Recombinant dust mite allergen protein drug mixture and its application

Abstract

The present invention relates to a recombinant dust mite allergen protein composition, which comprises recombinant dust mite type I allergen DP1, DF1 protein and recombinant dust mite type II allergen DP2, DF2 protein, and they are mixed at an activity ratio of 1:1:1:1 or at a mass ratio of (1-5): (1-20): (1-10): (1-20). Compared with existing products, the present invention has the following advantages: (1) recombinant allergens have the advantages of high purity and controllable quality; (2) recombinant proteins have good specificity; (3) compared with natural extracts, they have better immunogenicity, reduce the risk of severe allergic side effects, and improve the effect of desensitization treatment. Secondly, the recombinant allergen composition of the present invention not only has better activity than natural allergen proteins of the same proportion, but also has higher activity than the corresponding natural single component compared with a single component, and has a good prospect for product development and application.

Description

Technical Field

[0001] The present invention relates to a drug combination for treating dust mite allergy, in particular to an improved recombinant dust mite allergen protein drug mixture and application thereof. Background Art

[0002] Among the many allergens that cause allergic diseases, dust mites are the most prominent. Dust mites have a positive rate of approximately 70-80% in specific immunodiagnostics of patients with allergic diseases. Dust mites are numerous and widespread in human living and working environments. Their excreta, metabolites, and mite bodies all have strong allergenic properties. According to statistics, approximately 10% of the global population is allergic to dust mites, and approximately 80% of extrinsic asthma is caused by dust mites.

[0003] At present, dust mite allergen extracts are mainly used in clinical immunotherapy for dust mite allergies. For example, the "Changdi" dust mite drops of Zhejiang Wowu Biological, which was launched in 2006, are extracts of dust mite metabolic culture; Actair house dust mite sublingual tablets, which were launched in Japan in 2015, are used to treat allergic rhinitis caused by house dust mites (or household dust mites), and also use four components of natural type I and II household dust mites and type I and II dust mites; Odactra, which was approved by the FDA for marketing in 2017, also uses four components of natural type I and II household dust mites and type I and II dust mites. It can be seen that the use of mixtures has become a trend in dust mite desensitization treatment.

[0004] However, due to the complex composition of natural allergen extracts, maintaining a consistent composition is extremely difficult, and they are easily contaminated by exogenous toxic substances and pathogenic microorganisms, affecting their reproducibility and safety. Long-term use during treatment can easily lead to local reactions such as redness, swelling, nodules, and necrosis, as well as systemic reactions such as shock, edema, bronchospasm, urticaria, angioedema, and generalized erythema. Furthermore, using crude extracts for diagnosis cannot clearly determine the patient's response to each component of the allergen, which can easily lead to misdiagnosis. The quality of allergens is crucial for the diagnosis and treatment of allergic diseases. Allergens used for immunotherapy should be pure, not crude extracts. Furthermore, dust mite allergens are primarily found in feces and mite bodies. Extraction methods are time-consuming, cumbersome, and costly, and cannot prevent the occurrence of side effects during immunotherapy. Summary of the Invention

[0005] In order to overcome the above shortcomings, the purpose of the present invention is to provide a recombinant dust mite allergen protein drug mixture, and optimize the recombinant dust mite allergen protein drug mixture to obtain a drug mixture ratio with optimal activity.

[0006] First, the present invention provides a recombinant dust mite allergen protein pharmaceutical mixture, which contains recombinant dust mite type I allergen DP1, DF1 protein and recombinant dust mite type II allergen DP2, DF2 protein, and the four dust mite allergen proteins DP1:DP2:DF1:DF2 are mixed at an activity ratio of 1:1:1:1 or at a mass ratio of (1-5):(1-20):(1-10):(1-20).

[0007] Preferably, the four dust mite allergen proteins DP1:DP2:DF1:DF2 in the recombinant dust mite allergen protein pharmaceutical mixture are mixed in a mass ratio of 1:(1-20):1:(1-20) respectively.

[0008] More preferably, the four dust mite allergen proteins in the recombinant dust mite allergen protein pharmaceutical mixture are mixed at a mass ratio of 1:5:1:5.

[0009] Preferably, the recombinant dust mite type I allergen DP1, DF1 proteins and the recombinant dust mite type II allergen DP2, DF2 proteins are obtained by expressing the genes shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, through a Pichia pastoris expression system.

[0010] More preferably, the recombinant dust mite type I allergen protein is prepared by the following method:

[0011] 1. The above-mentioned gene was constructed into the plasmid pPICZα, and transferred into Pichia pastoris X33 competent cells, and cultured to obtain a host monoclonal engineered bacterium; 2. The monoclonal engineered bacterium was selected for high-density fermentation and purified by three steps of ion exchange chromatography, anion exchange chromatography, and hydrophobic chromatography.

[0012] Preferably, the above-mentioned high-density fermentation process, when expressing the recombinant dust mite type I allergen DP1 and DF1 proteins, is as follows: the fermentation medium used is 40% BSM medium, the induction temperature of the high-density fermentation induction stage is: 25-27°C, the pH value is 6.5±0.2, and the methanol flow acceleration of the high-density fermentation induction stage is 30 mL / h -1 L -1 , and maintain DO no higher than 40%. The fermentation temperature of the bacterial growth stage of high-density fermentation was 27°C, pH = 5.5 ± 0.2, rotation speed 300 rpm, DO value 100%, and PTM1 (2.4 ml / L) was added; the rate-limiting growth stage of high-density fermentation began at 30 ml / h -1 L -1 50% glycerol was added at a rate of 1000 ml / h, and the feeding rate was increased to 60 ml / h. -1 L -1 .

[0013] Preferably, when expressing recombinant dust mite type I allergen DP1 and DF1 proteins, the three-step purification process after high-density fermentation uses SP FF as the purification medium for cation exchange chromatography; the preferred pH range is 4.0-6.5. More preferably, the pH of the sample and buffer in the cation exchange chromatography system is 5.0.

[0014] The purification filler for anion exchange chromatography is QFF; the preferred pH range is 6.0-8.0; the preferred conductivity is 2.0 mS / cm-20.0 mS / 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.

[0015] The purification filler for hydrophobic chromatography is phenyl FF; the preferred pH range is 6.5-8.5; the preferred ammonium sulfate concentration is 1.0-2.0 M. More preferably, the ammonium sulfate concentration of the sample and equilibration buffer in the hydrophobic chromatography is 2.0 M and the pH is 8.5.

[0016] Preferably, the high-density fermentation process is as follows when expressing recombinant dust mite type II allergen DP2 and DF2 proteins: the fermentation medium used is 60% BSM medium, the induction temperature of the high-density fermentation induction stage is 20-22°C, the pH value is 6.0±0.2; the methanol flow acceleration of the high-density fermentation induction stage is 30 mL / h -1 L -1 , and maintain DO no higher than 40%; the rate-limiting growth phase of high-density fermentation begins at 30ml / h -1 L -1 50% glycerol was added at a rate of 1000 ml / h, and the feeding rate was increased to 60 ml / h. -1 L -1 .

[0017] Preferably, when expressing recombinant dust mite type II allergen DP2 and DF2 proteins, the three-step purification process after high-density fermentation uses SP FF as the purification medium for cation exchange chromatography; the preferred pH range is 4.0-6.0. More preferably, the pH of the sample and buffer is 6.0.

[0018] The purification filler for anion exchange chromatography is QFF; the preferred pH range is 6.0-9.0; the preferred conductivity is 2.0 mS / cm-20.0 mS / cm. More preferably, the sample and buffer solution have a pH of 7.5 and a conductivity of 10.0 mS / cm.

[0019] The purification filler for hydrophobic chromatography is phenyl FF; the preferred pH range is 6.0-8.0; and the preferred ammonium sulfate concentration is 1.0-2.0 M. More preferably, the ammonium sulfate concentration of the sample and equilibration buffer is 1.0 M, and the pH is 6.0.

