ⁇ -Art v 1-02.
  • T4 ligase (M0202S, purchased from New England Biolabs) was connected to the pGAPZ ⁇ A plasmid (purchased from Invitrogen), and transformed into DH5 ⁇ competent cells (CB101, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), Incubate overnight at 37°C in LB solid medium (purchased from Invitrogen). The next day, the positive clones were picked for sequencing and compared, and they were completely consistent with the expected sequence, that is, the expression plasmids after codon optimization of Art v 1 were obtained, which were denoted as pGAPZ ⁇ -Art v 1-01 and pGAPZ ⁇ -Art v 1-02.
  • Embodiment 4 Art v 1 expression plasmid transforms and the screening of engineering strain
  • YPDS+Zeocin-resistant solid medium preparation Invitrogen company Pichia expression vectors for constitutive expression and purification of recombinant proteins instructions provided, including yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, Sorbet Alcohol 182g/L, Zeocin final concentration 0.1mg/ml.
  • Electrocompetent cells were prepared according to the instructions of Invitrogen's Easy SelectPichia Expression Kit.
  • the plasmids pPICZ-Art v 1-01, pPICZ-Art v 1-02, pPICZ ⁇ -Art v 1-01 and pPICZ ⁇ -Art v 1-02 obtained in Step 1 of Example 2 and Step 1 of Example 3 were respectively treated with Sac I restriction endonuclease (purchased from New England Biolabs) was digested and linearized, and after ethanol precipitation, the linearized vector was electrotransformed into Pichia pastoris X33 competent cells, spread on YPDS solid medium, and cultured at 30°C until transformation The child grows.
  • Sac I restriction endonuclease purchased from New England Biolabs
  • the electroporation competent cells were prepared.
  • the plasmids pGAPZ-Art v 1-01, pGAPZ-Art v 1-02, pGAPZ ⁇ -Art v 1-01 and pGAPZ ⁇ -Art v 1-02 obtained in Example 2 step 2 and Example 3 step 2 were respectively used with Avr II restriction endonuclease (R0174S, purchased from New England Biolabs) was digested and linearized, and after ethanol precipitation, the linearized vector was electrotransformed into Pichia pastoris X33 competent cells, spread on YPDS solid medium, and cultured at 30°C until the transformants grow out.
  • Avr II restriction endonuclease R0174S, purchased from New England Biolabs
  • Embodiment 5 Induced expression and identification of Art v 1 genetic engineering strain
  • 5-a and b are the induced expression identification diagrams of pPICZ-Art v 1-01 and pPICZ ⁇ -Art v 1-02 plasmid engineering strains respectively; the expression identification diagrams of strains with other construction methods are not listed in full, and the expression levels are shown in Table 1.
  • Table 1 The results show , Art v 1 protein was expressed in engineering strains with different construction methods, and the pPICZ ⁇ -Art v 1-01 (no Ste 13 protease site between ⁇ -factor and the target gene) strain with the highest expression was 200mg/ L.
  • BMGY+zeocin medium preparation Invitrogen Company Easy SelectPichia Expression Kit instructions provided, including yeast extract 10g/L, peptone 20g/L, K 2 HPO 4 3g/L, KH 2 PO 4 11.8g/L, YNB 13.4g/L L, biotin 4 ⁇ 10 -4 g/L, glycerol 10g/L, Zeocin final concentration 0.1mg/ml.
  • BMMY+Zeocin medium preparation provided by Invitrogen’s Easy SelectPichia Expression Kit, in which yeast extract 10g/L, peptone 20g/L, K 2 HPO 4 3g/L, KH 2 PO 4 11.8g/L, YNB 13.4g/L L, biotin 4 ⁇ 10 -4 g/L, methanol 5 mL/L, Zeocin final concentration 0.1 mg/ml.
  • FIG. 6a and b are the induced expression identification diagrams of pGAPZ-Art v1-01 and pGAP ⁇ -Art v 1-02 plasmid engineering strains respectively; the expression identification diagrams of strains with other construction methods are not all listed The expression levels are shown in Table 1.
  • YPD+Zeocin resistance medium preparation Invitrogen company Pichia expression vectors for constitutive expression and purification of recombinant proteins instructions provided, including yeast extract 10g/L, peptone 20g/L, glucose 20g/L, Zeocin final concentration 0.1mg/ml .
  • the signal peptide and the target protein are separated by the Ste 13 signal cleavage cleavage sequence (the amino acid sequence is EAEA).
  • Embodiment 6 the purification of recombinant Art v 1 protein
  • the culture scale was expanded to 1 L by the culture method in Example 5, and a fermentation broth was prepared, and the sample was purified by ion exchange and hydrophobic chromatography.
  • the chromatography medium is selected as Hitrap SP HP, Hitrap Phenyl HP, and the specific steps are as follows:
  • Fermentation broth pretreatment The fermentation broth was centrifuged at low temperature and high speed to collect the supernatant, concentrated by ultrafiltration with 3KD membrane bag, replaced by ultrafiltration with 25mM PB buffer at pH7.0, and filtered with a 0.45 ⁇ m membrane filter.
  • Cation chromatography equilibrate the SP HP chromatography column with equilibrium buffer, then use the purification system to pass the ultrafiltered fermentation broth in the previous step through the separation filler, and then elute with the elution buffer to collect the elution peak;
  • the buffer is 25mM PB, pH7.0
  • the elution buffer is 25mM PB, 1.0M NaCl, pH7.0; as shown in Figure 7, the target protein is mainly in the elution peak 3.
  • Ultrafiltration replacement collect the target protein peak of hydrophobic chromatography, and replace the buffer solution with pH 7.0, 25mM PB.
  • the final yield of recombinantly expressed Art v 1 protein in the form of pGAPZ ⁇ -Art v 1-01 (with a Kex 2 protease cleavage site between the signal peptide and the target protein, without Ste 13 site) is 94.5mg/ L, the yield is 45%.
