WO2019242662A1 - 重组尘螨变应原蛋白药物合剂及其应用 - Google Patents

重组尘螨变应原蛋白药物合剂及其应用 Download PDF

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WO2019242662A1
WO2019242662A1 PCT/CN2019/091968 CN2019091968W WO2019242662A1 WO 2019242662 A1 WO2019242662 A1 WO 2019242662A1 CN 2019091968 W CN2019091968 W CN 2019091968W WO 2019242662 A1 WO2019242662 A1 WO 2019242662A1
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protein
recombinant
dust mite
allergen
fermentation
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French (fr)
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马永
范宇
吴诚
赵利利
江辰阳
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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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Priority to EP19821864.6A priority Critical patent/EP3831401A4/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to a medicine combination for treating dust mite allergy, in particular to an improved recombinant dust mite allergen protein medicine mixture and application thereof.
  • dust mites are the most important allergens.
  • the positive rate of dust mites in patients with allergic diseases is about 70-80%.
  • dust mites There are many types of dust mites, which are widely present in human living and working environments. Their excreta, metabolites and mites all have strong allergic prototypes. According to statistics, about 10% of the world ’s population is allergic to dust mites, and about 80% are outside. Originating asthma is caused by dust mites.
  • dust mite allergen extracts are used clinically to treat dust mite allergies.
  • the "Changdi" dust mite drops of Zhejiang Wowu Biological which was launched in 2006, are extracts of dust mite metabolism cultures; 2015 Actair House Dust Mite Sublingual Tablets listed in Japan are used to treat allergic rhinitis caused by House Dust Mites (or House Dust Mites).
  • Natural I and II House Dust Mites and Type I and II Dust Mites are also used.
  • Odactra which was approved by the FDA in 2017, also uses four components of natural dust mites of type I and II and dust mites of type I and II. It can be seen that the use of mixtures has become a trend for dust mite desensitization .
  • the composition of the natural allergen extract is very complex, it is very difficult to constant its composition, and it is easily contaminated by exogenous toxic substances and pathogenic microorganisms, which affects its repeatability and safety. It may easily lead to long-term use in the treatment process. Local reactions such as flushing, swelling, induration, and necrosis, and systemic reactions such as shock, edema, bronchospasm, urticaria, angioedema, and systemic erythema.
  • the use of crude extracts for diagnosis does not make it clear that patients are allergic to each group. The degree of response is likely to cause misdiagnosis. The quality of allergens is important for the diagnosis and treatment of allergic diseases.
  • Allergens used for immunological diagnosis and treatment should be pure products, not crude extracts, and dust mite allergens mainly exist in feces and mites.
  • the extraction method takes a long time, the process is tedious, and the cost is high, and side reactions in the immunotherapy cannot be avoided.
  • the object of the present invention is to provide a recombinant dust mite allergen protein drug mixture, and to optimize a recombinant dust mite allergen protein drug mixture to obtain a drug mixture with the best activity.
  • the present invention provides a recombinant dust mite allergen protein drug mixture, which comprises a recombinant dust mite type I allergen DP1, DF1 protein and a recombinant dust mite type II allergen DP2, DF2 protein, and four kinds of dust Mite allergen proteins DP1: DP2: DF1: DF2 are respectively mixed at an active ratio of 1: 1: 1 or at a mass ratio of (1 to 5): (1 to 20): (1 to 10): (1 to 20) )mixing.
  • the four dust mite allergen proteins DP1: DP2: DF1: DF2 in the recombinant dust mite allergen protein drug mixture are respectively mixed at a mass ratio of 1: (1-20): 1: (1-20) .
  • the four dust mite allergen proteins in the recombinant dust mite allergen protein drug mixture are mixed at a mass ratio of 1: 5: 1: 5.
  • the recombinant dust mite type I allergen DP1, DF1 protein and the recombinant dust mite type II allergen DP2, DF2 protein are respectively composed of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
  • the gene represented by SEQ ID NO: 4 was expressed by the Pichia pastoris expression system.
  • the recombinant dust mite type I allergen protein is prepared by the following method:
  • the main fermentation process when expressing the recombinant dust mite type I allergen DP1, DF1 protein is as follows: the fermentation medium used is 40% BSM medium, and the induction temperature at the induction stage of high-density fermentation is : 25 ⁇ 27 °C, pH value is 6.5 ⁇ 0.2, the acceleration of methanol flow during the induction stage of high-density fermentation is 30mL / h -1 L -1 , and the DO is not higher than 40%.
  • PTM1 (2.4ml / L) is added; the rate-limiting growth stage of high-density fermentation begins to be supplemented at a rate of 30ml / h -1 L -1 50% glycerol, the post-feeding speed is adjusted to 60ml / h -1 L -1 .
  • the recombinant dust mite type I allergen DP1, DF1 protein is expressed, a three-step purification process after high-density fermentation, wherein the purification filler of cation exchange chromatography is SPFF; the preferred pH range is 4.0-6.5. More preferably, the pH of the sample and buffer in the cation exchange layer system is 5.0.
  • the purification packing for anion exchange chromatography is QFF; the preferred pH range is 6.0-8.0; the preferred conductivity is 2.0mS / cm-20.0mS / cm. More preferably, the pH of the sample and buffer in anion exchange chromatography is 8.0, and the conductivity is 20.0 mS / cm.
  • the purification packing for hydrophobic chromatography is phenylFF; the preferred pH range is 6.5-8.5; the preferred ammonium sulfate concentration is 1.0-2.0M. More preferably, the concentration of the sample and the equilibration buffer ammonium sulfate in the hydrophobic chromatography is 2.0M, and the pH is 8.5.
  • the main fermentation process when expressing the recombinant dust mite type II allergen DP2 and DF2 proteins is as follows: the fermentation medium used is 60% BSM medium, and the induction temperature at the induction stage of high-density fermentation is : 20 ⁇ 22 °C, pH value 6.0 ⁇ 0.2; the methanol flow acceleration during the induction stage of high-density fermentation is 30mL / h -1 L -1 , and the DO is not higher than 40%; the rate-limiting growth stage of high-density fermentation begins 50% glycerol was added at a rate of 30 ml / h -1 L -1 , and the feed rate was adjusted to 60 ml / h -1 L -1 afterwards.
  • the fermentation medium used is 60% BSM medium
  • the induction temperature at the induction stage of high-density fermentation is : 20 ⁇ 22 °C, pH value 6.0 ⁇ 0.2
  • the methanol flow acceleration during the induction stage of high-density fermentation is
  • the recombinant dust mite type II allergen DP2, DF2 protein is expressed, a three-step purification process after high-density fermentation, wherein the purification filler of cation exchange chromatography is SPFF; the preferred pH range is 4.0-6.0 . More preferably, the sample and buffer pH is 6.0.
  • the purification packing of anion exchange chromatography is QFF; the preferred pH range is 6.0-9.0; the preferred conductivity value is 2.0mS / cm-20.0mS / cm. More preferably, the sample and buffer have a pH of 7.5 and a conductance of 10.0 mS / cm.
  • the purification filler for hydrophobic chromatography is phenylFF; the preferred pH range is 6.0-8.0; the preferred ammonium sulfate concentration is 1.0-2.0M. More preferably, the sample and the equilibration buffer have an ammonium sulfate concentration of 1.0 M and a pH of 6.0.
  • the present invention has the following advantages: first, the recombinant allergen protein is compared with the crude extract: (1) the recombinant allergen has higher purity and contains no non-allergens compared with the crude extract; Original components, enzymes, enzyme inhibitors, and toxic proteins, etc .; (2) Recombinant proteins have good specificity, and the components in the crude extract are complex.
  • the recombinant allergen protein drug mixture of the present invention also found a most suitable composition ratio, so that the composition is not only more than the same ratio of natural allergen protein (single recombinant allergen).
  • the original protein has substantially the same activity as the natural protein), and has better activity compared with the single component, and the activity is higher than the most active allergen protein.
  • Figure 1 shows the results of HPLC analysis of DP1 / DF1 protein purity.
  • Figure 1-a shows the results of RP-HPLC analysis of DP1 protein
  • Figure 1-b shows the results of RP-HPLC analysis of DF1 protein.
  • Figure 2 shows the results of amino acid coverage analysis of DP1 / DF1 protein.
  • Figure 2-a shows the analysis result of the amino acid coverage of the DP1 protein
  • Figure 2-b shows the analysis result of the amino acid coverage of the DF1 protein.
  • Figure 3 shows the results of HPLC analysis of DP2 / DF2 protein purity.
  • Figure 3-a shows the results of RP-HPLC analysis of DP2 protein
  • Figure 3-b shows the results of RP-HPLC analysis of DF2 protein.
  • Figure 4 shows the results of amino acid coverage analysis of DP2 / DF2 protein.
  • Figure 4-a shows the analysis result of the amino acid coverage of the DP2 protein
  • Figure 4-b shows the analysis result of the amino acid coverage of the DF2 protein.
  • FIG. 5 is a graph showing the relationship between the single component recombinant protein (r) and the native protein (n) Log10 (fold dilution) versus the inhibition rate;
  • Figure 5-a is a graph of the relationship between the log10 (dilution multiple) of the natural DP1 protein and the inhibition rate
  • Fig. 5-b is a graph showing the relationship between the log10 (dilution multiple) of the recombinant DP1 protein and the inhibition rate;
  • Figure 5-c is a graph of the relationship between the log10 (dilution multiple) of the natural DP2 protein and the inhibition rate
  • Fig. 5-d is a graph showing the relationship between the log10 (dilution multiple) of the recombinant DP2 protein and the inhibition rate;
  • Figure 5-e is a graph of the relationship between the log10 (dilution multiple) of the natural DF1 protein and the inhibition rate;
  • Fig. 5-f is a graph showing the relationship between the log10 (dilution multiple) of the recombinant DF1 protein and the inhibition rate;
  • Figure 5-g is a graph of the relationship between the log10 (dilution multiple) of the natural DF2 protein and the inhibition rate
  • Fig. 5-h is a graph showing the relationship between the log10 (dilution multiple) of recombinant DF2 protein and the inhibition rate;
  • Figure 6 is the percentage of activity of different weight ratio (mass ratio) combination agents compared with single component recombinant protein
  • Figure 6-a shows the comparison of the activity of different proportions (mass ratio) of the weight combination agent with the single component rDP1;
  • Figure 6-b shows the comparison of the activity of different proportions (mass ratio) of the weight combination agent with the single component rDP2;
  • Figure 6-c shows the comparison of the activity of different proportions (mass ratio) of the weight combination agent with the single component rDF1;
  • Figure 6-d shows the comparison of the activity of different proportions (mass ratios) of the weight combination agent with the single component rDF2;
  • Figure 7 is a comparison of the activity of four protein recombination agents of different proportions (mass ratio) with two allergens of class I and II;
  • Figure 8 is a comparison of the activity of the isoactive heavy combination with Odactra
  • FIG. 9 shows the detection results of cytokine content in the spleen cell stimulation supernatant after desensitization of each administration group
  • Figure 9-a shows the detection results of IL-4 content in the spleen cell stimulation supernatant after desensitization of each administration group
  • Figure 9-b shows the detection results of IL-5 content in the spleen cell stimulation supernatant after desensitization of each administration group
  • Figure 9-c shows the detection results of IL-13 content in the spleen cell stimulation supernatant after desensitization of each administration group
  • Figure 9-d shows the detection results of IL-10 content in the spleen cell stimulation supernatant after desensitization of each administration group
  • Example 1 Codon optimization of recombinant proDP1, construction of expression plasmids, and construction of host engineering strains
  • Step 1 Recombination codon optimization
  • the inventors Based on the published DNA sequence of DP1 (ENA accession number: FM177224.1) of the EMBL-EBI, the inventors obtained the proDP1 gene (the The gene adds the alpha-factor signal peptide and Kozak sequence GCCACCATGG on the basis of the wild-type gene, and is codon optimized).
