CN109852652B - Preparation and application of recombinant dust mite I-type allergen Der p1 and Der f1 proteins - Google Patents
Preparation and application of recombinant dust mite I-type allergen Der p1 and Der f1 proteins Download PDFInfo
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Abstract
The invention relates to a preparation method of recombinant I-type allergen Der p1 and Der f1 proteins, by optimizing fermentation parameters and adopting specific culture and fermentation control conditions, compared with non-optimized fermentation conditions, the activity of a fermentation product is improved by 50%, the yield is improved by more than 20%, and the components of a fermentation medium are simple, the cost is low, and the cost of a fermentation process is reduced; the yield is improved by 30% through optimizing the purification process. And the host residual protein and the residual DNA completely meet the pharmaceutical grade requirement, which means that the recombinant I-type allergen Der p1 and Der f1 proteins and the preparation method thereof have industrialized application prospects.
Description
Technical Field
The invention belongs to the field of bioengineering genes, and relates to an industrialized production method and application of recombinant I-type allergen Der p1 and Der f1 proteins.
Background
Dust mites are of a wide variety and widely exist in human life and working environments, and excreta, metabolites and mite bodies of the dust mites have strong allergenicity, and about 10% of population dust mites are allergic to dust mites worldwide and about 80% of exogenous asthma is caused by dust mites.
Currently, the crude extractive solution of the allergen of the dust mites is mainly used for treating allergic diseases caused by the dust mites clinically, for example, changdi dust mite drops of Zhejiang Megash organisms on the market in 2006 are the extractive solution of the metabolic culture of the dust mites. Dust mite allergens mainly exist in excrement and mite bodies, and an extraction method is long in time consumption, complex in process and high in cost; in addition, the natural allergen extract has very complex composition, constant components, and is difficult to be polluted by exogenous toxic substances and pathogen microorganisms. The long-term use of the dust mite allergen crude extract can easily cause local reactions such as red sickness, swelling, induration, necrosis and the like and systemic reactions such as shock, edema, bronchospasm, urticaria, angioedema, systemic erythema and the like. In addition, the crude extract is used for diagnosis, the reaction degree of the patient to each component of the allergen cannot be clarified, and misdiagnosis is easy to occur.
The quality of the allergen is critical for diagnosis and treatment of allergic diseases, and the allergen used for immunodiagnosis should be pure and not be a crude extract. The recombinant allergen has the following advantages compared with the crude extract: (1) The recombinant allergen has higher purity, and does not contain non-allergen components, enzymes, enzyme inhibitors, toxic proteins and the like compared with the crude extract; (2) The recombinant protein has single component and better specificity, the components in the crude extract are complex, and the patient can only react with partial components in the crude extract, so that the specificity is poor; (3) Compared with the natural extract, the recombinant allergen reduces the antigen epitope bound by IgE, effectively reduces IgE-mediated allergic reaction, simultaneously reserves the structural domain necessary for allergen T cell recognition, has better immunogenicity, reduces the risk of immunotherapy and improves the effect of desensitization therapy.
Dust mite allergens have a complex composition, about 30 species, of which type i and type ii allergens are the predominant allergen components. The most comprehensive allergen research of recombinant type I house dust mite allergens (Der P1, hereinafter referred to as DP 1) and recombinant type I dust mite allergens (Der f1, hereinafter referred to as DF 1) is the research of Japanese scholars Toshiro Takai and the like in 2005, and the paper shows that when the DP1 and DF1 are expressed in a Pichia pastoris system, protein propeptide is required to be added, otherwise, the DP1 and DF1 cannot be expressed in a eukaryotic expression system, and mature DP1 and DF1 proteins consistent with the amino acid sequence of natural proteins are obtained through an activation process, and the activity of the proteins before and after the activation is proved to be consistent with the activity of the natural proteins and higher than that of the proteins before the activation by the research of the DP1 and DF1 protein activation method; however, the method described in the paper has low yield of mature protein, is not suitable for large-scale preparation, and does not optimize the expression and fermentation process of the DP1 and DF1 proteins; in addition, no further studies have been reported for a while.
By combining the research results, the research on fermentation and purification of DP1 and DF1 is less so far, and the yield, structure, quality and the like are difficult to meet the clinical use requirements; the fermentation process and the purification process of the DP1 and DF1 proteins are optimized, so that the requirements of fermentation level, purification yield and quality all meet the clinical use requirements of the recombinant desensitization medicine.