  • Embodiment 7 the purification of native Art v 1 protein
  • Ultrafiltration replacement combine the eluted peak 3 in step 2, concentrate the sample, replace the buffer with PBS solution with pH 7.4, and freeze it at -20°C until use.
  • Embodiment 8 LC-MS detection Art v 1 protein N amino acid sequence and molecular weight
  • LC-MS molecular weight can accurately reflect whether the primary sequence of biological macromolecules is correct, including whether the N and C terminal sequences are missing, whether there are post-translational modifications such as glycosylation, oxidation and deamidation, and is the most important analysis of biological macromolecules
  • the signal peptide and the target protein are separated by the Ste 13 signal cleavage cleavage sequence (the amino acid sequence is EAEA).
  • Embodiment 9 Art v 1 protein peptide mass map analysis
  • Peptide mass atlas is one of the most important identification methods in protein research. In theory, each protein has different peptides after digestion. The mass of these peptides is the peptide atlas of this protein. Then the measured amino acid sequence is compared with the known By comparing the sequences, we can know whether the amino acid primary structure of the analyzed protein is correct,
  • Embodiment 10 Art v 1 protein disulfide bond detection
  • Embodiment 11 Art v 1 protein purity HPLC detection
  • Electrophoretic purity identification of purified samples using Agilent 1260 HPLC, column Sepax Zenix SEC-80, mobile phase 20mM PB+300mM NaCl (pH7.0) buffer, flow rate 0.5ml/min, isocratic elution, column temperature 25.0 °C, 280nm to detect the purity of the sample by SEC-HPLC; the results in Figure 12 show that the SEC-HPLC purity of the recombinant Art v 1 protein obtained by the pGAPZ-Art v 1-01 construction method is 99.16%, and the purity reaches the pharmaceutical standard.
  • Embodiment: 12 Art v 1 protein activity detection
  • Sample preparation use blocking solution (2%BSA/PBST) to dilute the recombinant protein or natural protein to the initial concentration of 500ug/ml (S1), and then follow the 5-fold gradient dilution, a total of 10 gradients (S2-S11), The samples of each dilution were mixed 1:1 with the positive serum (diluted 30 times), and the mixed samples were incubated overnight at 4°C.
  • blocking solution 2%BSA/PBST
  • mice anti-human IgE-HRP secondary antibody diluted 1:1500 to each well, 100 ⁇ l per well, 300 rpm, and incubate at 37°C for 1 hour.

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Abstract

提供了重组艾蒿花粉Ⅰ型变应原（Art v1）及其编码基因和表达纯化方法。通过不同基因序列优化、不同分泌信号肽、表达载体和菌株的组合得到的Art v1蛋白表达量>200mg/L，纯度>99%，活性与天然蛋白相当，可用于蒿属花粉过敏性疾病的脱敏免疫治疗和过敏原诊断。