  • the nucleotide sequence is shown in SEQ ID No: 1.
  • the inventor Based on the published DNA sequence of DF1 (GenBank accession number: AB034946.1) of the GenBank, the inventor obtained the proDF1 gene of the present invention after codon optimization and modification after combining the requirements of the expression system of the present invention and multiple verifications (the gene is in On the basis of the wild-type gene, an alpha-factor signal peptide and a Kozak sequence GCCACCATGG are added, and the codon is optimized), and the nucleotide sequence is shown in SEQ ID No: 2.
  • the DP2 gene of the present invention was obtained after codon optimization and modification after combining the requirements of the expression system of the present invention and multiple verifications.
  • the nucleotide sequence of the DP2 gene of the present invention is as follows: SEQ ID No: 3 is shown.
  • Step 2 Construction of expression plasmid containing proDP1 gene
  • the codon-optimized proDP1 was introduced into the XhoI restriction site sequence at the 5 'end, and the XbaI restriction site sequence was introduced at the 3' end, and the whole gene was synthesized.
  • the synthesized gene fragment was constructed into the pUC57 plasmid (by Nanjing Kingsray Technology Co., Ltd.), a long-term storage plasmid was obtained, which was recorded as pUC57-proDP1 plasmid.
  • the pUC57-proDP1 plasmid was used as a template for PCR amplification.
  • the primer sequences used were as follows:
  • Upstream primer (SEQ ID No: 5):
  • the total reaction volume was 50 ⁇ L, in which 2.5 ⁇ L of each primer was added at a concentration of 10 ⁇ mol / L, and 1 ⁇ L of dNTP was used at a concentration of 10 mmol / L.
  • the DNA polymerase used was Q5 (# M0491L, purchased from New England BioLabs), 2 U / ⁇ L, added 0.5 ⁇ L.
  • 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. The results showed that the product size was consistent with the expected size (1000bp).
  • XhoI (# R0146S, purchased from New England BioLabs) and Xba I (# R01445S, purchased from New England BioLabs) were double-digested and electrophoresed on 1% agarose to obtain the gene product using a DNA gel recovery kit. (DP214, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) was purified.
  • T4 ligase (# M0202S, purchased from New England BioLabs) was ligated into pPICZ ⁇ A plasmid (V173-20, purchased from Invitrogen) and transformed into DH5 ⁇ competent cells (CB101, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) In a LB solid medium containing bleomycin (purchased from Invitrogen), the cells were cultured overnight at 37 ° C. The next day, the positive clones were picked and sequenced. The alignment was completely consistent with the expected sequence, that is, the proDP1 codon optimized expression plasmid was obtained and recorded as pPICZ ⁇ -proDP1.
  • Step 3 Construction of a Pichia host engineered strain containing the recombinant proDP1 gene
  • YPDS solid medium preparation Provided by Invitrogen's EasySelectPichia Expression Kit instructions, where yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L, and sorbitol 182g / L.
  • Electrocompetent cells were prepared according to the method of Easy Select Pichia Expression Kit of Invitrogen.
  • the plasmid pPICZ ⁇ -proDP1 obtained in step 2 was digested with Sac I restriction enzyme (# R0156S, purchased from New England Biolabs) and linearized. After ethanol precipitation, the linearized vector was electrotransformed into Pichia X33 competent cells. The medium was spread on YPDS solid medium and cultured at 30 ° C until the transformants grew.
  • proDF1, DP2, DF2 expression plasmids and host engineering strains are consistent with the above method, and will not be repeated here.
  • Step 1 Recombinant strain activation
  • glycerol seed produced in the working seed bank frozen at -80 ° C after the above examples in YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L) Streak and incubate at 30 ° C for 3-5 days.
  • YPD solid medium yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L
  • Step 2 Primary seed liquid culture
  • YPD liquid culture medium yeast extract 10g / L, peptone 20g / L, glucose 20g / L
  • culture at 30 ° C, 220rpm to OD 600 ⁇ 6.0, and observe under a microscope Without any bacteria, the first-stage seed liquid for fermentation is obtained.
  • Step 3 Secondary seed liquid culture
  • Step 4 The fermentation process
  • the seed liquid obtained in step 3 is introduced into the fermentor at a ratio of 1:15 (V / V, seed liquid / fermentation medium).
  • the carbon source is consumed, the dissolved oxygen value rises rapidly, and the wet weight of the bacteria reaches 100g / L. At this time, it enters the rate-limiting growth stage.
  • the first 2h of this stage is 30ml / h -1 L -1 50% glycerol was added at a rate of 50%, and the feeding speed was increased to 60ml / h -1 L -1 after 2h. After 4 hours of supplementation, the wet weight of the bacteria was about 200 g / L. When the feeding was stopped, the DO increased, indicating that the carbon source was depleted and entered the induction stage.
  • Step 1 Pretreatment of fermentation broth
  • the fermentation broth obtained in the above example was centrifuged at high speed to obtain a supernatant; diatomaceous earth was added to assist filtration to obtain a clear fermentation broth sample overnight; and the solution was diluted with 10KD membrane ultrafiltration to reduce the conductivity to less than 5 mS / cm.
  • the acetic acid of the fermented broth after the above treatment was adjusted to pH 4.0, and then applied to a SPFF chromatography column with a column type of BPG140 / 100, a column bed volume of 2800ml, an equilibrium buffer solution of 50mM NaAc, pH4.0, and an elution buffer solution of 50mM NaAc, 1.0M NaCl, pH 4.0, linear elution according to 0-100%, the target protein of DP1 / DF1 mainly concentrated on the second elution peak.
  • the second elution peak in the first step of purification step 2 was combined, and diluted with 10KD membrane ultrafiltration to conductance ⁇ 2.0mS / cm.
  • the sample in step 1 to 20 mM Tris, adjust the pH to 6.0, and load it onto a Q FF anion exchange chromatography column with a Hiscale 50/40 column size and a column bed volume of 500 ml.
  • the equilibration buffer is 20 mM NaH 2 PO 4 , pH 6.0, and washed.
  • the debuffering solution was 20 mM NaH 2 PO 4 , 1.0 M NaCl, pH 6.0, and the penetration was collected, and 0-100% linearly eluted.
  • the target protein was mainly concentrated in the penetration.
  • the optimal conditions are pH 8.0 and conductivity 20.0 mS / cm.
  • the target protein has the highest resolution from impurities. Most of the target protein remains in the penetration, and the impurities are bound to the medium to achieve complete separation. The yield of DP1 / DF1 protein during penetration was greatly improved.
  • ammonium sulfate concentration of 2.0M and pH8.5 are the optimal conditions.
  • the DP1 / DF1 protein obtained after optimization is the most pure. High, minimal impurities, the purity of the obtained protein can reach more than 95%.
  • the yield of the purification process shown in Table 1 is about 30% higher than that before the optimization, and the activity (determined by the ELISA method) is also improved compared to the level before the process optimization.
  • Example 3 The gradient elution peaks in Example 3 were collected and concentrated with a Vivaflow50 tangential flow ultrafiltration membrane package (VF05P9-50cm 2 , Sartorius), and the dialysis bag replacement buffer solution was pH 7.4PBS solution; the Pierce BCA protein concentration kit was used to determine the protein concentration .
  • VF05P9-50cm 2 a Vivaflow50 tangential flow ultrafiltration membrane package
  • the dialysis bag replacement buffer solution was pH 7.4PBS solution
  • Pierce BCA protein concentration kit was used to determine the protein concentration .
  • the purified DP1 / DF1 sample was diluted to 1 mg / ml and filtered through a 0.22um filter to obtain a sample.
  • the mobile phase is 20mM PB, pH 7.5, flow rate 1ml / min, injection 10ul, RP column analysis of purity, Figure 1-a, Figure 1-b shows the purity test results, the purity of DP1 / DF1 protein purified in three steps is greater than 95 %.
  • Amino acid coverage analysis is one of the very important indicators in the research of the quality of recombinant protein drugs. Only when the primary structure of the amino acid is completely the same can the product have the same biological activity as the natural protein.
  • the inventor commissioned Shanghai Zhongke New Life Biotechnology Co., Ltd. to analyze the amino acid coverage of the DP1 / DF1 protein.
  • Figure 2-a and Figure 2-b show that the amino acid coverage is greater than 98%, which meets the requirements of relevant policies and regulations.
  • rDP1, rDF1 recombinant DP1, DF1 protein
  • nDP1 natural DP1 protein
  • Blocking wash 3 times with PBST (pH 7.4, 0.15M PBS + 0.05% Tween20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37 ° C for 2h.
  • Secondary antibody incubation wash 4 times with PBST, pat dry, add 100ul of secondary antibody dilution solution (rat anti-human IgE-HRP 1: 1500 dilution) to each well, and incubate at 37 ° C for 1h.
  • secondary antibody dilution solution rat anti-human IgE-HRP 1: 1500 dilution
  • Termination and reading 50ul 2M H 2 SO 4 was added to each well to stop the reaction, and the reading was taken at a wavelength of 450nm.
  • the experimental results are shown in Table 2.
  • the calculated rDP1 activity value was 79616.0BU / ml; the specific activity was 9.95E + 04BU / mg. Simultaneously, nDF1 was detected as a control.
  • the experimental results are shown in Table 2. This indicates that yeast expresses rDP1, and rDF1 has similar biological activity compared with natural proteins.