Disclosure of Invention
The method optimizes key quality parameters in the fermentation and purification processes of the DP1 and DF1 proteins according to the genetic engineering strains screened by the methods provided in patent application numbers 201611270412.2 (DP 1) and 201611267250.7 (DF 1) of the company; because of the high sequence homology of the DP1 and DF1 genes, the structure and the property of the proteins are greatly similar, and the inventor discovers that the DP1 and DF1 proteins have extremely high similarity in the purification process in the research.
The invention provides a preparation method of recombinant dust mite I allergen protein, which comprises a fermentation process and a purification process, wherein the fermentation process comprises recombinant yeast strain activation, seed liquid culture and high-density fermentation, a fermentation culture medium is 40-60% BSM culture medium, and the induction temperature in the induction stage of the high-density fermentation is as follows: the pH value is 5.5-6.5 at 23-27 ℃.
Preferably, the fermentation medium used is 40% BSM medium, and the induction temperature in the induction stage of the high-density fermentation is: the pH value is 6.5 plus or minus 0.2 at the temperature of 25-27 ℃.
More preferably, the methanol flow acceleration in the induction stage of the high-density fermentation is 30mL/h -1 L -1 And maintains DO not higher than 40%.
More preferably, the fermentation temperature in the cell proliferation stage of the high-density fermentation is 27 ℃, the pH=5.5+ -0.2, the rotation speed is 300rpm, the DO value is 100%, and PTM1 (2.4 ml/L) is added.
More preferably, the rate-limiting growth phase of the high-density fermentation begins at 30ml/h -1 L -1 Is added with 50 percent of glycerol, and the feeding speed is adjusted to be 60ml/h -1 L -1 。
Preferably, the purification process of the preparation method of the recombinant dust mite I type allergen protein comprises three steps of purification, including cation exchange chromatography, anion exchange chromatography and hydrophobic chromatography.
Preferably, the purification packing for cation exchange chromatography is SP FF; the preferred pH range is 4.0-6.5. More preferably, the sample and buffer pH in the cation exchange layer is 5.0.
Preferably, the purification packing of the anion exchange chromatography is qff; the preferred pH range is 6.0-8.0; preferred conductivity values are 2.0mS/cm to 20.0mS/cm. More preferably, the pH of the sample and buffer in anion exchange chromatography is 8.0 and the conductivity is 20.0mS/cm.
Preferably, the purification filler for hydrophobic chromatography is phenyl FF; the preferred pH range is 6.5-8.5; the preferred ammonium sulfate concentration is 1.0-2.0M. More preferably, the sample and equilibration buffer ammonium sulfate concentration in the hydrophobic chromatography is 2.0m and the ph is 8.5.
After the technical scheme is adopted, the invention has the following advantages: the fermentation parameters are optimized, so that the prepared fermentation broth is higher in activity and yield, the adopted fermentation medium is simple in components and low in cost, and the cost of a fermentation process is reduced; the fermentation process adopts specific culture and fermentation control conditions, and compared with an unoptimized inorganic culture medium, the activity is improved by 50 percent (slightly higher than the activity of DP1 and DF1 extracted naturally), and the yield is improved by more than 20 percent; the yield is improved by 30% by optimizing the purification process. And the residual protein and the residual DNA of the host completely meet the pharmacopoeia requirements, which means that DP1 and DF1 fermented by the inventor on a large scale completely meet the pharmaceutical grade requirements.
Drawings
FIG. 1 recombinant DP1 Pichia pastoris Strain SDS-PAGE electrophoresis of first batch fermentation in example 1 and fermentation broth OD 600 And a trend chart of the wet weight of the cells.
Wherein FIG. 1-a is an SDS-PAGE electrophoresis of the recombinant DP1 Pichia pastoris strain of example 1 in a first fermentation run, lane M is a (10-230 kDa) wide-range protein loading Marker; lane 1 is fed methanol 0h fermentation supernatant, lane 2 is fed methanol 6h fermentation supernatant, lane 3 is fed methanol 12h fermentation supernatant, lane 4 is fed methanol 18h fermentation supernatant, lane 5 is fed methanol 24h fermentation supernatant, lane 6 is fed methanol 30h fermentation supernatant, lane 7 is fed methanol 36h fermentation supernatant, and lane 8 is fed methanol 42h fermentation supernatant, wherein the arrow indicates the recombinant DP1 band. FIG. 1-b is recombinant DP1 Pichia pastorisStrain example 1 medium and small Scale high Density fermentation broth OD in first batch fermentation 600 And a trend chart of the wet weight of the cells.