Description

重组艾蒿花粉I型变应原及其制备方法和应用 技术领域
本发明属于生物制药技术领域，涉及一种表征及活性与天然变应原一致的重组艾蒿花粉I型变应原及其制备方法和应用。
背景技术
花粉是季节性过敏的主要诱因之一，不同于食物过敏，花粉过敏通过空气传播，常常难以避免，可诱发鼻炎、皮炎和哮喘等一系列过敏反应，严重影响患者生活质量。在美国约7％的成人和9％的儿童受花粉过敏的困扰(NIAID，National Institute of Allergy and Infectious Diseases)，欧洲的患病率估计高达40％(G.D’Amato，2007)。近年来随着我国退耕还林和绿地面积不断增加，花粉过敏发病率逐年上升，高发病区可达5％。
蒿属(Genus Artemisia)植物花粉是引起夏秋季花粉症的重要变应原(又称过敏原)之一。国内一项2008-2010年215210次变应原特异性IgE检测结果显示，在吸入性变应原中，阳性率最高的为蒿花粉。杨琼梁等2015年研究结果表明中国北方地区最主要的致敏花粉为蒿属花粉。
蒿属(Artemisia L.)是菊科植物中物种数量最多的大属之一，全球约有300种以上的蒿属植物，广泛分布于北半球温带、温寒带及亚热带地区。黄花蒿和艾蒿是常见的花粉致敏植物，其中黄花蒿(学名Artemisia annus)可用于提取抗疟疾特效药青蒿素，是我国最常见的蒿属植物之一，也是我国研究最早的蒿属过敏植物。艾蒿(学名Artemisia vulgaris)又名北艾，广泛分布于中国、蒙古、俄罗斯、欧洲、美国、加拿大等地，是研究最深入的致敏花粉之一。其他主要的蒿属致敏植物还有大籽蒿(学名Artemisia sieversiana)、茵陈蒿(学名Artemisia capillaris)、野艾蒿(学名Artemisia lavabdykufikua)、沙蒿(学名Artemisia desertorum)、艾(学名Artemisia argyi)等。不同蒿属花粉的主要致敏蛋白均为I型和III型变应原，其中I型变应原属于防御素蛋白(Defensin-like protein)家族，分子量约12kD，不同蒿属植物的I型变应原蛋白序列保守；III型变应原属于非特异性脂质转运蛋白(nsLTP)，在不同蒿属植物花粉中变化度相对较高。
针对过敏性疾病，WHO提出“四位一体”疗法：避免接触变应原，对症药物治疗，特异性免疫治疗，医生及患者教育。其中，避免接触变应原是指在确定变应原的基础上，做好环境防控，尽量避免接触变应原。在过敏性疾病的治疗过程中很好地避免接触变应原不仅可以降低患者发生过敏的几率，还可提高药物疗效，帮助患者较快脱敏。特异性免疫治疗，即脱敏治疗，是唯一可能影响过敏性疾病自然进程，改变免疫应答机制的“对因”疗法，其通过对患者使用逐渐增加剂量的变应原，提高患者对该变应原的耐受性，减轻由暴露于该变应原而引发的症状，最终达到耐受乃至免疫耐受的目的。
2021年浙江我武用于治疗黄花蒿/艾蒿花粉过敏引起的变应性鼻炎的脱敏药物黄花蒿花粉变应原舌下滴剂获批上市，主要成分为黄花蒿(Artemisia annus)花粉变应原蛋白提取物，其专利CN101905022A指出“以蒿属花粉为原料，将花粉脱脂、浸提、浓缩，制得蒿属花粉变应原浸提液”。然而，天然变应原提取物由于原料来源及生产方法的限制不可避免存在质量方面的问题，比如存在未定义的非过敏性物质、污染物以及变应原含量和生物活性的高度变异性(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.)。欧洲过敏与临床免疫学会2018年发布的过敏性鼻炎变应原免疫治疗指南(EAACI Guidelines on allergen immunotherapy：allergic rhinoconjunctivitis(2018))也指出：混合变应原存在许多潜在缺点，包括稀释效应、由于某些变应原的酶活性导致的潜在变应原降解以及充分证明变应原组合的功效的困难。经EMA、HMA、FDA审批上市的标准化脱敏药物基本都限定了主要致敏蛋白，如治疗尘螨过敏的ODACTRA含有Der P1、Der P2、Der F1、Der F2四种主要致敏蛋白，治疗梯牧草过敏的GRAZAX含有主要致敏蛋白Phl P5，治疗豚草过敏的RAGWIZAX含有主要致敏蛋白Amb a1。另一方面，采用天然提取物进行变应原诊断存在灵敏度及特异度不高的问题，且无法明确患者对变应原组分的反应程度，易导致误诊。
目前还没有重组表达的蒿属花粉过敏原蛋白药物上市或开展临床试验。
发明内容
申请人希望提供一种主要致敏蛋白明确的重组艾蒿花粉变应原，以提升产品质量可控性，保证蒿属花粉过敏性疾病的脱敏免疫治疗药物的精准性和过敏原诊 断的准确性，为重组艾蒿花粉变应原的成药性奠定基础。
本发明的一个目的是提供一种用于治疗蒿属花粉过敏的蛋白，其为重组Art v 1蛋白。Art v 1是艾蒿的I型变应原蛋白，为主要致敏蛋白，由N端的防御素结构域和C端的富羟脯氨酸部分组成的糖蛋白，属于防御素蛋白(Defensin-like protein)家族。研究表明不同蒿属植物的I型变应原蛋白均属于防御素蛋白，且序列高度保守，各亚型与sIgE抗体的结合能力相似，其免疫活性主要由N端防御素结构域决定。所述重组Art v 1蛋白的氨基酸序列、二硫键及分子量与天然蛋白完全一致，具有与天然蛋白相似的生物学活性。
优选的，该Art v 1蛋白的氨基酸序列如SEQ ID NO：4所示。
本发明的重组Art v 1蛋白的氨基酸序列、分子量、氨基酸覆盖率和二硫键配对方式与天然Art v 1蛋白完全一致，具有与天然Art v 1相似的生物学活性，相比天然艾蒿花粉提取物，避免了不同来源的天然花粉主要变应原含量及活性批次间差异、工艺及质量更加稳定可控、还可避免天然花粉中其他成分相互作用对主要变应原的降解以及产生其他致敏反应等，满足现代生物制品的安全有效质量可控的要求，可用于过敏性鼻炎、哮喘等蒿属花粉过敏性疾病的治疗和诊断，提高蒿属花粉脱敏免疫治疗的精准性以及蒿属花粉变应原诊断的准确性，具有良好的药用前景。
本发明的另一个目的是提供一种编码Art v 1蛋白的DNA序列，其碱基序列如SEQ ID NO：13所示。该序列针对毕赤酵母表达系统进行了密码子优化，其更利于Art v 1在毕赤酵母中表达。