  • Host residual protein refers to the host protein remaining in biological products.
  • the protein composition of this type is complex and varied, and it will vary depending on the production process and purification process. Residual HCP in genetically engineered products is an important factor affecting the purity of products. Repeated use of genetically engineered products containing HCPs can cause allergic reactions in the body, which has a potential "adjuvant effect" and may also cause the body to produce antibodies to drugs, which in turn affects The efficacy of the drug. HCP residues in biological products reflect not only the consistency between batches, but also an important indicator of the quality of biological products.
  • the inventors used the Pichia HCP detection kit (F140, CYGNUS) to test the prepared samples. Table 3 shows that the DP1 and DF1 protein HCP contents purified in three steps were far lower than the recombinant biological products (yeast) specified in the 2015 Pharmacopoeia. ) HCP ceiling.
  • the DNA fragments of the host cells may still remain in the product. These residual DNA may bring infectious or tumorigenic risks, may cause insertion mutations, cause inactivation of tumor suppressor genes, Oncogenes are activated, etc.
  • genomic DNA derived from microorganisms is rich in CpG and unmethylated sequences, which increases the immunogenicity risk of recombinant protein drugs in vivo. Therefore, the limits of residual DNA by WHO and drug registration regulatory agencies in various countries The requirements are very strict.
  • the methods for detecting DNA residues mainly include DNA probe hybridization, fluorescent dye method and qPCR method. The first two methods have technical defects and it is difficult to achieve the sensitivity of impurity limit detection.
  • Step 1 Recombinant strain activation
  • Glycerol seed stored in -80 ° C working seed bank obtained in Example 1 was frozen in YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L) Streak and incubate at 30 ° C for 3-5 days.
  • YPD solid medium yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L
  • Step 2 Primary seed liquid culture
  • YPD liquid medium yeast extract 10g / L, peptone 20g / L, glucose 20g / L
  • culture at 30 ° C, 220rpm to OD 600 ⁇ 6.0, and under the microscope Observing no bacteria, the first-stage seed liquid for fermentation was obtained.
  • Step 3 Secondary seed liquid culture
  • Step 4 The fermentation process
  • the seed liquid obtained in step 3 is introduced into the fermentor at a ratio of 1:15 (V / V, seed liquid / fermentation medium).
  • the first 2h of this stage is 30ml / h -1 L -1 50% glycerol was added at a rate of 50%, and the feeding speed was increased to 60ml / h -1 L -1 after 2h. After 4 hours of supplementation, the wet weight of the bacteria was about 200 g / L. When the feeding was stopped, the DO increased, indicating that the carbon source was depleted and entered the induction stage.
  • the fermentation broth obtained in the above example was centrifuged at high speed to obtain a supernatant; diatomaceous earth was added to aid filtration to obtain a clear fermentation broth sample overnight; and the solution was diluted with a 3KD membrane ultrafiltration to reduce the conductivity to less than 5 mS / cm.
  • the acetic acid of the fermented broth after the above treatment was adjusted to pH 4.0, and then applied to a SPFF chromatography column with a column type of BPG140 / 100, a column bed volume of 2800ml, an equilibrium buffer solution of 50mM NaAc, pH4.0, and an elution buffer solution of 50mM NaAc, 1.0M NaCl, pH 4.0, linear elution according to 0-100%, the target protein of DP2 / DF2 mainly concentrated on the second elution peak.
  • the second elution peak in step 2 of the example was combined and diluted by ultrafiltration with a 3KD membrane package to a conductance ⁇ 2.0 mS / cm.
  • Step 2 Second step anion exchange chromatography
  • the sample in step 1 to 20 mM Tris, adjust the pH to 6.0, and load it onto a Q FF anion exchange chromatography column with a Hiscale 50/40 column size and a column bed volume of 500 ml.
  • the equilibration buffer is 20 mM NaH 2 PO 4 , pH 6.0, and washed.
  • the debuffering solution was 20 mM NaH 2 PO 4 , 1.0 M NaCl, pH 6.0, and the penetration was collected, and 0-100% linearly eluted.
  • the target protein was mainly concentrated in the penetration.
  • the concentration of ammonium sulfate is 1.0M and the pH is the best.
  • the DP2 protein obtained after optimization is higher in purity and has the largest amount of protein. There is no target protein in the penetration and all are bound to the filler.
  • the optimized yield of DP2 protein after optimization shown in Table 5 is 30% higher than that before optimization, and the activity (determined by ELISA method) is also improved compared with that before the optimization of the process.
  • Example 6 The gradient elution peaks in Example 6 were collected and concentrated with a Vivaflow 50 tangential flow ultrafiltration membrane package (VF05P9-50cm 2 , Sartorius), and the dialysis bag replacement buffer solution was pH 7.4 PBS solution; the Pierce BCA protein concentration kit was used to determine the protein concentration .
  • a Vivaflow 50 tangential flow ultrafiltration membrane package (VF05P9-50cm 2 , Sartorius)
  • the dialysis bag replacement buffer solution was pH 7.4 PBS solution
  • the Pierce BCA protein concentration kit was used to determine the protein concentration .
  • the purified DP2 / DF2 sample was diluted to 1 mg / ml and filtered through a 0.22um filter to obtain a sample.
  • the mobile phase was 20mM PB, pH 7.5, flow rate 1ml / min, injection 10ul, and purity analysis by RP column.
  • Figure 3-a, 3-b are the purity test results. The purity of DP2 and DF2 protein purified in three steps is greater than 95% .
  • Amino acid coverage analysis is one of the very important indicators in the research of the quality of recombinant protein drugs. Only when the primary structure of the amino acid is completely the same can the product have the same biological activity as the natural protein. The inventor entrusted Shanghai Zhongke New Life Biotechnology Co., Ltd. to analyze the amino acid coverage of DP2 and DF2 proteins. Figures 4-a and 4-b show that the amino acid coverage is 100%, which meets the requirements of relevant policies and regulations.
  • Host residual protein refers to the host protein remaining in biological products.
  • the protein composition of this type is complex and varied, and it will vary depending on the production process and purification process.
  • Residual HCP in genetically engineered products is an important factor affecting the purity of products.
  • Repeated use of genetically engineered products containing HCPs can cause allergic reactions in the body, which has a potential "adjuvant effect", and may also cause the body to produce antibodies to drugs, which in turn affects The efficacy of the drug.
  • HCP residues in biological products reflect not only the consistency between batches, but also an important indicator of the quality of biological products.
  • the inventors used the Pichia yeast HCP test kit (F140, CYGNUS) to test the prepared samples. Table 6 shows that the DP2 and DF2 protein HCP contents purified in three steps were far lower than the recombinant biological products (yeast) specified in the 2015 Pharmacopoeia. ) HCP ceiling.
  • the DNA fragments of the host cells may still remain in the product. These residual DNA may bring infectious or tumorigenic risks, may cause insertion mutations, cause inactivation of tumor suppressor genes, Oncogenes are activated, etc.
  • genomic DNA derived from microorganisms is rich in CpG and unmethylated sequences, which increases the immunogenicity risk of recombinant protein drugs in vivo. Therefore, the limits of residual DNA by WHO and drug registration regulatory agencies in various countries The requirements are very strict.
  • the methods for detecting DNA residues mainly include DNA probe hybridization, fluorescent dye method and qPCR method. The first two methods have technical defects and it is difficult to achieve the sensitivity of impurity limit detection.
  • the FDA has stipulated qPCR in the latest version of the USP.
  • the inventors used qPCR to detect residual DNA in the sample (SK030205P100, Huzhou Shenke Biotechnology Co., Ltd.).
  • the results in Table 7 show that the residual DNA content of the samples purified in three steps is far away. Residual DNA content of recombinant biological products (yeast) is lower than the maximum limit specified in the 2015 Pharmacopoeia.
  • the competitive inhibition ELISA method was used to determine the biological activity of rDF1 / rDF2 / rDP1 / rDP2 and compare it with natural nDF1 / nDF2 / nDP1 / nDP2.
  • the specific steps are (taking rDP2 as an example):
  • Blocking wash 3 times with PBST (pH7.4, 0.1M, PBS + 0.05% Tween20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37 ° C for 2h.
  • PBST pH7.4, 0.1M, PBS + 0.05% Tween20
  • Sample Dilution Dilute rDP2 / nDP2 sample 1000 times, and then perform 3 times gradient dilution, a total of 7 dilutions. Take the samples of each dilution and the appropriate dilution of the serum pool serum (more than 15 phadia100 detection d1 / d2 specific IgE values> 100Kua / L patient serum pool) and mix them in equal volumes, that is, 125ul sample + 125ul serum. The samples were incubated at 4 ° C overnight. Positive control: 125ul dilution + 125ul serum.
  • Color development wash 4 times with PBST, pat dry, add 100ul TMB I color development solution to each well, and develop color at 37 ° C for 10 min.
  • Termination and reading 50ul 2M H 2 SO 4 is added to each well to stop the reaction, and the reading is taken at a wavelength of 450nm.
  • the counterweight combination was mixed in the following proportions to compare the biological activity of rDF1 / rDF2 / rDP1 / rDP2 with a single component.
  • the specific steps are (taking rDP1 as an example):
  • Blocking wash 3 times with PBST (pH7.4, 0.1M, PBS + 0.05% Tween20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37 ° C for 2h.
  • PBST pH7.4, 0.1M, PBS + 0.05% Tween20
  • Sample Dilution Dilute 1000 times the recombination agent and the rDP1 sample for comparison, and then perform 3 times gradient dilution for a total of 7 dilutions. Take the samples of each dilution and the appropriate dilution of the serum pool serum (more than 15 phadia100 detection d1 / d2 specific IgE values> 100Kua / L patient serum pool) and mix them in equal volumes, that is, 125ul sample + 125ul serum. The samples were incubated at 4 ° C overnight. Positive control: 125ul dilution + 125ul serum.
  • the counterweight combination was mixed in the following proportions to compare the biological activity of rDF1 / rDF2 / rDP1 / rDP2 with a single component.
  • the specific steps are (taking rDP1 as an example):
  • Blocking wash 3 times with PBST (pH7.4, 0.1M, PBS + 0.05% Tween20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37 ° C for 2h.
  • PBST pH7.4, 0.1M, PBS + 0.05% Tween20
  • Sample dilution Mix the recombination agent in 1 with the rDP1 + rDF1, rDP2 + rDP2 samples (combined according to the ratio of 20: 1, 5: 1, 1: 1, 1: 5, 1; 20) to 1000. Dilution, followed by 3 times gradient dilution, a total of 7 dilutions. Take the samples of each dilution and the appropriate dilution of the serum pool serum (more than 15 phadia100 detection d1 / d2 specific IgE values> 100Kua / L patient serum pool) and mix them in equal volumes, that is, 125ul sample + 125ul serum. The samples were incubated at 4 ° C overnight. Positive control: 125ul dilution + 125ul serum.