[0020] Compared with existing products, the present invention has the following advantages: First, compared with crude extracts: (1) recombinant allergens have higher purity and do not contain non-allergenic components, enzymes, enzyme inhibitors and toxic proteins, etc. compared with crude extracts; (2) recombinant proteins have good specificity. The components in crude extracts are complex, and patients may only react to some of them, resulting in poor specificity, while recombinant allergens have a single component and good specificity; (3) recombinant allergens can replace natural extracts, reduce the antigenic epitopes bound by IgE compared with natural extracts, effectively reduce IgE-mediated allergic reactions, and at the same time retain the structural domains necessary for allergen T cell recognition, have good immunogenicity, reduce the risk of immunotherapy, and improve the effect of desensitization therapy. Secondly, after repeated exploration by the applicant, the recombinant allergen protein pharmaceutical composition of the present invention has also found the most appropriate composition ratio, so that the composition not only has better activity than the natural allergen protein of the same ratio (the activity of the recombinant allergen protein alone is basically the same as that of the natural one), but also has higher activity than the most active allergen protein among the single components. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the HPLC analysis results of DP1 / DF1 protein purity.

[0022] in Figure 1-a This is the RP-HPLC analysis result of DP1 protein; Figure 1-b The figure shows the results of RP-HPLC analysis of DF1 protein.

[0023] Figure 2 shows the results of amino acid coverage analysis of DP1 / DF1 protein.

[0024] in Figure 2-a This is the analysis result of amino acid coverage of DP1 protein; Figure 2-b The results of amino acid coverage analysis of DF1 protein are shown.

[0025] FIG3 shows the HPLC analysis results of DP2 / DF2 protein purity.

[0026] in Figure 3-a This is the RP-HPLC analysis result of DP2 protein; Figure 3-b The figure shows the RP-HPLC analysis results of DF2 protein.

[0027] Figure 4 shows the results of amino acid coverage analysis of DP2 / DF2 protein.

[0028] in Figure 4-a This is the analysis result of amino acid coverage of DP2 protein; Figure 4-b The results of amino acid coverage analysis of DF2 protein are shown in Figure 2.

[0029] FIG5 is a graph showing the relationship between the inhibition rate of single-component recombinant protein (r) and native protein (n) Log10 (dilution multiple);

[0030] in Figure 5-a This is a graph showing the relationship between Log10 (dilution factor) of natural DP1 protein and inhibition rate;

[0031] in Figure 5-b This is a curve diagram showing the relationship between the Log10 (dilution factor) of the recombinant DP1 protein and the inhibition rate;

[0032] in Figure 5-c This is a graph showing the relationship between Log10 (dilution factor) of natural DP2 protein and inhibition rate;

[0033] in Figure 5-d This is a curve diagram showing the relationship between the Log10 (dilution factor) of the recombinant DP2 protein and the inhibition rate;

[0034] in Figure 5-e This is a graph showing the relationship between Log10 (dilution factor) of natural DF1 protein and inhibition rate;

[0035] in Figure 5-f This is a curve diagram showing the relationship between the Log10 (dilution factor) of the recombinant DF1 protein and the inhibition rate;

[0036] in Figure 5-g This is a graph showing the relationship between Log10 (dilution multiple) of natural DF2 protein and inhibition rate;

[0037] in Figure 5-h This is a curve diagram showing the relationship between the Log10 (dilution multiple) of the recombinant DF2 protein and the inhibition rate;

[0038] FIG6 shows the activity percentages of recombinant agents at different ratios (mass ratio) compared with single-component recombinant proteins;

[0039] in Figure 6-a Comparison of the activity of recombinant agents with different proportions (mass ratio) and single-component rDP1;

[0040] in Figure 6-b Comparison of the activity of recombinant agents with different proportions (mass ratio) and single-component rDP2;

[0041] in Figure 6-c Comparison of the activity of recombinant agents with different ratios (mass ratio) and single-component rDF1;

[0042] in Figure 6-d Comparison of the activity of recombinant agents with different proportions (mass ratio) and single-component rDF2;

[0043] Figure 7Comparison of the activity of four protein recombinant preparations in different proportions (mass ratio) with two allergens of categories I and II;

[0044] Figure 8 For the comparison of the activity of the recombinant formulation with that of Odactra;

[0045] Figure 9 The results of cytokine content detection in the supernatant of spleen cell stimulation after desensitization in each drug-treated group;

[0046] in Figure 9 -a is the IL-4 content detection result in the supernatant of spleen cell stimulation after desensitization in each drug-treated group;

[0047] in Figure 9 -b is the IL-5 content detection results in the supernatant of spleen cell stimulation after desensitization in each drug-treated group;

[0048] in Figure 9 -c is the IL-13 content detection result in the supernatant of spleen cell stimulation after desensitization in each drug-treated group;

[0049] in Figure 9 -d is the IL-10 content detection result in the supernatant of spleen cell stimulation after desensitization in each drug-treated group; DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific examples. It should be understood that the examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] Example 1 Codon optimization of recombinant proDP1, construction of expression plasmid and construction of host engineering strain

[0052] Step 1: Recombinant codon optimization

[0053] Based on the DNA sequence of DP1 published by EMBL-EBI (ENA accession number: FM177224.1), the inventors obtained codon optimization and modification to obtain the proDP1 gene of the present invention (this gene adds an alpha-factor signal peptide and a Kozak sequence GCCACCATGG to the wild-type gene and is codon-optimized), and the nucleotide sequence is shown in SEQ ID No: 1.

[0054] Based on the DF1 DNA sequence published in GenBank (GenBank accession number: AB034946.1), the inventors obtained the proDF1 gene of the present invention after codon optimization and modification in combination with the requirements of the expression system of the present invention and multiple verifications (this gene adds an alpha-factor signal peptide and Kozak sequence GCCACCATGG on the basis of the wild-type gene, and is codon-optimized), the nucleotide sequence of which is shown in SEQ ID No: 2.

[0055] Based on the DNA sequence of DP2 published in GenBank (GenBank accession number: AAF86462), the inventors obtained the DP2 gene of the present invention after codon optimization and modification in combination with the requirements of the expression system of the present invention and multiple verifications. The nucleotide sequence is shown in SEQ ID No: 3.

[0056] Based on the DNA sequence of DF2 published in GenBank (GenBank accession number: EF139432.1), the inventors optimized the gene codons in combination with the requirements of the expression system of the present invention and after multiple verifications to obtain the DF2 gene of the present invention, the nucleotide sequence of which is shown in SEQ ID No: 4.

[0057] Step 2: Construction of expression plasmid containing proDP1 gene

[0058] The codon-optimized proDP1 was introduced with an XhoI restriction site sequence at the 5' end and an XbaI restriction site sequence at the 3' end, and whole gene synthesis was performed. The synthesized gene fragment was constructed into the pUC57 plasmid (provided by Nanjing GenScript Technology Co., Ltd.) to obtain a long-term storage plasmid, which was recorded as pUC57-proDP1 plasmid.

[0059] PCR amplification was performed using the pUC57-proDP1 plasmid as a template. The primer sequences used are as follows:

[0060] Upstream primer (SEQ ID No: 5):

[0061] M13F:TGT AAA ACG ACG GCC AGT

[0062] Downstream primer (SEQ ID No: 6):

[0063] M13R:CAG GAA ACA GCT ATG AC

[0064] The total reaction volume was 50 μL, with 2.5 μL of each primer at a concentration of 10 μmol / L and 1 μL of dNTPs at a concentration of 10 mmol / L. 0.5 μL of Q5 DNA polymerase (#M0491L, purchased from New England BioLabs) was used at 2 U / μL. 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, which showed that the product size was consistent with the expected size (1000 bp). After double digestion with XhoI (#R0146S, purchased from New England BioLabs) and XbaI (#R01445S, purchased from New England BioLabs), the resulting gene product was purified by 1% agarose gel electrophoresis using a DNA gel recovery kit (DP214, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.). The plasmid was ligated into the pPICZα A plasmid (V173-20, purchased from Invitrogen) using T4 ligase (#M0202S, purchased from New England BioLabs), transformed into DH5α competent cells (CB101, purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and cultured overnight at 37°C in LB solid medium supplemented with bleomycin (purchased from Invitrogen). The next day, positive clones were sequenced and aligned to the expected sequence, resulting in the codon-optimized proDP1 expression plasmid, designated pPICZα-proDP1.