本发明的另一个目的是提供一种有利于毕赤酵母表达系统表达Art v 1蛋白的分泌信号肽设计形式，其不仅提高Art v 1蛋白表达量，而且获得的重组Art v 1蛋白与天然蛋白相比分子表征完全一致。所述信号肽为酵母α-factor信号肽、黑曲霉素信号肽、酸性磷酸酶信号肽(PHO)、酿酒酵母信号肽(SUC2)和Art v 1蛋白自身信号肽，优选的，所述信号肽为α-factor信号肽(SEQ ID NO：11)和自身信号肽(SEQ ID NO：12)。发明人发现，在毕赤酵母中使用不同分泌信号肽引导Art v 1分泌表达对于最终重组Art v 1蛋白分子的均一性和表达量有显著影响，优选的信号肽更利于Art v 1蛋白在毕赤酵母系统中正确高效地表达，产生的重组Art v 1不仅蛋白表达量提高，而且与天然Art v 1的一级结构、分子量、氨基酸覆盖率和二硫键配对方式完全一致。
本发明的另一个目的是提供一种含有上述编码优化后Art v 1基因的载体，优选的，所述的载体为pAO815，pPIC9，pPIC9K，pPIC3.5，pPIC3.5K，pPICZαA、B、C或pGAPZαA、B、C，更优选为pPICZ A或pGAPZ A。
本发明的另一个目的是提供一种包含上述所述载体的毕赤酵母菌株，优选的，所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H，更优选为KM71或X33菌株。
本发明的重组Art v 1蛋白编码基因更利于毕赤酵母表达，且发明人发现，以上不同分泌信号肽、表达载体和菌株的组合得到的工程菌株Art v 1蛋白表达量有较大差异，优选的组合方式得到的Art v 1克隆表达量最高可达210mg/L，经过纯化的重组Art v 1蛋白具有与天然蛋白完全一致的氨基酸序列、二硫键及分子量，在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
本发明的另一个目的是提供一种Art v 1蛋白的表达方法，所述方法包含下述步骤：
A.当使用常用的pPICZ系列或者pGAPZ系列作为载体时，外源基因表达框中不包含信号肽，因此可用于构建含有自身信号肽的Art v 1表达框。具体来说就是，将含有自身信号肽的Art v 1人工合成序列(设计含有起始密码子和终止密码子)克隆到对应载体的多克隆位点(例如EcoRI和NotI)，使得开放阅读框位于启动子下游和终止子上游，构建成重组表达载体。
当使用常用的pPICZα系列或者pGAPZα系列作为载体时，外源基因表达框中包含α-factor信号肽及信号肽切割位点：Kex2(氨基酸序列为KR)、Ste 13(氨基酸序列为EAEA)。可以将Art v 1目标基因克隆到Kex2序列后(该设计方式α-factor信号肽通过Kex2蛋白酶切除)，亦可以克隆到Ste 13序列后(该设计方式α-factor信号肽可Kex2和(或)Ste 13蛋白酶同时发挥作用切除)。以pPICZα A载体为例，若将Art v 1基因序列定位于Kex2后，可通过将目标基因克隆到XhoI和NotI位点之间实现；若将Art v 1基因序列定位于Ste 13后，可通过将目标基因克隆到EcoRI和NotI位点之间实现。通过举例操作方式构建使用α-factor信号肽分泌表达Art v 1的重组表达载体。
B.将步骤A的载体线性化后转入毕赤酵母菌株中，通过抗性压力筛选方法筛选获得表达量高的克隆，并在合适的条件下培养进一步验证表达量或者开展下游纯化；
C.回收纯化蛋白质。
上述所述载体优选为pPICZαA或pGAPZαA。
上述所述毕赤酵母菌株优选为KM71或X33菌株。
本发明的另一个目的是提供一种重组Art v 1蛋白纯化方法，所述纯化方法如下：
A.将Art v 1发酵液低温高速离心收集上清，3KD膜包超滤浓缩，25mM PB，pH7.0缓冲液置换，0.45μm滤膜过滤。
B.第一步阳离子层析，用平衡缓冲液平衡层析柱，接着运用纯化系统将步骤A中得到的发酵液通过分离填料，然后运用洗脱缓冲液梯度洗脱，收集洗脱峰；平衡缓冲液为25mM PB，pH7.0，洗脱缓冲液为25mM PB，1.0M NaCl，pH7.0。
C.第二步将B中收集得到的Art v 1蛋白峰用平衡缓冲液稀释，平衡缓冲液平衡层析柱，将稀释后的Art v 1蛋白溶液上疏水层析填料，收集洗脱峰；平衡缓冲液为1.0M(NH 4) 2SO 4，25mM PB，pH7.0，洗脱缓冲液为25mM PB，pH7.0。
D.第三步将C中收集到的目的蛋白峰超滤置换，缓冲液为25mM PB，pH7.0；过滤除菌后即得到Art v 1蛋白原液。
经过优化发酵培养工艺和纯化方法，本发明最终制备的重组Art v 1在纯度、工艺杂质残留、分子表征等多个方面达到了人用重组DNA制品要求，SEC-HPLC纯度>99％，表达量达到210mg/L，且具有与天然蛋白完全一致的氨基酸序列、二硫键及分子量，在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当，具有良好的药用前景。相比天然提取的过敏原产品，重组表达的过敏原分子具有较多优势，如避免不同来源的天然花粉主要变应原含量及活性批次间差异、变应原工艺及质量更加稳定可控、避免天然花粉中其他成分相互作用对主要变应原的降解以及产生其他致敏反应等，满足现代生物制品的安全有效质量可控的需求；此外重组变应原蛋白如用于过敏诊断试剂盒的开发可准确鉴别引发机体过敏的变应原蛋白。
附图说明
图1表示优化前后Art v 1基因序列对比图。
其中未优化序列对应的为天然Art v 1基因核苷酸序列；Art v 1-01为第一种优化的核苷酸序列，Art v 1-02为第二种优化的核苷酸序列。
图2-a，2-b，2-c为密码子优化前后Art v 1基因在毕赤酵母表达系统中平均GC碱基含量分布区域图。
其中，图2-a表示Art v 1天然基因核苷酸序列在毕赤酵母表达系统中平均GC碱基含量为50.71％；图2-b表示Art v 1-01密码子在毕赤酵母表达系统中平均GC碱基含量为55.48％；图2-c表示Art v 1-02密码子在毕赤酵母表达系统中平均GC碱基含量为56.31％。
图3为密码子优化后Art v 1-01和Art v 1-02基因(含自身信号肽)PCR产物的琼脂糖凝胶电泳图。