  • the inhibition rate% (positive value-sample value / positive value) was taken as the abscissa, and the log10 dilution factor was used as the ordinate to perform a quadratic curve fitting.
  • the 50% inhibition rate was substituted into the curve equation, and the dilution factor ⁇ 100 was calculated as the biological activity value (BU / ml).
  • Specific activity (BU / mg) activity value / protein concentration.
  • the comparative rDP1 activity is defined as 100%, and the ratio of the activity of various recombination agents to rDP1 is calculated (the method of comparing recombination agents with rDP2, rDF1, and rDF2 is the same as rDP1).
  • the competitive inhibition ELISA method was used to compare the activity of the counterweight combination according to the determination in Example 8 with the biological activity of Odactra.
  • Blocking wash 3 times with PBST (pH7.4, 0.1M, PBS + 0.05% Tween20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37 ° C for 2h.
  • PBST pH7.4, 0.1M, PBS + 0.05% Tween20
  • Sample Dilution Dilute 1000 times the recombination agent and the Odactra sample (calculated according to the indicated quantity 30ug / piece) in the comparison, and then make 3 times gradient dilution for a total of 7 dilutions. Take the samples of each dilution and the appropriate dilution of the serum pool serum (more than 15 phadia100 detection d1 / d2 specific IgE values> 100Kua / L patient serum pool) and mix them in equal volumes, that is, 125ul sample + 125ul serum. The samples were incubated at 4 ° C overnight. Positive control: 125ul dilution + 125ul serum.
  • mice were intraperitoneally injected with HDM (mite extract, purchased from LSL company, according to DF 0.45 ⁇ g + DP 0.40 ⁇ g) + aluminum adjuvant for about 4 consecutive weeks. After the sensitization was completed, an HDM solution (according to DF 0.90 ⁇ g + DP 0.80 ⁇ g) was dripped into the nasal cavity for 7 consecutive days. After the challenge, the detection indicators include: sneeze and penh value (the animals were stimulated with methacholine Mch at 3.125 / 6.25 / 12.5 / 25 / 50mg / ml, and the whole body plethysmograph WBP was used to detect the animals) .
  • HDM mite extract, purchased from LSL company, according to DF 0.45 ⁇ g + DP 0.40 ⁇ g
  • an HDM solution according to DF 0.90 ⁇ g + DP 0.80 ⁇ g
  • the detection indicators include: sneeze and penh value (the animals were stimulated with meth
  • mice with higher values were selected for grouping, and divided into PBS treatment group, recombination agent group (DP1, DF1, DP2, DF2 mass ratio 1: 1: 1: 1: 1) 25 ⁇ g / piece, positive Group 24 ⁇ g / head (based on Odactra active ingredient).
  • Sublingual desensitization treatment twice a week (between two and three days), for 8 consecutive weeks. Nasal challenge was performed again for 7 days in the same way.
  • the detection indicators include: sneeze, penh value and spleen cytokines.
  • the levels of IL-4, 5, and 13 in the desensitized recombination group were lower than those in the positive group, and they were significantly reduced compared with the PBS group (P ⁇ 0.05);
  • the levels of IL-10 in the group were comparable, and they were significantly lower than those in the PBS group. This indicates that the cytokines shifted from Th2 type to Th2 and Th1 balance, and the allergy in model mice was improved in terms of cytokine levels or desensitization mechanisms. .
  • the recombination group and the positive group have significantly improved the respiratory symptoms and mechanisms such as penh value, sneeze number, and cytokine level, which indicates that the recombination animal body Efficacy test activity is equivalent.

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Abstract

本发明公开了一种重组尘螨变应原蛋白组合物，其包含重组尘螨Ⅰ型变应原DP1、DF1蛋白和重组尘螨II型变应原DP2、DF2蛋白，并分别按活性比1:1:1:1混合或按质量比(1～5)：(1～20)：(1～10)：(1～20)混合。本发明与现有产品相比：(1)重组变应原具有高纯度、质量可控等优势；(2)重组蛋白具有较好的特异性；(3)与天然提取液相比具有较好的免疫原性，降低发生严重过敏副反应的风险，提高脱敏治疗的效果。其次，本发明的重组变应原组合物不仅比相同比例的天然变应原蛋白具有更好的活性，并且与单一组分相比活性也高于相应天然单一组份。

Description

重组尘螨变应原蛋白药物合剂及其应用 技术领域
本发明涉及一种治疗尘螨过敏的药物组合，尤其涉及一种改良的重组尘螨变应原蛋白药物合剂及其应用。
背景技术
在引起过敏性疾病的众多变应原中，尘螨是最主要的变应原。尘螨在过敏性疾病患者特异性免疫诊断中阳性率约为70-80％。尘螨种类繁多，广泛存在于人类生活和工作环境中，其排泄物、代谢物及螨体均具有较强的变应原型，据统计全球约10％的人口尘螨过敏，约80％的外源性哮喘由尘螨引起。
目前，临床上主要采用尘螨变应原提取液免疫治疗尘螨过敏，例如2006年上市的浙江我武生物的“畅迪”粉尘螨滴剂即为粉尘螨代谢培养物的提取液；2015年日本上市的Actair屋尘螨舌下片剂，用于治疗屋尘螨(或称为户尘螨)引起的过敏性鼻炎，采用的也是天然I、II型户尘螨和I、II型粉尘螨的四种组分；2017年FDA批准上市的Odactra也是采用天然I、II型户尘螨和I、II型粉尘螨的四种组分，可见采用合剂已经成为尘螨脱敏治疗的一种趋势。
然而由于天然变应原提取液的组成非常复杂，恒定其组分非常困难，且容易受到外源性有毒物质、病原体微生物的污染，影响其重复性与安全性，在治疗过程中长期使用易导致红晕、肿胀、硬结、坏死等局部反应和休克、水肿、支气管痉挛、荨麻疹、血管性水肿、全身性红斑等全身反应，此外，采用粗提液进行诊断，无法明确患者对变应原各组分的反应程度，易致误诊。变应原的质量对变态反应疾病的诊断和治疗至关重要，用于免疫诊治的变应原应该是纯品而不宜为粗提取液，而且尘螨变应原主要存在于排泄物和螨体中，采用提取方法耗时长，过程繁琐，成本较高，无法避免免疫治疗中副反应的发生。
发明内容
为了克服以上缺点，本发明的目的是提供一种重组尘螨变应原蛋白药物合剂，并且针对重组尘螨变应原蛋白药物合剂优化得到一种活性最优的药物合剂配比。
首先，本发明提供了一种重组尘螨变应原蛋白药物合剂，其包含重组尘螨Ⅰ型变应原DP1、DF1蛋白和重组尘螨II型变应原DP2、DF2蛋白，并且四种尘螨变应原蛋白DP1：DP2：DF1：DF2分别按活性比1:1:1:1混合或按质量比(1～5)：(1～20)：(1～10)：(1～20)混合。