[0065] Step 3: Construction of Pichia pastoris host engineering strain containing recombinant proDP1 gene

[0066] YPDS solid medium was prepared as provided in the Invitrogen Easy Select Pichia Expression Kit instructions, containing 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 15 g / L agarose, and 182 g / L sorbitol.

[0067] Prepare electrocompetent cells according to the Invitrogen Easy Select Pichia Expression Kit instructions. Linearize the plasmid pPICZα-proDP1 obtained in Step 2 with Sac I restriction endonuclease (#R0156S, purchased from New England Biolabs). After ethanol precipitation, the linearized vector was electroporated into Pichia pastoris X33 competent cells, plated onto YPDS solid medium, and cultured at 30°C until transformants emerged.

[0068] The construction of other expression plasmids and host engineering strains of proDF1, DP2, and DF2 was consistent with the above method and will not be repeated here.

[0069] Example 2 Large-scale (30 L) high-density fermentation experiment of recombinant DP1 / DF1

[0070] The following is an explanation of DP1 large-scale (30L) high-density fermentation.

[0071] Step 1: Activation of recombinant strains

[0072] The glycerol bacteria seeds in the working seed bank frozen at -80°C produced after the above example were streaked onto YPD solid medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agarose 15 g / L) and cultured in a constant temperature and humidity chamber at 30°C for 3-5 days.

[0073] Step 2: Primary seed liquid culture

[0074] Pick the monoclonal colony on the plate in step 1 and culture it in YPD liquid medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) at 30°C and 220 rpm until the OD 600 ≈6.0, and no foreign bacteria are observed under a microscope, thus obtaining the first-level seed liquid for fermentation.

[0075] Step 3: Secondary seed liquid culture

[0076] The first-level seed solution obtained in step 2 was inoculated into a Sartorius B-plus fermenter and cultured in 60% BSM, pH = 5.5 ± 0.2, 27 ° C, 300 rpm, and the dissolved oxygen was maintained at 25% by ventilation and rotation speed conditions until the OD 600 ≈40, the wet weight reaches about 80g / L, and no foreign bacteria are observed under a microscope, thus obtaining the secondary seed liquid for fermentation.

[0077] Step 4: Fermentation

[0078] The Sartorius Cplus bioreactor was cleaned, and the pH meter probe of the fermenter was calibrated with standard solutions of pH = 7.0 and pH = 4.0, respectively. Then, according to the design interval of the key process parameters in Example 1, the fermentation medium was determined to be 40% BSM. 20 L of fermentation medium was prepared and poured into the fermenter. It was sterilized online at 121°C for 20 min. After the temperature dropped to 50°C, the pH was adjusted to 5.5 ± 0.2 with concentrated ammonia water.

[0079] The seed solution obtained in step 3 was inoculated into the fermentation tank at a ratio of 1:15 (V / V, seed solution / fermentation medium). The fermentation temperature of the initial recombinant Pichia yeast cell proliferation stage was 27°C, pH = 5.5 ± 0.2, rotation speed = 300 rpm, DO value 100%, and PTM1 (2.4 ml / L) was added. This stage lasted about 20 hours. After the carbon source was consumed, the dissolved oxygen value rose rapidly and the wet weight of the cell reached 100 g / L. At this time, the rate-limiting growth stage was entered. The first 2 hours of this stage were 30 ml / h. -1 L -1 50% glycerol was added at a rate of 100 ml / h. After 2 h, the feeding rate was increased to 60 ml / h. -1 L -1 After 4 hours of feeding, the wet weight of the cells was about 200 g / L. The feeding was stopped and the DO increased, indicating that the carbon source was exhausted and the induction stage began. The pH was adjusted to 6.0 ± 0.2 and the methanol flow rate was 30 ml / h. -1 L -1 Induce the expression of recombinant DF1; maintain DO no higher than 40%, induction temperature at 25-27°C, pH = 6.5 ± 0.2, and perform fermentation expression. After the fermentation begins, take samples at regular intervals to measure the OD of the fermentation liquid. 600 Absorption value and wet weight of bacteria, during the fermentation of recombinant Pichia pastoris, the fermentation broth OD 600 The concentration of nitric oxide (NO3) and nitric oxide (NO3) can reach above 300, and the wet weight of the bacteria can reach 400 g / L. The fermentation is completed after 40 hours of induction.

[0080] The above fermentation process of recombinant DP1 is also applicable to recombinant DF1.

[0081] Example 3 Recombinant DP1 / DF1 Purification Process

[0082] 1. First step purification of recombinant DP1 / DF1 fermentation broth supernatant

[0083] Step 1: Pretreatment of fermentation broth

[0084] The fermentation broth obtained in the above example was centrifuged at high speed to obtain a supernatant; diatomaceous earth was added to aid filtration and a clarified fermentation broth sample was obtained overnight; and the broth was diluted by ultrafiltration using a 10KD membrane package to reduce the conductivity to below 5 mS / cm.

[0085] Step 2: Cation exchange chromatography

[0086] The supernatant of the fermentation broth after the above treatment was adjusted to pH 4.0 with acetic acid and applied to an SP FF chromatography column with a column type of BPG140 / 100 and a column bed volume of 2800 ml. The equilibration buffer was 50 mM NaAc, pH 4.0, and the elution buffer was 50 mM NaAc, 1.0 M NaCl, pH 4.0. The elution was performed according to a 0-100% linear elution method, and the DP1 / DF1 target protein was mainly concentrated in the second elution peak.

[0087] The other conditions remained unchanged, and the pH of the sample and buffer was adjusted to 5.0.

[0088] The other conditions remained unchanged, and the pH of the sample and buffer was adjusted to 6.5.

[0089] By comparison, it was found that pH 5.0 was the best condition, and the purity of DP1 protein was the highest after purification, while impurities were present under the other conditions.

[0090] 2. Second step purification of recombinant DP1 / DF1 protein:

[0091] Step 1: Ultrafiltration

[0092] The second elution peak in the first purification step 2 was combined and diluted by ultrafiltration with a 10KD membrane package until the conductivity was <2.0mS / cm.

[0093] Step 2: Anion exchange chromatography

[0094] Add 20mM Tris to the sample in step 1, adjust the pH to 6.0, and apply it to a Q FF anion exchange chromatography column with a Hiscale 50 / 40 column type and a bed volume of 500ml. The equilibration buffer is 20mM NaH2PO4, pH 6.0, and the elution buffer is 20mM NaH2PO4, 1.0M NaCl, pH 6.0. The breakthrough is collected and the linear elution is 0-100%. The target protein is mainly concentrated in the breakthrough.

[0095] The other conditions remained unchanged, and the pH of the sample and buffer solution was adjusted to 7.0, and the conductivity was 10.0 mS / cm.

[0096] The other conditions remained unchanged, and the pH of the sample and buffer solution was adjusted to 8.0, and the conductivity to 20.0 mS / cm.

[0097] By comparison, the optimal conditions were pH 8.0 and conductivity 20.0 mS / cm. After optimization, the separation degree between the target protein and impurities was the highest. Most of the target protein remained in the penetration, and the impurities were bound to the medium, achieving complete separation and greatly improving the yield of DP1 / DF1 protein in the penetration.

[0098] 3. Third step purification of recombinant DP1 / DF1 protein

[0099] Ammonium sulfate was added to the sample after the second step purification to a final concentration of 1.0 M, the pH was adjusted to 6.5, and the sample was loaded onto a Phenyl FF chromatography column with a Hiscale 50 / 40 column type and a bed volume of 500 ml. The equilibrium buffer was 20 mM NaH2PO4, 1.0 M (NH4)2SO4, pH 6.0, and the elution buffer was 20 mM NaH2PO4, pH 6.0. Elution was performed at 25, 50%, 70%, and 100% isocratic rates.

[0100] The other conditions remained unchanged, and the concentration of ammonium sulfate in the sample and equilibrium buffer was adjusted to 1.0 M, pH 7.5.

[0101] The other conditions remained unchanged, and the concentration of ammonium sulfate in the sample and equilibrium buffer was adjusted to 2.0 M, pH 8.5.

[0102] After comparison, it was found that the optimal conditions were 2.0 M ammonium sulfate concentration and pH 8.5. The DP1 / DF1 protein obtained after optimization was pure The purity of the protein can reach more than 95%.