其中，泳道1为200bp DNA Ladder；泳道2为Art v 1-01基因PCR产物；泳道3为Art v 1-02基因PCR产物。
图4为密码子优化后Art v 1-01和Art v 1-02基因(无自身信号肽)PCR产物的琼脂糖凝胶电泳图。
其中，泳道1为Art v 1-01基因PCR产物；泳道2为200bp DNA Ladder；泳道3为Art v 1-02基因PCR产物。
图5为Art v 1-01，02基因在pPIC体系中4种工程菌中的表达鉴定图。
其中，图5-a为pPICZ-Art v 1-01工程菌株表达72小时后，菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker；泳道2-9为通过Zeocin筛选出来的Art v 1-01基因各阳性单克隆宿主工程菌株培养菌液上清。
图5-b为pPICZα-Art v 1-02工程菌株表达72小时后，菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker；泳道2-10为通过Zeocin筛选出来的Art v 1-02基因各阳性单克隆宿主工程菌株培养菌液上清。
图6为Art v 1-01,02基因在pGAP体系中4种工程菌中的表达鉴定图。
图6-a为pGAPZ-Art v 1-01工程菌株表达48小时后，菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker；泳道2-10为通过Zeocin筛选出来的Art v 1-01基因单克隆工程菌株培养菌液上清。
其中，图6-b为pGAPZα-Art v 1-02工程菌株表达48小时后，菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker；泳道2-10为通过Zeocin筛选出来的Art v 1-02基因单克隆工程菌株培养菌液上清。
图7为重组Art v 1发酵液第一步阳离子层析纯化色谱图及电泳鉴定图。
其中图7-a为重组Art v 1发酵液上清第一步阳离子层析纯化色谱图；有三个 洗脱峰。
图7-b为重组Art v 1发酵液上清第一步阳离子层析电泳图；泳道1为10-94KD非预染蛋白Marker；泳道2为Art v 1发酵液纯化前样品；泳道3为Art v 1发酵液第一步阳离子层析穿透样品；泳道4为Art v 1发酵液第一步阳离子层析纯化洗脱峰1；泳道5为Art v 1发酵液第一步阳离子层析纯化洗脱峰2；泳道6为Art v 1发酵液第一步阳离子层析纯化洗脱峰3。
图8为重组Art v 1蛋白第二步疏水层析纯化色谱图及电泳鉴定图；
其中图8-a为重组Art v 1蛋白第二步疏水层析纯化色谱图，仅有一个洗脱峰。
图8-b为重组Art v 1蛋白第二步疏水层析电泳鉴定图；其中泳道1为10-94KD非预染蛋白Marker；泳道2为重组Art v 1蛋白第二步疏水层析纯化洗脱峰1。
图9为天然Art v 1蛋白阳离子层析色谱图及电泳鉴定结果；
其中图9-a为天然Art v 1蛋白阳离子层析色谱图，有3个洗脱峰。
图9-b为天然Art v 1蛋白阳离子层析电泳图；泳道1为10-94KD非预染蛋白Marker；泳道2为洗脱峰1；泳道3为洗脱峰2；泳道4为洗脱峰3。
图10为重组Art v 1蛋白肽段覆盖率检测结果。
图11为Art v 1蛋白二硫键鉴定结果；其中图11-a为重组Art v 1蛋白二硫键鉴定结果，图11-b为天然Art v 1蛋白二硫键鉴定结果。
图12为重组Art v 1蛋白SEC-HPLC纯度检测结果。
图13为重组Art v 1蛋白与天然Art v 1蛋白对阳性血清中IgE抗体的抑制曲线
具体实施方式
下面结合具体实施例，进一步阐述本发明，应理解，引用实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1：Art v 1基因密码子优化
发明人根据NCBI已公开的Art v 1的DNA序列(Genbank登录号：AF493943，含自身信号肽)，如SEQ ID NO：1所示，对该基因进行密码子优化后得到含有自身信号的两条基因序列：Art v 1-01和Art v 1-02，核苷酸序列分别如SEQ ID NO：2和SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示。密码子优化前后碱基序列对比如图1所示。
GC含量会影响基因的表达水平，GC含量理想分布区域为30％-70％，在这个 区域外的出现任何峰都会不同程度地影响转录和翻译效率。由图2-a、图2-b和图2-c的Art v 1基因的GC碱基平均含量分布区域图对比可知，图2-a中Art v 1基因GC碱基平均含量为50.71％，图2-b中优化后Art v 1-01的GC碱基平均含量为55.48％，图2-c中优化后Art v 1-02的GC碱基平均含量为56.31％；经过优化后GC平均含量得到了提高，Art v 1-01、Art v 1-02相差不大。
实施例2：含自身信号肽的Art v 1基因表达质粒构建
1、构建至pPIC表达质粒系统
将实施例1中密码子优化后的Art v 1-01、02基因在5’端引入EcoR I酶切位点序列，在3’端引入XhoI酶切位点序列，并进行全基因合成，将合成的基因片段，构建到pPICZ质粒(由南京金斯瑞科技有限公司提供)中，得到一种长期保存质粒，分别根据不同优化方式记为pPICZ-Art v 1-01和pPICZ-Art v 1-02质粒。
2、构建至pGAP表达质粒系统
分别以pPICZ-Art v 1-01和pPICZ-Art v 1-02质粒为模板，进行PCR扩增，所用引物序列如下：
上游引物5’AOX primer，序列如SEQ ID NO：5所示；下游引物3’AOX primer，序列如SEQ ID NO：6所示。
反应总体积50μL，其中浓度为10μmol/L引物各加2.5μL，浓度为10mmol/L的dNTP加1μL，所用DNA聚合酶为Q5(购自New England Biolabs公司)，2U/μL，加0.5μL。反应条件为98℃5秒、55℃45秒、72℃30秒，25个循环后，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(400bp)一致(结果如图3所示)。分别用Xho I(R0146S，购自New England Biolabs公司)和EcoR I(R3101S，购自New England Biolabs公司)双酶切后，1％琼脂糖电泳，得到的基因产物用DNA凝胶回收试剂盒(DP214，北京天根生化科技有限公司)纯化。