优选的，所述重组尘螨变应原蛋白药物合剂中四种尘螨变应原蛋白DP1：DP2：DF1：DF2分别按质量比1：(1～20)：1：(1～20)混合。
更优选的，所述重组尘螨变应原蛋白药物合剂中四种尘螨变应原蛋白按质量比1：5：1：5混合。
优选的，所述重组尘螨Ⅰ型变应原DP1、DF1蛋白和重组尘螨II型变应原DP2、DF2蛋白，分别由如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示的基因通过毕赤酵母表达系统表达得到。
更优选的，所述重组尘螨Ⅰ型变应原蛋白由以下方法制备得到：
1、将上述所述的基因构建到质粒pPICZα上，并转入毕赤酵母X33感受态细胞中，并培养获得宿主单克隆工程菌；2、挑取单克隆工程菌经过高密度发酵，并经离子交换层析、阴离子交换层析、疏水层析的三步纯化得到。
优选的，上述高密度发酵工序，其在表达重组尘螨Ⅰ型变应原DP1、DF1蛋白时主要发酵工艺如下：所用发酵培养基为40％BSM培养基，高密度发酵的诱导阶段诱导温度为：25～27℃，pH值为6.5±0.2，高密度发酵的诱导阶段甲醇流加速度为30mL/h -1L -1，并维持DO不高于40％，高密度发酵的菌体增值阶段发酵温度为27℃，pH＝5.5±0.2，转速300rpm，DO值100％，添加PTM1(2.4ml/L)；高密度发酵的限速生长阶段开始以30ml/h -1L -1的速率补加50％甘油，后补料速度上调为60ml/h -1L -1。
优选的，其在表达重组尘螨Ⅰ型变应原DP1、DF1蛋白时，高密度发酵后的三步纯化工艺，其中阳离子交换层析的纯化填料为SP FF；优选的pH范围4.0-6.5。更优选的，阳离子交换层系中样品和缓冲液pH为5.0。
阴离子交换层析的纯化填料为Q FF；优选的pH范围6.0-8.0；优选的电导值2.0mS/cm-20.0mS/cm。更优选的，阴离子交换层析中样品和缓冲液pH为8.0，电导为20.0mS/cm。
疏水层析的纯化填料为phenyl FF；优选的pH范围6.5-8.5；优选的硫酸铵浓度为1.0-2.0M。更优选的，疏水层析中样品和平衡缓冲液硫酸铵浓度为2.0M，pH为8.5。
优选的，上述高密度发酵工序，其在表达重组尘螨II型变应原DP2、DF2蛋白时主要发酵工艺如下：所用发酵培养基为60％BSM培养基，高密度发酵的诱导阶段诱导温度为：20～22℃，pH值为6.0±0.2；高密度发酵的诱导阶段甲醇流加速度为30mL/h -1L -1，并维持DO不高于40％；高密度发酵的限速生长阶段开始以30ml/h -1L -1的速率补加50％甘油，后补料速度上调为60ml/h -1L -1。
优选的，其在表达重组尘螨II型变应原DP2、DF2蛋白时，高密度发酵后的三步纯化工艺，其中，阳离子交换层析的纯化填料为SP FF；优选的pH范围4.0-6.0。更优选的，样品和缓冲液pH为6.0。
其中，阴离子交换层析的纯化填料为Q FF；优选的pH范围6.0-9.0；优选的电导值 2.0mS/cm-20.0mS/cm。更优选的，样品和缓冲液pH7.5，电导10.0mS/cm。
其中，疏水层析的纯化填料为phenyl FF；优选的pH范围6.0-8.0；优选的硫酸铵浓度为1.0-2.0M。更优选的，样品和平衡缓冲液硫酸铵浓度1.0M，pH6.0。
本发明与现有产品相比具有以下优势：首先，重组变应原蛋白与粗提液相比：(1)重组变应原具有更高的纯度，与粗提液相比不含非变应原成分、酶类、酶抑制剂和毒性蛋白等；(2)重组蛋白具有较好的特异性，粗提液中成分复杂，患者可能只与其中部分成分反应，特异性差，而重组变应原成分单一，特异型好；(3)重组变应原活性可替代天然提取液，与天然提取液相比减少IgE结合的抗原表位，有效降低IgE介导的过敏反应，同时保留变应原T细胞识别所必须的结构域，具有较好的免疫原性，减少免疫治疗的危险性，提高脱敏治疗的效果。其次，本发明的重组变应原蛋白药物合剂经过申请人的反复摸索，也找到了一个最为合适的组合物比例，使得该组合物不仅比相同比例的天然变应原蛋白(单独的重组变应原蛋白与天然的相比活性基本持平)具有更好的活性，并且与单一组分相比活性也高于其中活性最高的变应原蛋白。
附图说明
图1为DP1/DF1蛋白纯度HPLC分析结果。
其中图1-a为DP1蛋白RP-HPLC分析结果；图1-b为DF1蛋白RP-HPLC分析结果。
图2为DP1/DF1蛋白氨基酸覆盖率分析结果。
其中图2-a为DP1蛋白氨基酸覆盖率分析结果；图2-b为DF1蛋白氨基酸覆盖率分析结果。
图3为DP2/DF2蛋白纯度HPLC分析结果。
其中图3-a为DP2蛋白RP-HPLC分析结果；图3-b为DF2蛋白RP-HPLC分析结果。
图4为DP2/DF2蛋白氨基酸覆盖率分析结果。
其中图4-a为DP2蛋白氨基酸覆盖率分析结果；图4-b为DF2蛋白氨基酸覆盖率分析结果。
图5为单一组分的重组蛋白(r)与天然蛋白(n)Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-a为天然DP1蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-b为重组DP1蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-c为天然DP2蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-d为重组DP2蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-e为天然DF1蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-f为重组DF1蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-g为天然DF2蛋白Log10(稀释倍数)对抑制率关系的曲线图；
其中图5-h为重组DF2蛋白Log10(稀释倍数)对抑制率关系的曲线图；
图6为不同比例(质量比)重组合剂与单组分重组蛋白比较时活性百分比；
其中图6-a为不同比例(质量比)重组合剂与单组分rDP1活性比较；
其中图6-b为不同比例(质量比)重组合剂与单组分rDP2活性比较；
其中图6-c为不同比例(质量比)重组合剂与单组分rDF1活性比较；
其中图6-d为不同比例(质量比)重组合剂与单组分rDF2活性比较；
图7为不同比例(质量比)四种蛋白重组合剂与I、II类的两种变应原活性比较；
图8为等活性重组合剂与Odactra活性比较；
图9为各给药组脱敏后脾细胞刺激上清中细胞因子含量检测结果；
其中图9-a为各给药组脱敏后脾细胞刺激上清中IL-4含量检测结果；
其中图9-b为各给药组脱敏后脾细胞刺激上清中IL-5含量检测结果；
其中图9-c为各给药组脱敏后脾细胞刺激上清中IL-13含量检测结果；
其中图9-d为各给药组脱敏后脾细胞刺激上清中IL-10含量检测结果；
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重组proDP1密码子优化改造、表达质粒构建及宿主工程菌株的构建
步骤1：重组密码子优化改造
发明人根据EMBL-EBI已公开的DP1的DNA序列(ENA登录号：FM177224.1)，在结合本发明表达体系需求及多次验证后，获得密码子优化改造后得到本发明的proDP1基因(该基因在野生型基因的基础上增加了alpha-factor信号肽和Kozak序列GCCACCATGG，并经密码子优化)，核苷酸序列如SEQ ID No：1所示。
发明人根据GenBank已公开的DF1的DNA序列(GenBank登录号：AB034946.1)，在结合本发明表达体系需求及多次验证后，获得密码子优化改造后得到本发明的proDF1基因(该基因在野生型基因的基础上增加了alpha-factor信号肽和Kozak序列GCCACCATGG，并经密码子优化)，核苷酸序列如SEQ ID No：2所示。
发明人根据GenBank已公开的DP2的DNA序列(GenBank登录号：AAF86462)，在结合本发明表达体系需求及多次验证后，获得密码子优化改造后得到本发明的DP2基因，核苷酸序列如SEQ ID No：3所示。
发明人根据GenBank已公开的DF2的DNA序列(GenBank登录号：EF139432.1)，在结合本 发明表达体系需求及多次验证后，对该基因进行密码子优化后得到本发明的DF2基因，核苷酸序列如SEQ ID No：4所示。
步骤2：含有proDP1基因的表达质粒构建
将密码子优化后的proDP1在5’端引入XhoI酶切位点序列，在3’端引入XbaI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pUC57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，记为pUC57-proDP1质粒。
以pUC57-proDP1质粒为模板，进行PCR扩增，所用引物序列如下：
上游引物(SEQ ID No：5)：
M13F:TGT AAA ACG ACG GCC AGT
下游引物(SEQ ID No：6)：
M13R:CAG GAA ACA GCT ATG AC
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(#M0491L，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(1000bp)一致。分别用XhoI(#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，购自北京天根生化科技有限公司)纯化。用T4连接酶(#M0202S，购自New England BioLabs公司)连接到pPICZαA质粒(V173-20，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proDP1密码子优化后的表达质粒，记为pPICZα-proDP1。
步骤3：含有重组proDP1基因毕赤酵母宿主工程菌株的构建
YPDS固体培养基配制：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，葡萄糖20g/L，琼脂糖15g/L，山梨醇182g/L。
按照Invitrogen公司Easy SelectPichia Expression Kit说明书的方法制备成电感受态细胞。将步骤2得到的质粒pPICZα-proDP1，用Sac I限制性内切酶(#R0156S，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。
其他proDF1、DP2、DF2的表达质粒、宿主工程菌株构建与上述方法一致，此处不再重复表述。
实施例2重组DP1/DF1大规模(30L)高密度发酵实验
下面先以DP1大规模(30L)高密度发酵进行说明
步骤1：重组菌株活化
取上述实施例后产生的冻存于-80℃的工作种子库中甘油菌种子于YPD固体培养基(酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂糖15g/L)划线，30℃恒温恒湿箱培养3-5天。