[0103] The purification process shown in Table 1 showed that the yield after optimization was about 30% higher than that before optimization, and the activity (determined by ELISA method) was also improved compared with the level before process optimization.

[0104] Table 1 Yield of DP1 protein before and after optimization of purification process

[0105]

[0106] The above purification process for recombinant DP1 is also applicable to recombinant DF1.

[0107] Example 4 Identification of recombinant DP1 / DF1 protein

[0108] 1. Concentrate and determine protein concentration

[0109] The gradient elution peaks in Example 3 were collected and then filtered through a Vivaflow 50 tangential flow ultrafiltration membrane (VF05P9-50cm 2 , Sartorius) concentration, the dialysis bag replaced the buffer solution with pH 7.4 PBS solution; the protein concentration was determined by Pierce BCA protein concentration kit.

[0110] 2. HPLC purity analysis of DP1 / DF1 protein

[0111] The purified DP1 / DF1 sample was diluted to 1 mg / ml and filtered through a 0.22 μm filter membrane. The mobile phase was 20 mM PB, pH 7.5, with a flow rate of 1 ml / min. 10 μl of sample was injected and the purity was analyzed using an RP column. Figure 1-a , Figure 1-b This is the purity test result. The purity of DP1 / DF1 protein after three-step purification is greater than 95%.

[0112] 3. Analysis of amino acid coverage of DP1 / DF1 protein

[0113] Amino acid coverage analysis is one of the most important indicators in the quality research of recombinant protein drugs. Only when the primary structure of amino acids is completely identical can the product be guaranteed to have the same biological activity as the natural protein. The inventor commissioned Shanghai Zhongke New Life Biotechnology Co., Ltd. to conduct amino acid coverage analysis on DP1 / DF1 protein. Figure 2-a , Figure 2-bThe results showed that the amino acid coverage rate was greater than 98%, which met the requirements of relevant policies and regulations.

[0114] 4. DP1 / DF1 protein activity analysis

[0115] The biological activity of recombinant DP1 and DF1 proteins (rDP1 and rDF1) was determined using competitive inhibition ELISA and compared with that of natural DP1 protein (nDP1). The specific steps are as follows (taking DP1 as an example):

[0116] (1) Coating: Dilute rDP1 and nDP1 to 2 μg / ml using coating solution (pH 9.6 0.15 M carbonate buffer), 100 μl / well, and incubate at 4°C overnight.

[0117] (2) Blocking: Wash three times with PBST (pH 7.4 0.15M PBS + 0.05% Tween 20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37°C for 2h.

[0118] (3) Sample dilution: dilute the rDP1 and nDP1 samples 30-fold, then perform a 3-fold gradient dilution for a total of 7 dilutions. Take samples of each dilution and serum from a serum pool of appropriate dilutions (a mixed pool of more than 15 patient serum samples with specific IgE values ​​greater than 100 Kua / L on d1 / d2 as detected by phadia100) and mix them in equal volumes, i.e., 125ul sample + 125ul serum. The mixed samples are incubated at 4°C overnight. Positive control: 125ul diluent + 125ul serum.

[0119] (4) Sample addition: Add the mixed incubated samples to the corresponding coated wells and incubate at 37°C for 90 minutes.

[0120] (5) Secondary antibody incubation: Wash 4 times with PBST, pat dry, add 100ul of secondary antibody dilution (mouse anti-human IgE-HRP 1:1500 dilution) to each well, and incubate at 37℃ for 1h.

[0121] (6) Color development: Wash 4 times with PBST, pat dry, add 100 μl TMBⅠ color development solution to each well, and develop at 37°C for 10 min.

[0122] (7) Termination and reading: Add 50ul 2M H2SO4 to each well to terminate the reaction and read the results at a wavelength of 450nm.

[0123] (8) Result processing: Using Excel software, a quadratic curve was fitted using the inhibition rate (% positive value - sample value / positive value) as the abscissa and the Log10 dilution factor as the ordinate. Substitute the 50% inhibition rate into the curve equation and calculate the biological activity value (BU / ml) as the dilution factor × 100. Specific activity (BU / mg) = activity value / protein concentration.

[0124] The experimental results are shown in Table 2. The calculated activity of rDP1 was 79616.0 BU / ml, and the specific activity was 9.95E+04 BU / mg. Similarly, nDF1 was tested as a control. The experimental results are shown in Table 2, indicating that yeast-expressed rDP1 has similar biological activity to the native protein.

[0125] Table 2: Comparison of activity values ​​between recombinant protein and native protein

[0126] sample 50% inhibition rate log10 Dilution multiple Activity value (BU / mL) Protein concentration (mg / mL) Specific activity (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

[0127] 5. DP1 / DF1 host protein content detection

[0128] Host cell protein (HCP) refers to the host protein remaining in biological products. This type of protein has complex components and a wide variety, and will change due to different production processes and purification processes. The residual HCP in genetically engineered products is an important factor affecting the purity of the products. Repeated use of genetically engineered products containing HCP can cause allergic reactions in the body, has a potential "adjuvant effect", and may also cause the body to produce antibodies to the drug, thereby affecting the efficacy of the drug. The residual amount of HCP in biological products reflects not only the consistency of product batches, but is also an important indicator for measuring the quality of biological products. The inventors used the Pichia pastoris HCP detection kit (F140, CYGNUS) to test the prepared samples. Table 3 shows that the HCP content of the DP1 and DF1 proteins purified in three steps is far below the maximum limit of HCP for recombinant biological products (yeast) specified in the 2015 edition of the Pharmacopoeia.

[0129] Table 3 DP1 and DF1 protein HCP detection results

[0130]

[0131] 6. Detection of residual DNA content of DP1 / DF1

[0132] Despite the multi-step purification process, recombinant protein drugs may still contain residual host cell DNA fragments. These residual DNA may pose infectious or tumorigenic risks, potentially causing insertional mutagenesis, inactivation of tumor suppressor genes, and activation of oncogenes. Furthermore, microbial genomic DNA is rich in CpG and unmethylated sequences, increasing the risk of immunogenicity in vivo. Consequently, the World Health Organization and national drug registration regulatory agencies have established very strict limits for residual DNA. Currently, the main methods for detecting residual DNA include DNA probe hybridization, fluorescent dyes, and quantitative polymerase chain reaction (qPCR). The first two methods have technical limitations and struggle to achieve the sensitivity required for impurity detection. The FDA has designated qPCR as the sole recommended method for residual DNA detection in the latest edition of the USP. Therefore, the inventors employed qPCR to detect residual DNA in samples (SK030205P100, Huzhou Shenke Biotechnology Co., Ltd.). The results in Table 4 show that the residual DNA content in samples purified through the three steps was well below the maximum limit for residual DNA in recombinant biological products (yeast) specified in the 2015 Pharmacopoeia.

[0133] Table 4. Detection results of residual DNA of DP1 and DF1 proteins

[0134]

[0135] Example 5 Large-scale (30 L) high-density fermentation experiment of recombinant DP2 / DF2

[0136] The following is an explanation of the large-scale (30L) high-density fermentation of DP2.

[0137] Step 1: Activation of recombinant strains

[0138] The glycerol bacteria seeds in the working seed bank stored at -80°C obtained in Example 1 were streaked onto YPD solid medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 15 g / L agarose) and cultured in a constant temperature and humidity chamber at 30°C for 3-5 days.

[0139] Step 2: Primary seed liquid culture

[0140] Pick the monoclonal colony on the solid medium in step 1 and culture it in YPD liquid medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) at 30°C and 220 rpm until the OD 600 ≈6.0, and no foreign bacteria are observed under a microscope, thus obtaining the first-level seed liquid for fermentation.

[0141] Step 3: Secondary seed liquid culture

[0142] The first-level seed solution obtained in step 2 was inoculated into a Sartorius B-plus fermenter and cultured in 60% BSM, pH = 5.5 ± 0.2, 27 ° C, 300 rpm, and the dissolved oxygen was maintained at 25% by ventilation and rotation speed conditions until the OD 600 ≈40, the wet weight reaches about 80g / L, and no foreign bacteria are observed under a microscope, thus obtaining the secondary seed liquid for fermentation.