T4连接酶(M0202S，购自New England Biolabs)连接到pGAPZ A质粒(购自Invitrogen公司)中，转化到DH5α感受态细胞(CB101，购自北京天根生化科技有限公司)中，在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序，比对，与预期序列完全一致，即得到Art v 1密码子优化后的表达质粒，记为pGAPZ-Art v 1-01和pGAPZ-Art v 1-02。
实施例3：含酵母α-factor信号肽的Art v 1基因表达质粒构建
以pPICZ-Art v 1-01质粒为模板，进行PCR扩增，得到无信号肽的Art v 1-01基因序列，如SEQ ID NO：13所示。所用引物序列：上游引物为SEQ ID NO：7；下游引物为SEQ ID NO：8。
以pPICZ-Art v 1-02质粒为模板，进行PCR扩增，得到无信号肽的Art v 1-02基因序列，如SEQ ID NO：14所示。所用引物序列：上游引物为SEQ ID NO：9；下游引物为SEQ ID NO：10。
反应总体积50μL，其中浓度为10μmol/L引物各加2.5μL，浓度为10mmol/L的dNTP加1μL，所用DNA聚合酶为Q5(购自New England Biolabs公司)，2U/μL，加0.5μL。反应条件为98℃5秒、55℃45秒、72℃30秒，25个循环后，产物经1.0％琼脂糖凝胶电泳分析，结果显示产物大小与预期大小(400bp)一致(结果如图4所示)。分别用Xho I(R0146S，购自New England Biolabs公司)和Xba I(R01445S，购自New England Biolabs公司)双酶切后，1％琼脂糖电泳，得到的基因产物用DNA凝胶回收试剂盒(DP214，北京天根生化科技有限公司)纯化。
1、构建至pPICZα表达质粒系统
T4连接酶(M0202S，购自New England Biolabs)连接到pPICZαA质粒(购自Invitrogen公司)中，转化到DH5α感受态细胞(CB101，购自北京天根生化科技有限公司)中，在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序，比对，与预期序列完全一致，即得到Art v 1密码子优化后的表达质粒，记为pPICZα-Art v 1-01和pPICZα-Art v 1-02。
2、构建至pGAPZα表达质粒系统
T4连接酶(M0202S，购自New England Biolabs)连接到pGAPZαA质粒(购自Invitrogen公司)中，转化到DH5α感受态细胞(CB101，购自北京天根生化科技有限公司)中，在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序，比对，与预期序列完全一致，即得到Art v 1密码子优化后的表达质粒，记为pGAPZα-Art v 1-01和pGAPZα-Art v 1-02。
实施例4：Art v 1表达质粒转化和工程菌株的筛选
YPDS+Zeocin抗性固体培养基配制：Invitrogen公司Pichia expression vectors for constitutive expression and purification of recombinant proteins说明书提供，其中 酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L，山梨醇182g/L，Zeocin终浓度0.1mg/ml。
1、pPIC体系表达质粒转化和工程菌株筛选
按照Invitrogen公司Easy SelectPichia Expression Kit说明书的方法制备成电感受态细胞。将实施例2步骤1和实施例3步骤1得到的质粒pPICZ-Art v 1-01、pPICZ-Art v 1-02、pPICZα-Art v 1-01和pPICZα-Art v 1-02，分别用Sac I限制性内切酶(购自New England Biolabs)酶切线性化，乙醇沉淀后将线性化载体，电转化毕赤酵母X33感受态细胞中，涂布于YPDS固体培养基，30℃培养直到转化子长出。
2、pGAP体系表达质粒转化和工程菌株筛选
按照Pichia expression vectors for constitutive expression and purification of recombinant proteins说明书的方法制备成电转化感受态细胞。将实施例2步骤2和实施例3步骤2得到的质粒pGAPZ-Art v 1-01、pGAPZ-Art v 1-02、pGAPZα-Art v 1-01和pGAPZα-Art v 1-02，分别用Avr II限制性内切酶(R0174S，购自New England Biolabs)酶切线性化，乙醇沉淀后将线性化载体，电转化毕赤酵母X33感受态细胞中，涂布于YPDS固体培养基，30℃培养直到转化子长出。
实施例5：Art v 1基因工程菌株诱导表达及鉴定
1、pPIC体系克隆筛选鉴定
挑取实施例4步骤1中获得的宿主单克隆工程菌于5mL BMGY培养基中，于50mL无菌离心管中30℃，至OD600＝1.0-2.0时，将菌液4000rpm离心10分钟，用BMMY培养基重悬后诱导表达，每隔24h补加甲醇至终浓度为1％，220rpm培养72小时，离心收集菌液上清，通过SDS-PAGE凝胶电泳分析，观察表达产物条带亮度，图5-a、b分别为pPICZ-Art v 1-01和pPICZα-Art v 1-02质粒工程菌株诱导表达鉴定图；其余构建方式菌株表达鉴定图未全部列出，表达量见表1，结果显示，Art v 1蛋白在不同构建方式的工程菌株中均得到了表达，其中表达量最高的pPICZα-Art v 1-01(α-factor与目标基因之间无Ste 13蛋白酶位点)菌株为200mg/L。