FIG. 2 SDS-PAGE electrophoresis of the second fermentation in example 1 of recombinant DP1 Pichia pastoris strain and fermentation broth OD 600 And a trend chart of the wet weight of the cells.
FIG. 2-a shows SDS-PAGE electrophoresis of a second fermentation of Pichia pastoris strain recombinant DP1, example 1, lane M shows a wide range of (10-230 kDa) protein loading markers; lane 1 is fed methanol 0h fermentation supernatant, lane 2 is fed methanol 6h fermentation supernatant, lane 3 is fed methanol 12h fermentation supernatant, lane 4 is fed methanol 18h fermentation supernatant, lane 5 is fed methanol 24h fermentation supernatant, lane 6 is fed methanol 30h fermentation supernatant, lane 7 is fed methanol 36h fermentation supernatant, and lane 8 is fed methanol 42h fermentation supernatant, wherein the arrow indicates the recombinant DP1 band. FIG. 2-b second batch fermentation of Small Scale high Density fermentation broth OD from example 1 for recombinant DP1 Pichia pastoris Strain 600 And a trend chart of the wet weight of the cells.
FIG. 3 SDS-PAGE electrophoresis of the third batch fermentation in example 1 of recombinant DP1 Pichia pastoris strain and fermentation broth OD 600 And a trend chart of the wet weight of the cells.
Wherein FIG. 3-a is an SDS-PAGE electrophoresis of a third fermentation of recombinant DP1 Pichia pastoris strain of example 1, lane M is a (10-230 kDa) wide-range protein loading Marker; lane 1 is fed methanol 0h fermentation supernatant, lane 2 is fed methanol 6h fermentation supernatant, lane 3 is fed methanol 12h fermentation supernatant, lane 4 is fed methanol 18h fermentation supernatant, lane 5 is fed methanol 24h fermentation supernatant, lane 6 is fed methanol 30h fermentation supernatant, lane 7 is fed methanol 36h fermentation supernatant, and lane 8 is fed methanol 42h fermentation supernatant, wherein the arrow indicates the recombinant DP1 band. FIG. 3-b is a third batch fermentation of a small scale high density broth OD from recombinant DP1 Pichia pastoris strain example 1 600 And a trend chart of the wet weight of the cells.
FIG. 4 SDS-PAGE electrophoresis of recombinant DP1 Pichia pastoris strain example 2 and fermentation broth OD 600 And change in wet weight of cellsTrend graph.
Wherein FIG. 4-a shows SDS-PAGE electrophoresis of recombinant DP1 Pichia pastoris strain example 2, lane 1 shows methanol fed-batch 0h fermentation supernatant, lane 2 shows methanol fed-batch 6h fermentation supernatant, lane 3 shows methanol fed-batch 12h fermentation supernatant, lane 4 shows methanol fed-batch 18h fermentation supernatant, lane 5 shows methanol fed-batch 24h fermentation supernatant, lane 6 shows methanol fed-batch 30h fermentation supernatant, lane 7 shows methanol fed-batch 36h fermentation supernatant, and lane 8 shows methanol fed-batch 42h fermentation supernatant, wherein the arrow indicates recombinant DP1 band. FIG. 4-b shows the OD of a small-scale high-density fermentation broth of recombinant DP1 Pichia pastoris strain example 2 600 And a trend chart of the wet weight of the cells.
FIG. 5 SDS-PAGE electrophoresis of recombinant DP1 Pichia pastoris strain example 3 and fermentation broth OD 600 And a trend chart of the wet weight of the cells.
Wherein FIG. 5-a is an SDS-PAGE electrophoresis of recombinant DP1 Pichia pastoris strain example 3, lane 1 is a fed-batch methanol 0h fermentation supernatant, lane 2 is a fed-batch methanol 2h fermentation supernatant, lane 3 is a fed-batch methanol 4h fermentation supernatant, lane 4 is a fed-batch methanol 8h fermentation supernatant, lane 5 is a fed-batch methanol 10h fermentation supernatant, lane 6 is a fed-batch methanol 15h fermentation supernatant, lane 7 is a fed-batch methanol 18h fermentation supernatant, lane 8 is a fed-batch methanol 22h fermentation supernatant, lane 9 is a fed-batch methanol 26h fermentation supernatant, wherein the arrow indicates the recombinant DP1 band
FIG. 5-b pilot-scale high density fermentation broth OD for recombinant DP1 Pichia pastoris strain example 3 600 And a trend chart of the wet weight of the cells.