步骤2：一级种子液培养
挑取步骤1中平板上单克隆菌落于YPD液体培养基(酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L)，30℃，220rpm培养至OD 600≈6.0，并在显微镜下观察无杂菌，即得到发酵用一级种子液。
步骤3：二级种子液培养
将步骤2中获得的一级种子液接种至赛多利斯B-plus发酵罐中，并以60％BSM，pH＝5.5±0.2，27℃，300rpm值培养，通过通气和转速条件使溶氧保持在25％，最终至OD 600≈40，湿重达到约80g/L，并在显微镜下观察无杂菌，即得到发酵用二级种子液。
步骤4：发酵过程
洗净赛多利斯Cplus生物反应器，用pH＝7.0和pH＝4.0的标准液分别校正发酵罐的pH计探头，然后按照实施例1中关键工艺参数的设计区间，确定发酵培养基为40％BSM进行配置发酵培养基20L，并倒入发酵罐中，121℃在线灭菌20min，待温度降到50℃后，用浓氨水调pH＝5.5±0.2。
将步骤3中得到的种子液按照1:15(V/V，种子液/发酵培养基)比例接入发酵罐中。初期重组毕赤酵母菌体增殖阶段发酵温度为27℃，pH＝5.5±0.2，转速＝300rpm培养，DO值100％，添加PTM1(2.4ml/L)。此阶段大约20h，当碳源消耗完毕后，溶氧值迅速上升，菌体湿重达到100g/L，此时进入限速生长阶段，此阶段的开始的2h以30ml/h -1L -1的速率补加50％甘油，2h后补料速度上调为60ml/h -1L -1。补加4h，菌体湿重约200g/L，停止补料，DO上升，表明碳源耗尽，进入诱导阶段。调节pH＝6.0±0.2，以甲醇流加速度为30ml/h -1L -1进行诱导表达重组DF1；维持DO不高于40％，诱导温度为25～27℃，pH＝6.5±0.2，进行发酵表达，发酵开始后每隔一段时间取样一次，测发酵液OD 600吸收值以及菌体湿重，在重组毕赤酵母发酵期间，发酵液OD 600均能达到300以上，且菌体湿重均能达到400g/L。诱导40h发酵结束。
重组DP1的上述发酵工艺对重组DF1同样适用。
实施例3重组DP1/DF1纯化工艺
一、重组DP1/DF1发酵液上清第一步纯化
步骤1：发酵液的预处理
按上述实施例中得到的发酵液高速离心，得到上清液；加入硅藻土助滤，过夜得到澄清的发酵液样品；用10KD膜包超滤稀释，降低电导至5mS/cm以下。
步骤2：阳离子交换层析
将上述处理后的发酵液上清乙酸调节pH4.0，上SP FF层析柱，柱型为BPG140/100，柱床体积2800ml，平衡缓冲液为50mM NaAc，pH4.0，洗脱缓冲液为50mM NaAc，1.0M NaCl，pH4.0，按照0-100％线性洗脱，DP1/DF1目的蛋白主要集中在第二个洗脱峰。
其余条件不变，调节样品和缓冲液pH5.0。
其余条件不变，调节样品和缓冲液pH6.5。
经对比得知其中pH5.0为最佳条件，纯化后DP1蛋白纯度最高，其余条件均有杂质存在。
二、重组DP1/DF1蛋白第二步纯化:
步骤1：超滤
合并上述第一步纯化步骤2中第二个洗脱峰，10KD膜包超滤稀释至电导<2.0mS/cm。
步骤2：阴离子交换层析
将步骤1中样品加入20mM Tris，调节pH6.0，上Q FF阴离子交换层析柱，柱型为Hiscale50/40，柱床体积500ml，平衡缓冲液为20mM NaH 2PO 4，pH6.0，洗脱缓冲液为20mM NaH 2PO 4，1.0M NaCl，pH6.0，收集穿透，0-100％线性洗脱，目的蛋白主要集中在穿透。
其余条件不变，调节样品和缓冲液pH7.0，电导10.0mS/cm。
其余条件不变，调节样品和缓冲液pH8.0，电导20.0mS/cm。
经对比可知最佳条件为pH8.0，电导20.0mS/cm，经优化后目的蛋白与杂质分离度最高，目的蛋白大部分留在穿透中，杂质结合到介质中，实现了完全分离，且大大提高了DP1/DF1蛋白在穿透中的收率。
三、重组DP1/DF1蛋白第三步纯化
将第二步纯化后的样品加入硫酸铵至终浓度1.0M，调节pH6.5，上Phenyl FF层析柱，柱型为Hiscale 50/40，柱床体积500ml，平衡缓冲液为20mM NaH 2PO 4，1.0M(NH 4) 2SO 4，pH6.0，洗脱缓冲液为20mM NaH 2PO 4，pH6.0，按照25，50％，70％，100％等度洗脱。
其余条件不变，调节样品和平衡缓冲液硫酸铵浓度1.0M，pH7.5。
其余条件不变，调节样品和平衡缓冲液硫酸铵浓度2.0M，pH8.5。
经对比可得硫酸铵浓度2.0M，pH8.5为最优条件，经优化后得到的DP1/DF1蛋白纯度最 高，杂质最少，得到的蛋白纯度可达95％以上。
表1所示纯化工艺经过优化后收率比优化前提高30％左右，而且活性(ELISA方法测定)与工艺优化前的水平相比也有所提高。
表1 DP1蛋白纯化工艺优化前后收率
Figure PCTCN2019091968-appb-000001
重组DP1的上述纯化工艺对重组DF1同样适用。
实施例4重组DP1/DF1蛋白鉴定
1、浓缩，测定蛋白浓度
将实施例3中梯度洗脱峰收集后用Vivaflow50切向流超滤膜包(VF05P9-50cm 2,Sartorius)浓缩，透析袋置换缓冲溶液为pH7.4PBS溶液；Pierce BCA蛋白浓度试剂盒测定蛋白浓度。
2、DP1/DF1蛋白HPLC纯度分析
将纯化得到的DP1/DF1样品稀释至1mg/ml，0.22um滤膜过滤，即得到样品。流动相为20mM PB，pH7.5，流速1ml/min，进样10ul，RP柱分析纯度，图1-a，图1-b为纯度检测结果，经三步纯化的DP1/DF1蛋白纯度大于95％。
3、DP1/DF1蛋白氨基酸覆盖率分析
氨基酸覆盖率分析是重组蛋白类药物质量研究中非常重要的指标之一，只有氨基酸一级结构的完全相同，才能保证产品具有与天然蛋白相同的的生物学活性。发明人委托上海中科新生命生物科技有限公司对DP1/DF1蛋白进行氨基酸覆盖率分析，图2-a，图2-b结果显示氨基酸覆盖率均大于98％，符合相关政策法规的要求。
4、DP1/DF1蛋白活性分析
利用竞争抑制ELISA方法，对重组DP1，DF1蛋白(rDP1，rDF1)生物学活性进行测定，并与天然DP1蛋白(nDP1)进行比较。具体步骤为(以DP1为例)：
(1)、包被：分别使用包被液(pH 9.6 0.15M碳酸盐缓冲液)将rDP1、nDP1稀释至2ug/ml，100ul/孔，4℃孵育过夜。
(2)、封闭：PBST(pH7.4 0.15M PBS+0.05％Tween20)洗3次，拍干，每孔加入200ul封闭液(1％BSA/PBST)，37℃孵育2h。
(3)、样品稀释：将rDP1、nDP1样品进行30倍稀释，后进行3倍梯度稀释，共7个稀释度。 取各个稀释度的样品和适当稀释度的血清库血清(15份以上phadia100检测d1/d2特异性IgE值＞100Kua/L病人血清混合库)等体积混合，即125ul样品+125ul血清，混合后的样品放入4℃孵育过夜。阳性对照：125ul稀释液+125ul血清。
(4)、加样：混合孵育好的样品加入相应的包被孔中，37℃孵育90min。
(5)、二抗孵育：PBST洗4次，拍干，每孔加入100ul二抗稀释液(鼠抗人IgE-HRP 1:1500稀释)，37℃孵育1h。
(6)、显色：PBST洗4次，拍干，每孔加入100ul TMBⅠ号显色液，37℃显色10min。
(7)、终止及读数：每孔加入50ul 2M H 2SO 4终止反应，450nm波长处读数。
(8)、结果处理：利用EXCEL软件，以抑制率％(阳性值-样品值/阳性值)作为横坐标，以Log10稀释倍数为纵坐标，做二次曲线拟合。将50％抑制率代入曲线方程，计算出稀释倍数×100作为生物学活性值(BU/ml)。比活(BU/mg)＝活性值/蛋白浓度。
实验结果如表2所示，计算得rDP1活性值为79616.0BU/ml；比活为9.95E+04BU/mg。同理检测nDF1作为对照，实验结果如表2所示，说明酵母表达rDP1，rDF1与天然蛋白相比具有相似的生物学活性。
表2：重组蛋白与天然蛋白活性值比较
样品 50％抑制率log10 稀释倍数 活性值(BU/mL) 蛋白浓度(mg/mL) 比活(BU/mg)
rDP1 2.901 796.1 79616.0 0.8 9.95E+04
nDP1 2.718 552.4 55239.6 1.0 5.52E+04
rDF1 2.513 325.8 32583.7 1.6 2.04E+04
nDF1 2.316 207.0 20701.4 1.5 1.38E+04
5、DP1/DF1宿主蛋白含量检测
宿主残留蛋白(Host Cell Protein，简称HCP)是指在生物制品中残留的宿主蛋白。这类蛋白成分复杂，种类繁多，且会因生产过程及纯化工艺的不同而发生变化。基因工程产品中残留的HCP是影响制品纯度的重要因素，反复使用含HCP的基因工程制品会引起机体的过敏反应，具有潜在的“佐剂效应”，也可能使机体对药物产生抗体，进而影响药物的疗效。HCP在生物制品中的残留量反映的不仅是制品批间的一致性，而且还是衡量生物制品质量的一个重要指标。发明人采用毕赤酵母HCP检测试剂盒(F140，CYGNUS)对制备的样品进行检测，表3显示经三步纯化的DP1，DF1蛋白HCP含量均远低于2015版药典规定的重组生物制品(酵母)HCP最高限度。
表3 DP1和DF1蛋白HCP检测结果
Figure PCTCN2019091968-appb-000002
6、DP1/DF1残留DNA含量检测
重组蛋白类药物虽然经过多步纯化，但制品中仍有可能残留宿主细胞的DNA片段，这些残留DNA可能带来传染性或致瘤性风险，可能会造成插入突变、导致抑癌基因失活、癌基因被激活等；此外，微生物来源的基因组DNA富含CpG和非甲基化序列，增加了重组蛋白类药物在体内的免疫原性风险，因此WHO和各国药物注册监管机构对残留DNA的限量要求非常严格。目前检测DNA残留量的方法主要有DNA探针杂交法、荧光染料法和qPCR法，其中前两种方法都存在技术缺陷，很难达到杂质限量检测的灵敏度，FDA在最新版USP中规定了qPCR为唯一推荐的残留DNA检测方法，因此发明者采用qPCR对样品中的残留DNA进行了检测(SK030205P100，湖州申科生物技术有限公司)，表4结果显示经三步纯化的样品残留DNA含量均远低于2015版药典规定的重组生物制品(酵母)残留DNA含量最高限度。
表4.DP1和DF1蛋白残留DNA检测结果
Figure PCTCN2019091968-appb-000003
实施例5重组DP2/DF2大规模(30L)高密度发酵实验
下面先以DP2的大规模(30L)高密度发酵进行说明
步骤1：重组菌株活化
取冻实施例1中得到的存于-80℃的工作种子库中甘油菌种子于YPD固体培养基(酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂糖15g/L)划线，30℃恒温恒湿箱培养3-5天。
步骤2：一级种子液培养
挑取步骤1中固体培养基上单克隆菌落于YPD液体培养基(酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L)，30℃，220rpm培养至OD 600≈6.0，并在显微镜下观察无杂菌，即得 到发酵用一级种子液。
步骤3：二级种子液培养