BMGY+zeocin培养基配制：Invitrogen公司Easy SelectPichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K 2HPO 4 3g/L，KH 2PO 4 11.8g/L，YNB 13.4g/L，生物素4×10 -4g/L，甘油10g/L，Zeocin终浓度0.1mg/ml。
BMMY+Zeocin培养基配制：Invitrogen公司Easy SelectPichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K 2HPO 4 3g/L，KH 2PO 4 11.8g/L，YNB13.4g/L，生物素4×10 -4g/L，甲醇5mL/L，Zeocin终浓度0.1mg/ml。
2、pGAP体系克隆筛选鉴定
挑取实施例4步骤2中获得的宿主单克隆工程菌于5mL YPD培养基中，于50mL无菌离心管中30℃，220rpm培养48小时，离心收集菌液上清，通过SDS-PAGE凝胶电泳分析，观察表达产物条带亮度，图6-a、b分别为pGAPZ-Art v1-01和pGAPα-Art v 1-02质粒工程菌株诱导表达鉴定图；其余构建方式菌株表达鉴定图未全部列出，表达量见表1，结果显示，Art v 1蛋白在不同构建方式的工程菌株中均得到了表达，其中表达量最高的pGAPZα-Art v 1-01菌株为210mg/L，最低的pGAPZα-Art v 1-02菌株表达量仅30mg/L。
YPD+Zeocin抗性培养基配制：Invitrogen公司Pichia expression vectors for constitutive expression and purification of recombinant proteins说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，Zeocin终浓度0.1mg/ml。
表1 不同构建方式表达的重组Art v 1蛋白表达量
Figure PCTCN2022144040-appb-000001
Figure PCTCN2022144040-appb-000002
注：
①信号肽与目标蛋白之间被Ste 13signal cleavage切割序列(氨基酸序列为EAEA)隔开。
②信号肽与目标蛋白之间含有一个Kex 2蛋白酶酶切位点，无Ste 13位点，无其他序列。
实施例6：重组Art v 1蛋白的纯化
利用实施例5中筛选到的表达克隆，利用实施例5中培养方法扩大培养规模至1L，制备得到发酵液，采用离子交换和疏水层析进行样品纯化。层析填料选择为Hitrap SP HP，Hitrap Phenyl HP，具体步骤如下：
1.发酵液预处理：将发酵液低温高速离心收集上清，3KD膜包超滤浓缩，25mM pH7.0的PB缓冲液超滤置换，0.45μm滤膜过滤。
2.阳离子层析：用平衡缓冲液平衡SP HP层析柱，接着用纯化系统将上一步中已超滤的发酵液通过分离填料，然后用洗脱缓冲液洗脱，收集洗脱峰；平衡缓冲液为25mM PB，pH7.0，洗脱缓冲液为25mM PB，1.0M NaCl，pH7.0；如图7所示，目的蛋白主要在洗脱峰3。
3.疏水层析：将上一步中收集得到的Art v 1蛋白峰用平衡缓冲液稀释，将Art v 1蛋白溶液上phenyl HP疏水层析填料，平衡缓冲液为1.0M(NH 4) 2SO 4，25mM PB，pH7.0，洗脱缓冲液为25mM PB，pH7.0，收集洗脱峰；如图8所示，仅有一个洗脱峰，目的蛋白在洗脱峰中。
4.超滤置换：收集疏水层析目的蛋白峰，将缓冲液置换为pH7.0，25mM PB。
通过以上纯化步骤，pGAPZα-Art v 1-01(信号肽与目标蛋白之间含有一个Kex 2蛋白酶酶切位点，无Ste 13位点)形式重组表达的Art v 1蛋白最终产量为94.5mg/L，收率为45％。
实施例7：天然Art v 1蛋白的纯化
1.粗提液制备：称取已脱脂艾蒿花粉(购自Stallergenes Greer)，配制pH7.0，50mM PB溶液，按照w/v＝1:10加入PB溶液，低温下提取48-72小时；低温 4000rpm离心收集上清即得到粗提液。
2.层析纯化：将步骤2中收集得到的粗提液上SP FF阳离子层析填料，平衡缓冲液为25mM PB，pH7.0，洗脱缓冲液为25mM PB，1.0M NaCl，pH7.0，收集洗脱峰电泳鉴定；如图9所示，天然Art v 1蛋白主要在洗脱峰3中。
3.超滤置换：合并步骤2中洗脱峰3，将样品浓缩，缓冲液置换为pH7.4 PBS溶液，冻存于-20℃以下待用。
实施例8：LC-MS检测Art v 1蛋白N氨基酸序列和分子量
LC-MS分子量可以精确反映生物大分子一级序列是否正确，包括N、C端序列是否缺失，是否存在糖基化、氧化和脱酰胺等翻译后修饰，是生物大分子最重要的一种分析手段之一；通过对纯化后的不同构建方式的重组Art v 1蛋白进行LC-MS分子量进行分析，结果如表2所示，使用α-factor信号肽进行分泌表达时，当信号肽与目标基因之间有Ste 13蛋白酶酶切位点间隔时，该位点对应的氨基酸序列不能完全切除，产生的目标蛋白N端有该位点氨基酸的残留。除此之外的构建形式产生的Art v 1蛋白的N端序列与理论完全一致，无残留。。
表2.不同构建方式表达纯化的重组Art v 1蛋白LC-MS分子量
Figure PCTCN2022144040-appb-000003
Figure PCTCN2022144040-appb-000004
注：
①信号肽与目标蛋白之间被Ste 13signal cleavage切割序列(氨基酸序列为EAEA)隔开。
②信号肽与目标蛋白之间含有一个Kex 2蛋白酶酶切位点，无Ste 13位点，无其他序列。
实施例9：Art v 1蛋白肽质量图谱分析
肽质量图谱是蛋白质研究中最重要的鉴定手段之一，理论上每个蛋白消化后都有不同肽段，这些肽段的质量就是这个蛋白的肽图谱，然后将测得的氨基酸序列与已知序列进行比对，就可以知道分析的蛋白的氨基酸一级结构是否正确，