FIG. 6 shows the result of the first purification step of the DP 1/broth supernatant pH 4.0.
Wherein FIG. 6-a shows a first step purification chromatogram of a DP1 fermentation broth; FIG. 6-b shows SDS-PAGE analysis of the first step of purification of the DP1 broth, lane 1 for the non-preptained Marker, lane 2 for the pre-purification sample, and lane 3 for the purification breakthrough sample; lanes 4-10 are collection tubes for the purification elution peak.
FIG. 7 shows the result of the first purification step of the supernatant pH5.0 of the DP1 fermentation broth.
Wherein FIG. 7-a is a first step purification chromatogram of DP1 broth; FIG. 7-b shows SDS-PAGE analysis of the first step of purification of the DP1 broth, lane 1 for the non-preptained Marker, lane 2 for the pre-purification sample, and lane 3 for the purification breakthrough sample; lanes 4-10 are collection tubes for the purification elution peak.
FIG. 8 shows the result of the first purification step of the supernatant pH6.5 of the DP1 fermentation broth.
Wherein FIG. 8-a is a first step purification chromatogram of a DP1 fermentation broth; FIG. 8-b shows SDS-PAGE analysis of the first step of purification of the DP1 fermentation broth, lane 1 is the pre-purification sample, lane 2 is the purification penetration sample, and lane 3 is the non-pre-dye Marker; lanes 4-10 are collection tubes for the purification elution peak.
FIG. 9 shows the result of the second purification step of the DP1 protein at pH 6.0.
Wherein FIG. 9-a shows a second step purification chromatogram of the DP1 protein; FIG. 9-b shows SDS-PAGE analysis of the second step of purification of DP1 protein, lane 1 is pre-purification, lane 2 is non-preptaining Marker, lane 3 is purification penetration, and lanes 4-6 are elution.
FIG. 10 shows the result of the second purification step of the DP1 protein at pH 7.0.
Wherein FIG. 10-a shows a second step purification chromatogram of the DP1 protein; FIG. 10-b shows SDS-PAGE analysis of the second step of purification of DP1 protein, lane 1, non-preptained protein Marker, lane 2, pre-purification sample, lane 3, purification penetration sample, and lanes 4-6, elution samples.
FIG. 11 shows the result of the second purification step of the DP1 protein at pH 8.0.
Wherein FIG. 11-a shows a second step purification chromatogram of the DP1 protein; FIG. 11-b shows a SDS-PAGE analysis of the second step of purification of DP1 protein, lane 1 is a Marker of non-preptained protein, lane 2 is a pre-purification sample, lane 3 is a purification penetration sample, and lane 4 is an elution sample.
FIG. 12 shows the result of the third purification step of the DP1 protein at pH6.5.
Wherein FIG. 12-a is a third step purification chromatogram of the DP1 protein; FIG. 12-b shows SDS-PAGE analysis of the third step of purification of DP1 protein, lane 1 is a Marker of non-pre-stained protein, lane 2 is a pre-purification sample, lane 3 is a purification penetration sample, and lanes 4-8 are samples of the collection tube of the elution peak.
FIG. 13 shows the result of the third purification step of the DP1 protein at pH7.5.
Wherein, FIG. 13-a shows the third step purification chromatogram of the DP1/DF1 protein; FIG. 13-b shows SDS-PAGE analysis of the third step of purification of DP1/DF1 protein, lane 1 shows a pre-purification sample, lane 2 shows a Marker of non-preptained protein, lane 3 shows a purification penetration sample, and lanes 4-9 show samples of the collection tubes of the elution peak.
FIG. 14 shows the result of the third step purification of the DP1 protein at pH 8.5.
Wherein, FIG. 14-a shows the third step purification chromatogram of the DP1/DF1 protein; FIG. 14-b shows SDS-PAGE analysis of the third step of purification of DP1/DF1 protein, lane 1 shows the pre-purification sample, lane 2 shows the purification penetration sample, lane 3 shows the elution collection tube, lane 4 shows the non-preptaining Marker, and lanes 5-10 show the elution peak samples of each collection tube.
FIG. 15 shows the results of HPLC analysis of the purity of the DP1/DF1 protein.