将步骤2中获得的一级种子液接种至赛多利斯B-plus发酵罐中，并以60％BSM，pH＝5.5±0.2，27℃，300rpm值培养，通过通气和转速条件是溶氧保持在25％，最终至OD 600≈40，湿重达到约80g/L，并在显微镜下观察无杂菌，即得到发酵用二级种子液。
步骤4：发酵过程
洗净赛多利斯Cplus生物反应器，用pH＝7.0和pH＝4.0的标准液分别校正发酵罐的pH计探头，然后按照实施例1中关键工艺参数的设计区间，确定发酵培养基为60％BSM进行配置发酵培养基20L，并倒入发酵罐中，121℃在线灭菌20min，待温度降到50℃后，用浓氨水调pH＝5.5±0.2。
将步骤3中得到的种子液按照1:15(V/V，种子液/发酵培养基)比例接入发酵罐中。初期重组毕赤酵母菌体增殖阶段发酵温度为27℃，pH＝6.5±0.2，转速＝300rpm培养，DO值100％，添加PTM1(2ml/L)。此阶段大约20h，当碳源消耗完毕后，溶氧值迅速上升，菌体湿重达到100g/L，此时进入限速生长阶段，此阶段的开始的2h以30ml/h -1L -1的速率补加50％甘油，2h后补料速度上调为60ml/h -1L -1。补加4h，菌体湿重约200g/L，停止补料，DO上升，表明碳源耗尽，进入诱导阶段。调节pH＝6.5±0.2，以甲醇流加速度为30ml/h -1L -1进行诱导表达重组DP2；维持DO不高于40％，诱导温度为20～22℃，pH＝6.0±0.2，进行发酵表达，发酵开始后每隔一段时间取样一次，测发酵液OD 600吸收值以及菌体湿重，在重组毕赤酵母发酵期间，发酵液OD 600均能达到300以上，且菌体湿重均能达到400g/L。诱导40h发酵结束。
重组DP2的上述发酵工艺对重组DF2同样适用。
实施例6重组DP2/DF2纯化工艺
一、重组DP2发酵液上清第一步纯化
步骤1、发酵液的预处理
按上述实施例中得到的发酵液高速离心，得到上清液；加入硅藻土助滤，过夜得到澄清的发酵液样品；用3KD膜包超滤稀释，降低电导至5mS/cm以下。
步骤2、阳离子交换层析
将上述处理后的发酵液上清乙酸调节pH4.0，上SP FF层析柱，柱型为BPG140/100，柱床体积2800ml，平衡缓冲液为50mM NaAc，pH4.0，洗脱缓冲液为50mM NaAc，1.0M NaCl，pH4.0，按照0-100％线性洗脱，DP2/DF2目的蛋白主要集中在第二个洗脱峰。
其余条件不变，调节样品和缓冲液pH5.0。
其余条件不变，调节样品和缓冲液pH6.0。
经对比得知，随着pH增加，得到的DP2蛋白纯度更高，杂质更少，利于后续的纯化处理，其中pH6.0为最佳条件。
二、重组DP2蛋白第二步纯化
步骤1、超滤
合并实施例步骤2中第二个洗脱峰，3KD膜包超滤稀释至电导<2.0mS/cm。
步骤2、第二步阴离子交换层析
将步骤1中样品加入20mM Tris，调节pH6.0，上Q FF阴离子交换层析柱，柱型为Hiscale50/40，柱床体积500ml，平衡缓冲液为20mM NaH 2PO 4，pH6.0，洗脱缓冲液为20mM NaH 2PO 4，1.0M NaCl，pH6.0，收集穿透，0-100％线性洗脱，目的蛋白主要集中在穿透。
其余条件不变，调节样品和缓冲液pH7.5，电导10.0mS/cm。
其余条件不变，调节样品和缓冲液pH9.0，电导20.0mS/cm。
经对比得知pH7.5，电导10.0mS/cm条件最佳，经优化后目的蛋白与杂质分离度更高，目的蛋白大部分留在穿透中，杂质结合到介质中，实现了完全分离，且大大提高了DP2蛋白在穿透中的收率。
三、重组DP2蛋白第三步纯化
将第二步纯化后的样品加入硫酸铵至终浓度1.0M，调节pH6.0，上Phenyl FF层析柱，柱型为Hiscale 50/40，柱床体积500ml，平衡缓冲液为20mM NaH 2PO 4，1.0M(NH 4) 2SO 4，pH6.0，洗脱缓冲液为20mM NaH 2PO 4，pH6.0，按照25，50％，70％，100％等度洗脱。
其余条件不变，调节样品和平衡缓冲液硫酸铵浓度1.0M，pH7.0。
其余条件不变，调节样品和平衡缓冲液硫酸铵浓度2.0M，pH8.0。
经对比得知，硫酸铵浓度1.0M，pH6.0条件最佳，经优化后得到的DP2蛋白纯度更高，蛋白量也最多，穿透中无目的蛋白，全部结合到填料中。
表5所示经过优化后DP2蛋白纯化收率比优化前提高30％，活性(ELISA方法测定)与工艺优化前相比也有所提高。
表5.DP2蛋白纯化工艺优化前后收率
Figure PCTCN2019091968-appb-000004
重组DP2的上述纯化工艺对重组DF2同样适用。
实施例7重组DP2/DF2蛋白鉴定
1、浓缩，测定蛋白浓度
将实施例6中梯度洗脱峰收集后用Vivaflow50切向流超滤膜包(VF05P9-50cm 2,Sartorius)浓缩，透析袋置换缓冲溶液为pH7.4PBS溶液；Pierce BCA蛋白浓度试剂盒测定蛋白浓度。
2、DP2/DF2蛋白HPLC纯度分析
将纯化得到的DP2/DF2样品稀释至1mg/ml，0.22um滤膜过滤，即得到样品。流动相为20mM PB，pH7.5，流速1ml/min，进样10ul，RP柱分析纯度，图3-a，3-b为纯度检测结果，经三步纯化的DP2、DF2蛋白纯度大于95％。
3、DP2/DF2蛋白氨基酸覆盖率分析
氨基酸覆盖率分析是重组蛋白类药物质量研究中非常重要的指标之一，只有氨基酸一级结构的完全相同，才能保证产品具有与天然蛋白相同的的生物学活性。发明人委托上海中科新生命生物科技有限公司对DP2，DF2蛋白进行氨基酸覆盖率分析，图4-a，4-b结果显示氨基酸覆盖率均为100％，符合相关政策法规的要求。
4、DP2/DF2宿主蛋白含量检测
宿主残留蛋白(Host Cell Protein，简称HCP)是指在生物制品中残留的宿主蛋白。这类蛋白成分复杂，种类繁多，且会因生产过程及纯化工艺的不同而发生变化。基因工程产品中残留的HCP是影响制品纯度的重要因素，反复使用含HCP的基因工程制品会引起机体的过敏反应，具有潜在的“佐剂效应”，也可能使机体对药物产生抗体，进而影响药物的疗效。HCP在生物制品中的残留量反映的不仅是制品批间的一致性，而且还是衡量生物制品质量的一个重要指标。发明人采用毕赤酵母HCP检测试剂盒(F140，CYGNUS)对制备的样品进行检测，表6显示经三步纯化的DP2，DF2蛋白HCP含量均远低于2015版药典规定的重组生物制品(酵母)HCP最高限度。
表6.DP2和DF2蛋白HCP检测结果
Figure PCTCN2019091968-appb-000005
5、DP2/DF2残留DNA含量检测
重组蛋白类药物虽然经过多步纯化，但制品中仍有可能残留宿主细胞的DNA片段，这些 残留DNA可能带来传染性或致瘤性风险，可能会造成插入突变、导致抑癌基因失活、癌基因被激活等；此外，微生物来源的基因组DNA富含CpG和非甲基化序列，增加了重组蛋白类药物在体内的免疫原性风险，因此WHO和各国药物注册监管机构对残留DNA的限量要求非常严格。目前检测DNA残留量的方法主要有DNA探针杂交法、荧光染料法和qPCR法，其中前两种方法都存在技术缺陷，很难达到杂质限量检测的灵敏度，FDA在最新版USP中规定了qPCR为唯一推荐的残留DNA检测方法，因此发明者采用qPCR对样品中的残留DNA进行了检测(SK030205P100，湖州申科生物技术有限公司)，表7结果显示经三步纯化的样品残留DNA含量均远低于2015版药典规定的重组生物制品(酵母)残留DNA含量最高限度。
表7.DP2和DF2蛋白残留DNA检测结果
Figure PCTCN2019091968-appb-000006
实施例8单一重组蛋白与天然蛋白活性比较
利用竞争抑制ELISA方法，对rDF1/rDF2/rDP1/rDP2生物学活性进行测定，并与天然nDF1/nDF2/nDP1/nDP2进行比较。具体步骤为(以rDP2为例)：
1、包被：分别使用包被液(pH 9.6 0.15M碳酸盐缓冲液)将rDP2/nDP2稀释至2ug/ml，100ul/孔，4℃孵育过夜。
2、封闭：PBST(pH7.4 0.15M PBS+0.05％Tween20)洗3次，拍干，每孔加入200ul封闭液(1％BSA/PBST)，37℃孵育2h。
3、样品稀释：将rDP2/nDP2样品进行1000倍稀释，后进行3倍梯度稀释，共7个稀释度。取各个稀释度的样品和适当稀释度的血清库血清(15份以上phadia100检测d1/d2特异性IgE值＞100Kua/L病人血清混合库)等体积混合，即125ul样品+125ul血清，混合后的样品放入4℃孵育过夜。阳性对照：125ul稀释液+125ul血清。
4、加样：混合孵育好的样品加入相应的包被孔中，37℃孵育90min。
5、二抗孵育：PBST洗4次，拍干，每孔加入100ul二抗稀释液(鼠抗人IgE-HRP 1:1500稀释)，37℃孵育1h。
6、显色：PBST洗4次，拍干，每孔加入100ul TMBⅠ号显色液，37℃显色10min。
7、终止及读数：每孔加入50ul 2M H 2SO 4终止反应，450nm波长处读数。
8、结果分析：利用EXCEL软件，以抑制率％(阳性值-样品值/阳性值)作为横坐标，以 Log10稀释倍数为纵坐标，做二次曲线拟合。将50％抑制率代入曲线方程，计算出稀释倍数×100作为生物学活性值(BU/ml)。比活(BU/mg)＝活性值/蛋白浓度。实验结果如表8与图5所示，计算得rDP2活性值为125675BU/ml；比活为1.40E+05BU/mg。同理检测nDF1/nDF2/nDP1/nDP2作为对照，实验结果如表8所示，说明酵母表达rDF1/rDF2/rDP1/rDP2与天然蛋白相比具有相似的生物学活性。
表8.重组蛋白与天然蛋白活性值比较
样品 50％抑制率log10 稀释倍数 活性值(BU/ml) 蛋白浓度(mg/ml) 比活(BU/mg)
rDP1 5.17 146808.10 14680809.4 1.1 1.29E+07
nDP1 4.73 53119.02 5311901.3 1.10 4.83E+06
rDF1 5.03 107398.94 10739894.1 1.56 6.88E+06
nDF1 5.17 148679.17 14867912.6 1.00 1.49E+07
rDP2 4.17 14859.32 1485935.6 1.00 1.49E+06
nDP2 4.25 17762.30 1776233.3 1.80 9.87E+05
rDF2 3.68 4830.64 483058.8 1.5 3.31E+05
nDF2 3.41 2588.28 258821.3 1.20 2.16E+05
实施例9重组合剂(等质量)与天然合剂(等质量)活性的比较
1.包被和稀释按照以下进行：
表9
Figure PCTCN2019091968-appb-000007
2.稀释时分别将重组与天然合剂样品按照等质量(1:1:1:1)混合成1.0mg/ml合剂，然后稀释1000倍至其实测定浓度；再往下3倍稀释7个梯度，重组合剂与天然合剂稀释样品与血清混合后加入均采用天然合剂包被的包被体系中进行检测。
3.其余操作步骤同实施例8基本一致。
4.结果分析：表10中结果所示，重组合剂与天然合剂相比，体外活性更高，与血清中IgE结合力更高。
表10.重组合剂与天然合剂活性比较
样品 50％抑制率log10 稀释倍数 活性值(BU/ml) 蛋白浓度(mg/ml) 比活(BU/mg)
天然合剂 3.55 3552.22 355222.15 1.14 3.55E+05