将实施例8中不同构建方式菌株表达的Art v 1纯品进行肽段分析，结果显示，除了α-factor与目标基因存在Ste 13酶切位点间隔的构建形式以外的其他设计构建形式所得的重组Art v 1蛋白与理论序列的覆盖率均为100％，说明我们构建和重组表达的Art v 1蛋白一级结构正确，图10所示为pGAPZ-Art v 1-01重组表达的Art v 1蛋白肽段覆盖率检测结果。
实施例10：Art v 1蛋白二硫键检测
二硫键是否能正确配对对蛋白质等生物大分子的高级结构和活性的保持至关重要；利用本公司Thermo Scientific Q Exactive LC-MS系统对经pGAPZα-Art v1-01构建方式得到的重组Art v 1蛋白和天然Art v 1蛋白进行了二硫键确定，结果如图11显示，其中图11-a为利用trypsin和chymotrypsin双酶切，鉴定到重组Art v 1蛋白C6/C53、C22/C47、C26/C49、C17/C37四对理论二硫键；图11-b为利用trypsin和chymotrypsin双酶切，鉴定到天然Art v 1蛋白C6/C53、C22/C47、C26/C49、C17/C37四对理论二硫键。
实施例11：Art v 1蛋白纯度HPLC检测
纯化后样品电泳纯度鉴定：采用Agilient 1260型HPLC，色谱柱Sepax Zenix SEC-80，流动相20mM PB+300mM NaCl(pH7.0)缓冲液，流速0.5ml/min，等度洗脱，柱温25.0℃，280nm检测样品SEC-HPLC纯度；图12结果显示经pGAPZ-Art v 1-01构建方式得到的重组Art v 1蛋白SEC-HPLC纯度为99.16％，纯度达到药用 标准。
实施例:12：Art v 1蛋白活性检测
1.将经pGAPZ-Art v 1-01构建方式得到的重组Art v 1蛋白和实施例7纯化制备的天然Art v 1蛋白分别用50mM NaH 2PO 4缓冲液pH7.2稀释至0.5μg/ml，100μl每孔，4℃包被过夜。
2.样品制备：使用封闭液(2％BSA/PBST)将重组蛋白或天然蛋白稀释到起始浓度500ug/ml(S1)，然后按照5倍梯度稀释，共10个梯度(S2-S11)，各稀释度样品与阳性血清(稀释30倍)进行1：1混合，混合后样品放入4℃孵育过夜。
3.次日取出ELISA板，PBST洗涤3次，每孔加200μl 2％BSA/PBST溶液，37℃封闭2h。
4.封闭结束后弃去封闭液，上述混合孵育好的样品按100ul每孔加入，300rpm，37℃孵育1.5h。
5.PBST洗涤3次，每孔加入1:1500稀释的鼠抗人IgE-HRP二抗，100μl每孔，300rpm，37℃孵育1h。
6.PBST洗涤3次，每孔加入100μl TMB II号显色液，37℃反应10min后每孔加入50μl终止液(2M H 2SO 4)，OD 450nm立即检测。
7.结果分析：如图13所示，重组Art v 1与天然Art v 1蛋白相比，对阳性血清中IgE抗体的抑制曲线基本一致，以天然Art v 1蛋白为标准品，重组Art v 1相对活性为118.7％，说明重组蛋白在体外与过敏患者血清中IgE特异性抗体的免疫反应活性与天然蛋白相当。

Claims (9)

  1. 一种用于治疗蒿属花粉过敏的蛋白，其为重组艾蒿花粉I型变应原蛋白，所述重组艾蒿花粉I型变应原蛋白的氨基酸序列、二硫键及分子量与天然蛋白一致，在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
  2. 权利要求1所述的用于治疗蒿属花粉过敏的蛋白，氨基酸序列如SEQ ID NO:4所示。
  3. 编码权利要求2所述的用于治疗蒿属花粉过敏的蛋白的核苷酸，碱基序列如SEQ ID NO:13所示。
  4. 含有权利要求3所述的用于治疗蒿属花粉过敏蛋白的核苷酸的载体，所述的载体为pAO815，pPIC9，pPIC9K，pPIC3.5，pPIC3.5K，pPICZαA、B、C，pGAPZαA、B、C，pPICZ A、B、C或pGAPZ A、B、C。
  5. 包含权利要求4所述载体的毕赤酵母菌株，所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H。
  6. 权利要求1或2所述治疗蒿属花粉过敏的蛋白的表达方法，所述方法包含下述步骤：
    A.构建含有权利要求4所述编码Art v1基因的载体：
    使用自身信号肽分泌表达时：将含有自身信号肽的Art v 1基因序列(设计含有起始密码子和终止密码子)克隆到权利要求4所述载体的启动子下游和终止子上游，构建形成表达框；
    使用载体中α-factor信号肽表达时：将Art v 1基因序列(不含有起始密码子，设计含有终止密码子)克隆到权利要求4所述载体的信号肽切割位点Kex2(氨基酸序列为KR)序列后，信号肽与目标蛋白之间含有一个Kex 2蛋白酶酶切位点，无Ste 13(氨基酸序列为EAEA)位点，无其他序列；
    B.将步骤A的载体线性化后转入毕赤酵母菌株中，并在合适的条件下培养；
    C.回收纯化蛋白质。
  7. 权利要求1或2所述治疗蒿属花粉过敏的蛋白的纯化方法，所述纯化方法如下：
    A.将Art v1发酵液低温高速离心收集上清，3KD膜包超滤浓缩，25mM PB，pH7.0缓冲液置换，0.45μm滤膜过滤；
    B.阳离子层析：用平衡缓冲液平衡层析柱，接着运用纯化系统将步骤A中得到的发酵液通过分离填料，然后运用洗脱缓冲液梯度洗脱，收集洗脱峰，平衡 缓冲液为25mM PB，pH7.0，洗脱缓冲液为25mM PB，1.0M NaCl，pH7.0；
    C.疏水层析：将B中收集得到的Art v1蛋白峰用平衡缓冲液稀释，平衡缓冲液平衡层析柱，将稀释后的Art v1蛋白溶液上疏水层析填料，收集洗脱峰，平衡缓冲液为1.0M(NH 4) 2SO 4，25mM PB，pH7.0，洗脱缓冲液为25mM PB，pH7.0；
    D.超滤置换：将C中收集到的目的蛋白峰超滤置换，缓冲液为25mM PB，pH7.0，过滤除菌后即得到Art v1蛋白原液。
  8. 权利要求1或2的蛋白在制备治疗蒿属花粉过敏性疾病的药物中的应用。
  9. 权利要求1或2的蛋白在制备检测蒿属花粉过敏原的诊断试剂中的应用。
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