Wherein FIG. 15-a shows the RP-HPLC analysis result of the DP1 protein; FIG. 15-b shows the result of RP-HPLC analysis of DF1 protein.
FIG. 16 shows the results of the analysis of the amino acid coverage of the DP1/DF1 protein.
Wherein FIG. 16-a shows the amino acid coverage analysis result of the DP1 protein; FIG. 16-b shows the results of the DF1 protein amino acid coverage analysis.
FIG. 17 shows the results of a first purification step of the supernatant of the DP1 300L fermentation.
Wherein FIG. 17-a is a first step purification chromatogram of DP1 broth; FIG. 17-b is a SDS-PAGE analysis of the first step of purification of the DP1 fermentation broth, lane 1 is the pre-purification sample, lane 2 is the non-preptaining Marker, and lane 3 is the purification breakthrough sample; lanes 4-10 are collection tubes for the purification elution peak.
FIG. 18 shows the result of the second purification step of the DP1 protein.
Wherein, FIG. 18-a is a second step purification chromatogram of DP1 protein; FIG. 18-b shows SDS-PAGE analysis of the second step of purification of DP1 protein, lane 1 is a pre-purification sample, lane 2 is a post-purification sample, and lane 3 is an elution sample.
FIG. 19 shows the result of the third purification step of the DP1 protein.
Wherein, FIG. 19-a shows the third step purification chromatogram of the DP1/DF1 protein; FIG. 19-b shows SDS-PAGE analysis of the third step of purification of DP1/DF1 protein, lane 1 is the pre-purification sample, lane 2 is the non-preptaining Marker, lane 3 is the purification penetration sample, and lanes 4-9 are samples of the collection tubes of the elution peak.
Detailed Description
The invention will be further illustrated with reference to specific examples, which are to be understood as illustrative only and are not intended to limit the scope of the invention.
EXAMPLE 1 recombinant DP1/DF1 Small-Scale (3L) high Density fermentation Process
Step 1: recombinant strain activation
And (3) streaking glycerol bacteria seeds frozen in a DP1 working seed library at the temperature of minus 80 ℃ on YPD solid culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20g/L and agarose 15 g/L), and culturing in a constant temperature and humidity box at the temperature of 30 ℃ for 3-5 days.
Step 2: seed liquid culture
The monoclonal colony on the plate is picked up and cultured in YPD liquid culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20 g/L) at 30℃and 220rpm to OD 600 And (3) about 6.0, and observing the sterile condition under a microscope to obtain the seed liquid for fermentation.
Step 3: fermentation process
The Saidoris B-plus bioreactor was cleaned, pH meter probes of the fermenter were calibrated with standard solutions of ph=7.0 and ph=4.0, respectively, then BSM medium was prepared as fermentation medium (BSM concentrations for different fermentation batches are shown in Table 1), poured into the tank, autoclaved at 121℃for 20min, and after the temperature had fallen to 50℃the ph=5.5.+ -. 0.2 was adjusted with concentrated ammonia.
And (3) inoculating the seed liquid obtained in the step (2) into a bioreactor according to the ratio of 1:15 (V/V, seed liquid/fermentation medium). The initial recombinant Pichia pastoris is cultured at a fermentation temperature of 27 ℃ at pH=5.5+ -0.2 and a rotation speed=300 rpm at a DO value of 100% and PTM1 (2.4 ml/L) is added. During this stage, DO is continuously decreased for about 22 hours, DO is maintained at 20% by increasing stirring speed, ventilation and oxygen ventilation, after the carbon source is consumed, dissolved oxygen value is rapidly increased, the wet weight of the thallus reaches 120g/L,at this time, a rate-limiting growth phase was entered, the beginning of which was at 30ml/h for 3h -1 L -1 The glycerol was fed at a rate of 50% and after 3 hours the feed rate was adjusted up to 60ml/h -1 L -1 . And supplementing for 6 hours, stopping feeding when the wet weight of the thalli is about 250g/L, and increasing DO to indicate that the carbon source is exhausted and entering an induction stage. (Induction conditions of different fermentation batches are shown in Table 1) and maintained at DO of not higher than 30%, fermentation expression is carried out, sampling is started at intervals after induction is started, meanwhile DO SPIKE is carried out in the initial period of induction, no accumulation of methanol in a fermentation tank is ensured, and OD of fermentation broth is measured 600 The absorption values and the wet weight of the cells are shown in FIGS. 1 to 3, which are graphs showing the trend of the change during the wet weight fermentation of three batches of cells.