重组合剂 4.17 14769.81 1476981.27 1.10 1.48E+06
实施例10重组合剂与单一组分重组蛋白活性的比较
利用竞争抑制ELISA方法，对重组合剂按照以下比例混合分别比较与单一组分的rDF1/rDF2/rDP1/rDP2的生物学活性。具体步骤为(以rDP1为例)：
1、包被：按质量比rDP1：rDP2：rDF1：rDF2＝20:1:20:1制成重组合剂，稀释到2ug/ml，100ul/孔，4℃孵育过夜。(其他重组合剂的比例按照相同的顺序)
2、封闭：PBST(pH7.4 0.15M PBS+0.05％Tween20)洗3次，拍干，每孔加入200ul封闭液(1％BSA/PBST)，37℃孵育2h。
3、样品稀释：将1中混合的重组合剂及进行比较的rDP1样品进行1000倍稀释，后进行3倍梯度稀释，共7个稀释度。取各个稀释度的样品和适当稀释度的血清库血清(15份以上phadia100检测d1/d2特异性IgE值＞100Kua/L病人血清混合库)等体积混合，即125ul样品+125ul血清，混合后的样品放入4℃孵育过夜。阳性对照：125ul稀释液+125ul血清。
4.其余操作步骤同实施例8基本一致。
5、结果分析：利用EXCEL软件，以抑制率％(阳性值-样品值/阳性值)作为横坐标，以Log10稀释倍数为纵坐标，做二次曲线拟合。将50％抑制率代入曲线方程，计算出稀释倍数×100作为生物学活性值(BU/ml)。比活(BU/mg)＝活性值/蛋白浓度。以比较的rDP1活性定义为100％，计算进行比较的各种重组合剂活性与rDP1的比值(重组合剂与rDP2、rDF1、rDF2活性比较方法同rDP1)，实验结果如图6所示，一定比例的重组合剂表现出明显高于单一重组组分的活性。
实施例11重组合剂与I类、II类重组变应原蛋白活性的比较
利用竞争抑制ELISA方法，对重组合剂按照以下比例混合分别比较与单一组分的rDF1/rDF2/rDP1/rDP2的生物学活性。具体步骤为(以rDP1为例)：
1、包被：按质量比rDP1：rDP2：rDF1：rDF2＝20:1:20:1制成重组合剂，稀释到2ug/ml，100ul/孔，4℃孵育过夜。(其他重组合剂的比例按照相同的顺序)
2、封闭：PBST(pH7.4 0.15M PBS+0.05％Tween20)洗3次，拍干，每孔加入200ul封闭液(1％BSA/PBST)，37℃孵育2h。
3、样品稀释：将1中混合的重组合剂及进行比较的rDP1+rDF1、rDP2+rDP2样品(按照20:1、5:1、1:1、1:5、1；20比例混合)进行1000倍稀释，后进行3倍梯度稀释，共7个稀释度。取各个 稀释度的样品和适当稀释度的血清库血清(15份以上phadia100检测d1/d2特异性IgE值＞100Kua/L病人血清混合库)等体积混合，即125ul样品+125ul血清，混合后的样品放入4℃孵育过夜。阳性对照：125ul稀释液+125ul血清。
4.其余操作步骤同实施例8基本一致。
5、结果分析：利用EXCEL软件，以抑制率％(阳性值-样品值/阳性值)作为横坐标，以Log10稀释倍数为纵坐标，做二次曲线拟合。将50％抑制率代入曲线方程，计算出稀释倍数×100作为生物学活性值(BU/ml)。比活(BU/mg)＝活性值/蛋白浓度。以比较的rDP1活性定义为100％，计算进行比较的各种重组合剂活性与rDP1的比值(重组合剂与rDP2、rDF1、rDF2活性比较方法同rDP1)，实验结果如图7所示，各比例的重组合剂几乎都表现出明显高于I类和II类重组变应原的活性。原因与实施例10类型，4种重组合剂产生的协同作用大于单独的I类或者II类重组变应原。
实施例12重组合剂与Odactra活性的比较
利用竞争抑制ELISA方法，对重组合剂按照实施例8中测定得到的活性比较与Odactra的生物学活性。
1、包被：按等活性比rDP1：rDP2：rDF1：rDF2＝1:1:1:1制成重组合剂，稀释到2ug/ml，100ul/孔，4℃孵育过夜。
2、封闭：PBST(pH7.4 0.15M PBS+0.05％Tween20)洗3次，拍干，每孔加入200ul封闭液(1％BSA/PBST)，37℃孵育2h。
3、样品稀释：将1中混合的重组合剂及进行比较的Odactra样品(按照标示量30ug/片计算)进行1000倍稀释，后进行3倍梯度稀释，共7个稀释度。取各个稀释度的样品和适当稀释度的血清库血清(15份以上phadia100检测d1/d2特异性IgE值＞100Kua/L病人血清混合库)等体积混合，即125ul样品+125ul血清，混合后的样品放入4℃孵育过夜。阳性对照：125ul稀释液+125ul血清。
4.其余操作步骤同实施例8基本一致。
5、结果分析：利用EXCEL软件，以抑制率％(阳性值-样品值/阳性值)作为横坐标，以Log10稀释倍数为纵坐标，做二次曲线拟合。将50％抑制率代入曲线方程，计算出稀释倍数×100作为生物学活性值(BU/ml)。比活(BU/mg)＝活性值/蛋白浓度。以比较的Odactra活性定义为100％，计算进行比较的重组合剂活性与Odactra的比值，实验结果如图8所示，等活性的重组合剂活性比Odcatra高出40％，说明等活性的重组合剂活性明显高于Odactra。
实施例13重组合剂与阳性药物Odactra脱敏效果比较
1.模型制备及脱敏治疗：BALB/c小鼠每周腹腔注射HDM(螨虫提取物，购自LSL公司，按DF 0.45μg+DP 0.40μg)+铝佐剂，连续约4周，末次致敏结束后鼻腔滴入HDM溶液(按DF0.90μg+DP 0.80μg)，连续7天。激发结束后，检测指标，包括：喷嚏、penh值(动物在乙酰甲胆碱Mch分别为3.125/6.25/12.5/25/50mg/ml浓度刺激下，使用BUXCO公司仪器全身体积描记仪WBP检测)等。根据各浓度下penh值平均值降序排列，选择数值较高动物入组，分成PBS治疗组、重组合剂组(DP1、DF1、DP2、DF2质量比1：1：1：1)25μg/只、阳性组24μg/只(以Odactra活性成分计)。舌下含服脱敏治疗，一周2次(2次之间间隔2～3天)，连续8周。同法再次进行鼻腔激发连续7天。激发结束后，检测指标，包括：喷嚏、penh值和脾细胞因子。
2.结果分析：表11所示，重组合剂、阳性组、正常组小鼠喷嚏数量均少于PBS组，有显著差异(p＜0.01)；此外，重组合剂组与阳性组小鼠在Mch浓度3.125～50mg/ml刺激下，penh值明显低于PBS组，重组合剂组与阳性组penh值接近。图9所示，脱敏后重组合剂组相比阳性组IL-4、5、13含量更低，与PBS组相比均出现了显著降低(P<0.05)；脱敏后重组合剂组和阳性组IL-10水平相当，与PBS组相比均出现了明显降低；说明细胞因子由Th2型偏多转向Th2和Th1平衡，在细胞因子水平或者脱敏机制上说明模型小鼠的过敏得到了改善。综上所述，在舌下含服脱敏治疗后，重组合剂组与阳性组在penh值、喷嚏数和细胞因子水平等呼吸道症状和机理上均有明显改善的作用，说明重组合剂的动物体内药效试验活性相当。
表11.penh值和喷嚏计数(mean±sem)
Figure PCTCN2019091968-appb-000008

Claims (9)

  1. 一种重组尘螨变应原蛋白药物合剂，其特征在于：所述药物合剂包含重组尘螨Ⅰ型变应原DP1、DF1蛋白和重组尘螨II型变应原DP21、DF2蛋白，并四种尘螨变应原蛋白DP1：DP2：DF1：DF2分别按活性比1:1:1:1混合或按质量比(1～5)：(1～20)：(1～10)：(1～20)混合。
  2. 如权利要求1所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述药物合剂中四种尘螨变应原蛋白DP1：DP2：DF1：DF2分别按质量比1：(1～20)：1：(1～20)混合。
  3. 如权利要求1所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述药物合剂中四种尘螨变应原蛋白DP1：DP2：DF1：DF2分别按质量比1：5：1：5混合。
  4. 如权利要求1～3任一项所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述重组尘螨Ⅰ型变应原DP1、DF1蛋白和重组尘螨II型变应原DP2、DF2蛋白，分别由如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示的基因通过毕赤酵母表达系统表达得到。
  5. 如权利要求4所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述重组尘螨Ⅰ型变应原蛋白由以下方法制备得到：
    首先，将权利要求4所述的基因构建到质粒pPICZα上，并转入毕赤酵母X33感受态细胞中，并培养获得宿主单克隆工程菌；其次，挑取单克隆工程菌经过高密度发酵，并经离子交换层析、阴离子交换层析、疏水层析的三步纯化得到。
  6. 如权利要求5所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述制备方法，在表达重组尘螨Ⅰ型变应原DP1、DF1蛋白时主要发酵工艺如下：所用发酵培养基为40％BSM培养基，高密度发酵的诱导阶段诱导温度为：25～27℃，pH值为6.5±0.2，高密度发酵的诱导阶段甲醇流加速度为30mL/h -1L -1，并维持DO不高于40％，高密度发酵的菌体增值阶段发酵温度为27℃，pH＝5.5±0.2，转速300rpm，DO值100％，添加PTM1(2.4ml/L)；高密度发酵的限速生长阶段开始以30ml/h -1L -1的速率补加50％甘油，后补料速度上调为60ml/h -1L -1。
  7. 如权利要求5所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述制备方法，在表达重组尘螨II型变应原DP2、DF2蛋白时主要发酵工艺如下：所用发酵培养基为60％BSM培养基，高密度发酵的诱导阶段诱导温度为：20～22℃，pH值为6.0±0.2；高密度发酵的诱导阶段甲醇流加速度为30mL/h -1L -1，并维持DO不高于40％；高密度发酵的限速生长阶段开始以30ml/h -1L -1的速率补加50％甘油，后补料速度上调为60ml/h -1L -1。
  8. 如权利要求5所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述制备方法，在纯化重组尘螨Ⅰ型变应原DP1、DF1蛋白时，
    其中阳离子交换层析的纯化填料为SPFF，pH为5.0；
    阴离子交换层析的纯化填料为Q FF，pH为8.0，电导为20.0mS/cm；
    疏水层析的纯化填料为phenyl FF，样品和平衡缓冲液硫酸铵浓度为2.0M，pH为8.5。
  9. 如权利要求5所述的重组尘螨变应原蛋白药物合剂，其特征在于：所述制备方法，在纯化重组尘螨II型变应原DP2、DF2蛋白时，
    其中，阳离子交换层析的纯化填料为SPFF，pH为6.0；
    阴离子交换层析的纯化填料为Q FF，pH7.5，电导10.0mS/cm；
    疏水层析的纯化填料为phenyl FF，样品和平衡缓冲液硫酸铵浓度1.0M，pH6.0。
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