[0143] Step 4: Fermentation

[0144] The Sartorius Cplus bioreactor was cleaned, and the pH meter probe of the fermenter was calibrated with standard solutions of pH = 7.0 and pH = 4.0, respectively. Then, according to the design interval of the key process parameters in Example 1, the fermentation medium was determined to be 60% BSM. 20 L of fermentation medium was prepared and poured into the fermenter. It was sterilized online at 121°C for 20 min. After the temperature dropped to 50°C, the pH was adjusted to 5.5 ± 0.2 with concentrated ammonia water.

[0145] The seed solution obtained in step 3 was inoculated into the fermentation tank at a ratio of 1:15 (V / V, seed solution / fermentation medium). The fermentation temperature of the initial recombinant Pichia yeast cell proliferation stage was 27°C, pH = 6.5 ± 0.2, rotation speed = 300 rpm, DO value 100%, and PTM1 (2 ml / L) was added. This stage lasted about 20 hours. After the carbon source was consumed, the dissolved oxygen value rose rapidly and the wet weight of the cell reached 100 g / L. At this time, the rate-limiting growth stage was entered. The first 2 hours of this stage were 30 ml / h. -1 L -1 50% glycerol was added at a rate of 100 ml / h. After 2 h, the feeding rate was increased to 60 ml / h. -1 L -1 After 4 hours of feeding, the wet weight of the bacteria reached about 200 g / L. The feeding was stopped and the DO increased, indicating that the carbon source was exhausted and the induction phase began. The pH was adjusted to 6.5 ± 0.2 and the methanol flow rate was 30 ml / h. -1 L -1 Induce expression of recombinant DP2; maintain DO no higher than 40%, induction temperature at 20-22°C, pH = 6.0 ± 0.2, and perform fermentation expression. After the fermentation begins, take samples at regular intervals to measure the OD of the fermentation liquid. 600 Absorption value and wet weight of bacteria, during the fermentation of recombinant Pichia pastoris, the fermentation broth OD 600 The concentration of nitric oxide (NO3) and nitric oxide (NO3) can reach above 300, and the wet weight of the bacteria can reach 400 g / L. The fermentation is completed after 40 hours of induction.

[0146] The above fermentation process of recombinant DP2 is also applicable to recombinant DF2.

[0147] Example 6 Recombinant DP2 / DF2 Purification Process

[0148] 1. First step purification of recombinant DP2 fermentation broth supernatant

[0149] Step 1: Pretreatment of fermentation broth

[0150] The fermentation broth obtained in the above example was centrifuged at high speed to obtain a supernatant; diatomaceous earth was added to aid filtration and a clarified fermentation broth sample was obtained overnight; and the broth was diluted by ultrafiltration using a 3KD membrane package to reduce the conductivity to below 5 mS / cm.

[0151] Step 2, cation exchange chromatography

[0152] The supernatant of the fermentation broth after the above treatment was adjusted to pH 4.0 with acetic acid and applied to an SP FF chromatography column with a column type of BPG140 / 100, a column bed volume of 2800 ml, an equilibration buffer of 50 mM NaAc, pH 4.0, and an elution buffer of 50 mM NaAc, 1.0 M NaCl, pH 4.0. The elution was performed according to a 0-100% linear elution method, and the DP2 / DF2 target protein was mainly concentrated in the second elution peak.

[0153] The other conditions remained unchanged, and the pH of the sample and buffer was adjusted to 5.0.

[0154] The other conditions remained unchanged, and the pH of the sample and buffer was adjusted to 6.0.

[0155] By comparison, it was found that as the pH increased, the obtained DP2 protein had higher purity and fewer impurities, which was conducive to subsequent purification treatment, among which pH 6.0 was the optimal condition.

[0156] 2. Second step purification of recombinant DP2 protein

[0157] Step 1: Ultrafiltration

[0158] The second elution peak in step 2 of Example was combined and diluted with 3KD membrane ultrafiltration to a conductivity of <2.0 mS / cm.

[0159] Step 2: Second step anion exchange chromatography

[0160] Add 20mM Tris to the sample in step 1, adjust the pH to 6.0, and apply it to a Q FF anion exchange chromatography column with a Hiscale 50 / 40 column type and a bed volume of 500ml. The equilibration buffer is 20mM NaH2PO4, pH 6.0, and the elution buffer is 20mM NaH2PO4, 1.0M NaCl, pH 6.0. The breakthrough is collected and the linear elution is 0-100%. The target protein is mainly concentrated in the breakthrough.

[0161] The other conditions remained unchanged, and the pH of the sample and buffer solution was adjusted to 7.5, and the conductivity was 10.0 mS / cm.

[0162] The other conditions remained unchanged, and the pH of the sample and buffer solution was adjusted to 9.0, and the conductivity to 20.0 mS / cm.

[0163] By comparison, it was found that pH 7.5 and conductivity 10.0 mS / cm were the best conditions. After optimization, the separation degree between the target protein and impurities was higher. Most of the target protein remained in the penetration, and the impurities were bound to the medium, achieving complete separation and greatly improving the yield of DP2 protein in the penetration.

[0164] 3. The third step of purification of recombinant DP2 protein

[0165] Ammonium sulfate was added to the sample after the second step purification to a final concentration of 1.0 M, the pH was adjusted to 6.0, and the sample was loaded onto a Phenyl FF chromatography column with a Hiscale 50 / 40 column type and a bed volume of 500 ml. The equilibrium buffer was 20 mM NaH2PO4, 1.0 M (NH4)2SO4, pH 6.0, and the elution buffer was 20 mM NaH2PO4, pH 6.0. Elution was performed at 25, 50%, 70%, and 100% isocratic rates.

[0166] The other conditions remained unchanged, and the concentration of ammonium sulfate in the sample and equilibrium buffer was adjusted to 1.0 M, pH 7.0.

[0167] The other conditions remained unchanged, and the concentration of ammonium sulfate in the sample and equilibrium buffer was adjusted to 2.0 M, pH 8.0.

[0168] By comparison, it was found that the optimal conditions were ammonium sulfate concentration of 1.0 M and pH 6.0. The DP2 protein obtained after optimization had higher purity and the highest protein amount. No target protein was found in the penetration, and all of it was bound to the filler.

[0169] As shown in Table 5, the purification yield of DP2 protein after optimization was increased by 30% compared with that before optimization, and the activity (determined by ELISA method) was also improved compared with that before process optimization.

[0170] Table 5. Yield of DP2 protein purification process before and after optimization

[0171]

[0172] The above purification process for recombinant DP2 is also applicable to recombinant DF2.

[0173] Example 7 Identification of recombinant DP2 / DF2 protein

[0174] 1. Concentrate and determine protein concentration

[0175] The gradient elution peaks in Example 6 were collected and then filtered through a Vivaflow 50 tangential flow ultrafiltration membrane (VF05P9-50cm 2, Sartorius) concentration, the dialysis bag replaced the buffer solution with pH 7.4 PBS solution; the protein concentration was determined by Pierce BCA protein concentration kit.

[0176] 2. HPLC purity analysis of DP2 / DF2 protein

[0177] The purified DP2 / DF2 sample was diluted to 1 mg / ml and filtered through a 0.22 μm filter membrane. The mobile phase was 20 mM PB, pH 7.5, with a flow rate of 1 ml / min. 10 μl of sample was injected and the purity was analyzed using an RP column. Figure 3-a 3-b is the purity test result. The purity of DP2 and DF2 proteins after three-step purification is greater than 95%.

[0178] 3. Analysis of amino acid coverage of DP2 / DF2 protein

[0179] Amino acid coverage analysis is one of the most important indicators in the quality research of recombinant protein drugs. Only when the primary structure of amino acids is completely identical can the product be guaranteed to have the same biological activity as the natural protein. The inventor commissioned Shanghai Zhongke New Life Biotechnology Co., Ltd. to conduct amino acid coverage analysis on DP2 and DF2 proteins. Figure 4-a ,4-b results showed that the amino acid coverage rate was 100%, which met the requirements of relevant policies and regulations.

[0180] 4. Detection of DP2 / DF2 host protein content

[0181] Host cell protein (HCP) refers to the host protein remaining in biological products. This type of protein has complex components and a wide variety, and will change due to different production processes and purification processes. The residual HCP in genetically engineered products is an important factor affecting the purity of the products. Repeated use of genetically engineered products containing HCP can cause allergic reactions in the body, has a potential "adjuvant effect", and may also cause the body to produce antibodies to the drug, thereby affecting the efficacy of the drug. The residual amount of HCP in biological products reflects not only the consistency of product batches, but is also an important indicator for measuring the quality of biological products. The inventors used the Pichia pastoris HCP detection kit (F140, CYGNUS) to test the prepared samples. Table 6 shows that the HCP content of the DP2 and DF2 proteins purified in three steps is far lower than the maximum limit of HCP for recombinant biological products (yeast) specified in the 2015 edition of the Pharmacopoeia.

[0182] Table 6. HCP detection results of DP2 and DF2 proteins

[0183]

[0184] 5. Detection of DP2 / DF2 residual DNA content

[0185] Despite the multi-step purification process, recombinant protein drugs may still contain residual host cell DNA fragments. These residual DNA may pose infectious or tumorigenic risks, potentially causing insertional mutagenesis, inactivation of tumor suppressor genes, and activation of oncogenes. Furthermore, microbial genomic DNA is rich in CpG and unmethylated sequences, increasing the risk of immunogenicity in vivo. Consequently, the World Health Organization and national drug registration regulatory agencies have established very strict limits for residual DNA. Currently, the main methods for detecting residual DNA include DNA probe hybridization, fluorescent dyes, and quantitative polymerase chain reaction (qPCR). The first two methods have technical limitations and struggle to achieve the sensitivity required for impurity detection. The FDA has designated qPCR as the sole recommended method for residual DNA detection in the latest edition of the USP. Therefore, the inventors employed qPCR to detect residual DNA in samples (SK030205P100, Huzhou Shenke Biotechnology Co., Ltd.). The results in Table 7 show that the residual DNA content in samples purified through the three steps was well below the maximum limit for residual DNA in recombinant biological products (yeast) specified in the 2015 Pharmacopoeia.

[0186] Table 7. Detection results of residual DNA of DP2 and DF2 proteins

[0187]

[0188] Example 8 Comparison of the activity of a single recombinant protein and a natural protein

[0189] The biological activities of rDF1 / rDF2 / rDP1 / rDP2 were determined using competitive inhibition ELISA and compared with those of natural nDF1 / nDF2 / nDP1 / nDP2. The specific steps are as follows (using rDP2 as an example):

[0190] 1. Coating: Dilute rDP2 / nDP2 to 2 μg / ml using coating solution (pH 9.6 0.15 M carbonate buffer), 100 μl / well, and incubate at 4°C overnight.

[0191] 2. Blocking: Wash three times with PBST (pH 7.4 0.15M PBS + 0.05% Tween 20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37°C for 2h.

[0192] 3. Sample Dilution: Dilute the rDP2 / nDP2 sample 1000-fold, then perform a 3-fold serial dilution for a total of seven dilutions. Mix equal volumes of each sample dilution with serum from a serum pool (a pool of at least 15 patient sera with specific IgE values ​​>100 Kua / L on day 1 / day 2 as measured by the Phadia 100) (125 μl sample + 125 μl serum). Incubate the mixed sample at 4°C overnight. Positive control: 125 μl diluent + 125 μl serum.

[0193] 4. Sample addition: Add the mixed incubated samples to the corresponding coated wells and incubate at 37°C for 90 minutes.

[0194] 5. Secondary antibody incubation: Wash 4 times with PBST, pat dry, add 100ul of secondary antibody dilution (mouse anti-human IgE-HRP 1:1500 dilution) to each well, and incubate at 37℃ for 1h.

[0195] 6. Color development: Wash 4 times with PBST, pat dry, add 100 μl of TMBⅠ color development solution to each well, and develop at 37°C for 10 min.

[0196] 7. Termination and reading: Add 50ul 2M H2SO4 to each well to terminate the reaction and read the reading at a wavelength of 450nm.

[0197] 8. Analysis of results: Using EXCEL software, a quadratic curve was fitted using the inhibition rate (% positive value - sample value / positive value) as the horizontal axis and the Log10 dilution factor as the vertical axis. Substitute the 50% inhibition rate into the curve equation and calculate the dilution factor × 100 as the biological activity value (BU / ml). Specific activity (BU / mg) = activity value / protein concentration. The experimental results are shown in Table 8 and Figure 5. The calculated activity value of rDP2 is 125675BU / ml; the specific activity is 1.40E+05BU / mg. Similarly, nDF1 / nDF2 / nDP1 / nDP2 were tested as a control. The experimental results are shown in Table 8, indicating that yeast-expressed rDF1 / rDF2 / rDP1 / rDP2 have similar biological activities to the natural protein.

[0198] Table 8. Comparison of activity values ​​between recombinant protein and native protein

[0199] sample 50% inhibition rate log10 Dilution multiple Activity value (BU / ml) Protein concentration (mg / ml) Specific activity (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

[0200] Example 9 Comparison of the activity of the recombinant preparation (equal mass) and the natural preparation (equal mass)

[0201] 1. Coating and dilution are carried out as follows:

[0202] Table 9

[0203]

[0204] 2. During dilution, the recombinant and natural mixture samples were mixed at equal mass (1:1:1:1) to form a 1.0 mg / ml mixture, and then diluted 1000-fold to the actual assay concentration; then diluted 3-fold in 7 steps, the recombinant and natural mixture diluted samples were mixed with serum and added to the coating system coated with the natural mixture for detection.

[0205] 3. The remaining steps are basically the same as those in Example 8.

[0206] 4. Result analysis: As shown in the results in Table 10, the recombinant preparation had higher in vitro activity and higher binding affinity to serum IgE than the natural preparation.

[0207] Table 10. Comparison of the activity of recombinant preparations and natural preparations

[0208]

[0209]

[0210] Example 10 Comparison of the activity of recombinant preparations and single component recombinant proteins

[0211] Using the competitive inhibition ELISA method, the biological activities of the recombinant agent were compared with those of the single components rDF1 / rDF2 / rDP1 / rDP2 by mixing them in the following ratios. The specific steps are as follows (using rDP1 as an example):

[0212] 1. Coating: Prepare a recombinant according to the mass ratio of rDP1:rDP2:rDF1:rDF2 = 20:1:20:1, dilute to 2ug / ml, add 100ul / well, and incubate at 4℃ overnight. (The ratios of other recombinant agents should be in the same order)

[0213] 2. Blocking: Wash three times with PBST (pH 7.4 0.15M PBS + 0.05% Tween 20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37°C for 2h.

[0214] 3. Sample Dilution: Dilute the recombinant reagent mixed in step 1 and the rDP1 sample for comparison 1000-fold, followed by a 3-fold serial dilution for a total of 7 dilutions. Mix equal volumes of each sample dilution with an appropriately diluted serum pool (a pool of serum from at least 15 patients with specific IgE values ​​>100 Kua / L on day 1 / day 2 as measured by the Phadia 100), i.e., 125 μl of sample + 125 μl of serum. Incubate the mixed sample at 4°C overnight. Positive control: 125 μl of diluent + 125 μl of serum.

[0215] 4. The remaining steps are basically the same as those in Example 8.

[0216] 5. Analysis of results: Using EXCEL software, a quadratic curve was fitted using the inhibition rate (% positive value - sample value / positive value) as the abscissa and the Log10 dilution factor as the ordinate. Substitute the 50% inhibition rate into the curve equation and calculate the dilution factor × 100 as the biological activity value (BU / ml). Specific activity (BU / mg) = activity value / protein concentration. The activity of rDP1 was defined as 100% and the ratio of the activity of the various recombinant agents to rDP1 was calculated (the method for comparing the activity of the recombinant agents with rDP2, rDF1, and rDF2 was the same as for rDP1). The experimental results are shown in Figure 6. A certain ratio of the recombinant agents exhibited significantly higher activity than that of the single recombinant components.

[0217] Example 11 Comparison of the activity of the recombinant agent with class I and class II recombinant allergen proteins

[0218] Using the competitive inhibition ELISA method, the biological activities of the recombinant agent were compared with those of the single components rDF1 / rDF2 / rDP1 / rDP2 by mixing them in the following ratios. The specific steps are as follows (using rDP1 as an example):

[0219] 1. Coating: Prepare a recombinant according to the mass ratio of rDP1:rDP2:rDF1:rDF2 = 20:1:20:1, dilute to 2ug / ml, add 100ul / well, and incubate at 4℃ overnight. (The ratios of other recombinant agents should be in the same order)

[0220] 2. Blocking: Wash three times with PBST (pH 7.4 0.15M PBS + 0.05% Tween 20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37°C for 2h.

[0221] 3. Sample Dilution: Dilute the recombinant reagent mixed in step 1 and the rDP1+rDF1 and rDP2+rDP2 samples for comparison (mixed at ratios of 20:1, 5:1, 1:1, 1:5, and 1:20) 1000-fold, followed by a 3-fold serial dilution for a total of 7 dilutions. Mix equal volumes of each sample with an appropriately diluted serum pool (a pool of 15 or more patient sera with specific IgE values ​​>100 Kua / L on day 1 / day 2 as determined by the Phadia 100 assay), i.e., 125 μl of sample + 125 μl of serum. Incubate the mixed sample at 4°C overnight. Positive control: 125 μl of diluent + 125 μl of serum.

[0222] 4. The remaining steps are basically the same as those in Example 8.

[0223] 5. Result analysis: Use EXCEL software to fit a quadratic curve using the inhibition rate (% positive value - sample value / positive value) as the horizontal axis and the Log10 dilution multiple as the vertical axis. Substitute the 50% inhibition rate into the curve equation and calculate the dilution multiple × 100 as the biological activity value (BU / ml). Specific activity (BU / mg) = activity value / protein concentration. Define the activity of rDP1 for comparison as 100%, and calculate the ratio of the activity of various recombinant agents for comparison to rDP1 (the method for comparing the activity of the recombinant agent with rDP2, rDF1, and rDF2 is the same as that of rDP1). The experimental results are as follows: Figure 7 As shown, the recombinant agents in each ratio almost all showed significantly higher activity than the recombinant allergens of Class I and Class II. The reason is similar to that in Example 10, the synergistic effect produced by the four recombinant agents is greater than that of the recombinant allergens of Class I or Class II alone.

[0224] Example 12 Comparison of the activity of the recombinant formulation and Odactra

[0225] The activity of the recombinant formulation determined in accordance with Example 8 was compared with the biological activity of Odactra using the competitive inhibition ELISA method.

[0226] 1. Coating: Prepare a recombinant preparation with equal activity ratio of rDP1:rDP2:rDF1:rDF2 = 1:1:1:1, dilute to 2ug / ml, add 100ul / well, and incubate at 4°C overnight.

[0227] 2. Blocking: Wash three times with PBST (pH 7.4 0.15M PBS + 0.05% Tween 20), pat dry, add 200ul blocking solution (1% BSA / PBST) to each well, and incubate at 37°C for 2h.

[0228] 3. Sample Dilution: Dilute the recombinant preparation mixed in step 1 and the Odactra sample for comparison (calculated based on the labeled amount of 30 μg / tablet) 1000-fold, followed by a 3-fold serial dilution for a total of 7 dilutions. Mix equal volumes of each sample dilution with serum from a serum pool (a pool of 15 or more patient sera with specific IgE values ​​>100 Kua / L on day 1 / day 2 as measured by the Phadia 100) (i.e., 125 μl of sample + 125 μl of serum). Incubate the mixed sample at 4°C overnight. Positive control: 125 μl of diluent + 125 μl of serum.

[0229] 4. The remaining steps are basically the same as those in Example 8.

[0230] 5. Result analysis: Use EXCEL software to fit a quadratic curve with inhibition rate % (positive value - sample value / positive value) as the horizontal axis and Log10 dilution multiple as the vertical axis. Substitute 50% inhibition rate into the curve equation and calculate the dilution multiple × 100 as the biological activity value (BU / ml). Specific activity (BU / mg) = activity value / protein concentration. Define the activity of Odactra to be compared as 100%, and calculate the ratio of the activity of the recombinant agent to Odactra. The experimental results are as follows: Figure 8 As shown, the activity of the recombinant agent with equal activity is 40% higher than that of Odcatra, indicating that the activity of the recombinant agent with equal activity is significantly higher than that of Odactra.

[0231] Example 13 Comparison of Desensitization Effects of Recombinant Agents and Positive Drug Odactra

[0232] 1. Model Preparation and Desensitization Treatment: BALB / c mice were intraperitoneally injected with HDM (a mite extract purchased from LSL, 0.45 μg DF + 0.40 μg DP) plus aluminum adjuvant weekly for approximately 4 weeks. After the final sensitization, HDM solution (0.90 μg DF + 0.80 μg DP) was administered intranasally for 7 consecutive days. Following challenge, parameters including sneezing and penh values ​​(measured using a Buxco whole-body plethysmography instrument under methacholine (Mch) concentrations of 3.125, 6.25, 12.5, 25, and 50 mg / ml, respectively) were measured. The animals with the highest penh values ​​at each concentration were ranked in descending order and divided into PBS-treated groups, a recombinant formulation group (DP1, DF1, DP2, and DF2, mass ratio 1:1:1:1) receiving 25 μg / mouse, and a positive control group receiving 24 μg / mouse (based on the active ingredient in Odactra). Sublingual desensitization therapy was administered twice weekly (with 2-3 days between sessions) for 8 consecutive weeks. Nasal provocation was repeated using the same method for 7 consecutive days. After provocation, indicators including sneezing, penh value, and spleen cytokines were measured.

[0233] 2. Analysis of results: As shown in Table 11, the number of sneezes in mice in the recombinant agent, positive group, and normal group was less than that in the PBS group, with a significant difference (p < 0.01); in addition, the penh values ​​of mice in the recombinant agent group and the positive group were significantly lower than those in the PBS group under stimulation with Mch concentrations of 3.125-50 mg / ml, and the penh values ​​of the recombinant agent group and the positive group were close. Figure 9As shown, after desensitization, the levels of IL-4, 5, and 13 in the recombinant agent group were lower than those in the positive group, and were significantly lower than those in the PBS group (P<0.05). After desensitization, the IL-10 levels in the recombinant agent group and the positive group were comparable, and both were significantly lower than those in the PBS group. This indicates that the cytokine profile shifted from a Th2-dominant profile to a balance between Th2 and Th1, suggesting that the allergy in the model mice was improved in terms of cytokine levels or desensitization mechanisms. In summary, after sublingual desensitization treatment, the recombinant agent group and the positive group showed significant improvements in respiratory symptoms and mechanisms, such as penh value, number of sneezes, and cytokine levels, indicating that the recombinant agent has comparable activity in animal efficacy tests.

[0234] Table 11. Penh values ​​and sneeze counts (mean ± sem)

[0235] Sequence Listing <110> Jiangsu Zhonghong Bioengineering Pharmaceutical Research Institute Co., Ltd. <120> Recombinant dust mite allergen protein pharmaceutical mixture and its application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 912 <212> DNA <213> Dermatophagoides pteronyssinus <400> 1 agaccatcct ccatcaagac tttcgaagag tacaagaagg ctttcaacaa gtcctacgct 60 actttcgagg acgaagaggc tgctagaaag aacttcttgg aatccgttaa gtacgttcag 120 tccaacggtg gtgctatcaa ccacttgtct gacttgtctt tggacgagtt caagaacaga 180 ttcttgatgt ccgctgaagc tttcgagcac ttgaaaacac agttcgactt gaacgctgaa 240 actaacgctt gttccatcaa cggtaacgct ccagctgaaa tcgacttgag acagatgaga actgttactc caatcagaat gcagggtggt tgtggttctt gttgggcttt ttctggtgtt gctgctactg aatccgctta cttggcttac agacaacagt ccttggactt ggctgaacaa gagttggttg actgtgcttc ccaacatggt tgtcacggtg acactattcc aagaggtatc 480 gagtacatcc agcacaacgg tgttgttcaa gaatcctact acagatacgt tgctagagag cagtcctgta destruction cgctcaaaga ttcggtatct ccaactactg tcagatctac 660. cccctaacg ctaacaagat cagagaggct ttggctcaaa ctcactccgc tatcgctgtt atcatcggta tcaaggactt ggacgctttc agacactacg acggtagaac tatcatccag 780. agagacaacg gttaccagcc aaactaccac gctgttaata tcgttggtta ctctaacgct caaggtgttg actactggat cgttagaaac tcctgggaca ctaactgggg tgataacggt tacggttact tcgctgctaa cattgacttg atgatgattg aagagtaccc atacgttgtt atcttgcatt aa <210> 2 <211> 912 <212> DNA <213> Dermatophagoides farinae <400> 2 agaccagcat ctatcaagac atttgaagag ttcaagaaag cttttaacaa gaactacgcc 60 actgttgaag aggaagaggt cgctagaaag aactttttgg aatctttgaa gtatgttgag 120 gcaaacaaag gtgctattaa tcacttgtct gatttgtccc ttgacgaatt caagaacaga 180 tacttgatga gtgctgaagc ctttgagcaa ttgaaaacac agttcgacct taacgccgaa 240 acctcagcat gtagaattaa ctcagttaat gtccctagtg agttggatct tagaagttttg 300 agaaccgtta ctcctattag aatgcaaggt ggatgtggtt cttgctgggc cttctccgga 360 gttgcagcta ccgaatctgc ttacttggcc tacagacaga cttcattgga tcttagtgaa 420 caagagttgg ttgactgtgc ttcccagcat ggttgccacg gagacactat tcctagaggt 480 attgaataca tccaacagaa cggagttgtc gaagagagat catacccata tgttgctaga 540 gagcaacagt gtagaagacc taactcacaa cactacggta ttagtaacta ctgccagatc 600 tatccacctg atgttaagca aattagagaa gcattgacac agacccatac tgcaattgct 660 gtcattatcg gaatcaagga tttgagagct ttccaacatt acgacggtag aacaattatc 720 caacacgata acggatacca gccaaattat catgctgtta acattgtcgg ttacggatcc 780 acccaaggtg ttgattattg gatcgtcaga aactcttggg atactacatg gggtgattct 840 ggttacggat atttccaagc tggaaacaat ttgatgatga ttgaacagta cccttatgtt 900 gtcatcatgt aa 912 <210> 3 <211> 441 <212> DNA <213> Dermatophagoides pteronyssinus <400> 3 atgatgtaca agatcttgtg tttgtctttg ttggttgctg ctgttgctag agaccaggtt 60 gacgttaagg actgtgctaa ccacgagatc aagaaggttt tggttccagg ttgtcacggt 120 tccgagccat gtattattca cagaggtaag ccattccagt tggaggctgt ttcgaagct 180 aaccagaaca ctaagactgc taagattgag atcaaggctt ccatcgacgg tttggaagtt 240 gacgttccag gtattgaccc aaacgcttgt cactacatga agtgtccatt ggttaagggt 300 cagcagtacg acatcaagta cacttggaat gttccaaaga tcgctccaaa gtccgagaac 360 gttgttgtta ctgttaaggt tatgggtgac gacggtgttt tggcttgtgc tattgctact 420 cacgctaaga tcagagacta a 441 <210> 4 <211> 444 <212> DNA <213> Dermatophagoides farinae <400> 4 atgatctcta agatcttgtg tttgtctttg ttggttgctg ctgttgttgc tgaccaggtt 60 gacgttaagg actgtgctaa caacgagatc aagaaagtta tggttgacgg ttgtcacggt 120 tccgacccat gtattattca cagaggtaag ccattcactt tggaggcttt gttcgacgct 180 aaccagaaca ctaagactgc taagatcgag attaaggctt ccttggacgg tttggagatt 240 gacgttccag gtatcgacac taacgcttgt cactttatga agtgtccatt ggttaagggt 300 cagcagtacg acatcaagta cacttggaat gttccaaaga tcgctccaaa gtccgagaac 360 gttgttgtta ctgttaagtt gatcggtgac aacggtgttt tggcttgtgc tattgctact 420 cacggtaaga tcagagatta atag 444 <210> 5[[ID=]31] <211> 18 <212> DNA ]><213> Artificial primer() <]400> 5 tgtaaaacga cggccagt 18 <210> 6 <211> 17 <212> DNA <213> Artificial primers () <400> 6 caggaaacag ctatgac 17

Claims

1. A recombinant dust mite allergen protein pharmaceutical composition, characterized in that: The allergen proteins in the drug mixture are composed of recombinant dust mite type I allergen DP1 and DF1 proteins and recombinant dust mite type II allergen DP2 and DF2 proteins. The four dust mite allergen proteins DP1: DP2: DF1: DF2 are mixed at an activity ratio of 1:1:1:1 or at a mass ratio of 5:1:10:1 or 1:1:1:1 or 1:5:1:5 or 1:20:1:

20. The recombinant dust mite type I allergen DP1 and DF1 proteins and the recombinant dust mite type II allergen DP2 and DF2 proteins are expressed by genes shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, through a Pichia pastoris expression system.

2. The recombinant dust mite allergen protein pharmaceutical composition according to claim 1, characterized in that: The recombinant dust mite type I allergen protein is prepared by the following method: First, the gene described in claim 1 is constructed into the plasmid pPICZα, and transferred into Pichia pastoris X33 competent cells, and cultured to obtain the host monoclonal engineered bacteria; secondly, the monoclonal engineered bacteria are selected for high-density fermentation, and purified by three steps of ion exchange chromatography, anion exchange chromatography, and hydrophobic chromatography.

3. The recombinant dust mite allergen protein pharmaceutical composition according to claim 2, characterized in that: The preparation method mainly involves the following fermentation process when expressing recombinant dust mite type I allergen DP1 and DF1 proteins: the fermentation medium used is 40% BSM medium, the induction temperature of the high-density fermentation induction stage is 25-27°C, the pH value is 6.5±0.2, and the methanol flow acceleration of the high-density fermentation induction stage is 30 mL / h. -1 L -1 , and maintain DO no higher than 40%. The fermentation temperature of the bacterial growth stage of high-density fermentation is 27℃, pH=5.5±0.2, rotation speed 300rpm, DO value 100%, and PTM12.4ml / L is added; the rate-limiting growth stage of high-density fermentation begins at 30ml / h -1 L -1 50% glycerol was added at a rate of 1.5, and the feeding rate was increased to 60 ml / h. -1 L -1 .

4. The recombinant dust mite allergen protein pharmaceutical composition according to claim 2, characterized in that: The preparation method mainly involves the following fermentation process when expressing recombinant dust mite type II allergen DP2 and DF2 proteins: the fermentation medium used is 60% BSM medium, the induction temperature of the high-density fermentation induction stage is 20-22°C, the pH value is 6.0±0.2; the methanol flow acceleration of the high-density fermentation induction stage is 30 mL / h -1 L -1 , and maintain DO no higher than 40%; the rate-limiting growth phase of high-density fermentation begins at 30ml / h -1 L -1 50% glycerol was added at a rate of 1.5, and the feeding rate was increased to 60 ml / h. -1 L -1 .

5. The recombinant dust mite allergen protein pharmaceutical composition according to claim 2, characterized in that: The preparation method, when purifying the recombinant dust mite type I allergen DP1 and DF1 proteins, The purification filler for cation exchange chromatography was SPFF, with a pH of 5.0; The purification filler for anion exchange chromatography was Q FF, pH 8.0, and conductivity 20.0 mS / cm; The purification medium for hydrophobic chromatography was phenyl FF, and the concentration of ammonium sulfate in the sample and equilibrium buffer was 2.0 M and the pH was 8.

5.

6. The recombinant dust mite allergen protein pharmaceutical composition according to claim 2, characterized in that: The preparation method, when purifying the recombinant dust mite type II allergen DP2 and DF2 proteins, Among them, the purification filler of cation exchange chromatography is SPFF, pH 6.0; The purification filler for anion exchange chromatography was Q FF, pH 7.5, conductivity 10.0 mS / cm; The purification medium for hydrophobic chromatography was phenyl FF, and the concentration of ammonium sulfate in the sample and equilibrium buffer was 1.0 M, pH 6.0.

Images