TABLE 1 recombinant DP1 Small-scale high-density fermentation process condition optimization
Wherein BSM fermentation medium refers to standard concentration determined in the fermentation specification of life, namely K 2 SO 4 18.2g/L，MgSO 4 14.9g/L， 4 KOH 4.13g/L，CaSO 0.93g/L，H 3 PO 4 26.7mL/L, 40g/L glycerol, 40% BSM fermentation medium means that the concentration of each component is reduced to 40% based on the standard concentration determined on the fermentation specification of life, namely K 2 SO 4 7.28g/L，MgSO 4 5.96g/L， 4 KOH 1.652g/L，CaSO 0.372g/L，H 3 PO 4 10.68mL/L, glycerin 16g/L.
The fermentation process described above for recombinant DP1 is equally applicable to recombinant DF 1.
EXAMPLE 2 large-scale (30L) high-Density fermentation verification of recombinant DP1/DF1
Step 1: recombinant strain activation
And (3) streaking the glycerinum seeds frozen in a working seed pool at the temperature of minus 80 ℃ on YPD solid culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20g/L and agarose 15 g/L), and culturing in a constant temperature and humidity box at the temperature of 30 ℃ for 3-5 days.
Step 2: first-stage seed liquid culture
The monoclonal colony on the plate in the step 1 is picked up and cultured in YPD liquid culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20 g/L) at 30 ℃ and 220rpm until OD 600 And (3) about 6.0, and observing the sterile condition under a microscope to obtain the primary seed liquid for fermentation.
Step 3: second-level seed liquid culture
Inoculating the primary seed solution obtained in step 2 into a Sadolis B-plus fermenter, culturing at 27deg.C and pH=5.5+ -0.2 with 60% BSM and 300rpm, maintaining dissolved oxygen at 25% by aeration and rotation speed, and finally reaching OD 600 And (4) obtaining the secondary seed liquid for fermentation, wherein the wet weight of the secondary seed liquid is about 80g/L, and the secondary seed liquid is obtained after observing the sterile under a microscope.
Step 4: fermentation process
Cleaning a Sidoris Cplus bioreactor, respectively correcting a pH meter probe of a fermentation tank by using standard solutions with pH=7.0 and pH=4.0, then determining that a fermentation medium is 40% BSM according to a design interval of key process parameters in the embodiment 1, preparing 20L of the fermentation medium, pouring the fermentation medium into the fermentation tank, sterilizing on line for 20min at 121 ℃, and adjusting the pH=5.5+/-0.2 by using concentrated ammonia water after the temperature is reduced to 50 ℃.
And (3) inoculating the seed liquid obtained in the step (3) into a fermentation tank according to the proportion of 1:15 (V/V, seed liquid/fermentation medium). The initial recombinant Pichia pastoris is cultured at a fermentation temperature of 27 ℃ at pH=5.5+ -0.2 and a rotation speed=300 rpm at a DO value of 100% and PTM1 (2.4 ml/L) is added. At this stage for about 20 hours, after the carbon source is consumed, the dissolved oxygen value is rapidly increased, the wet weight of the cells reaches 100g/L, at this time, the rate-limiting growth stage is started, and the initial 2 hours of the stage are 30ml/h -1 L -1 The rate of (2) is increased to 60ml/h after 2h by adding 50% glycerol -1 L - . And supplementing for 4 hours, stopping feeding when the wet weight of the thalli is about 200g/L, and increasing DO to indicate that the carbon source is exhausted and entering an induction stage. The pH was adjusted to be=6.0.+ -. 0.2 with a methanol flow acceleration of 30ml/h -1 L -1 Performing inducible expression recombinant DP1; maintaining DO not higher than 40%, inducing at 25-27deg.C and pH=6.5+ -0.2, fermenting, sampling at intervals after fermentation, and measuring OD of fermentation broth 600 Absorption values and cell wet weights, FIG. 4-b is a recombinant DP1 large scale fermentation broth OD 600 Absorption value and change trend graph of thallus wet weight fermentation period, and can be seen from graph, fermentation liquor OD during recombinant Pichia pastoris fermentation period 600 Can reach more than 300, and the wet weight of the thalli can reach 400g/L. After 40h of fermentation, the supernatant was collected by centrifugation and SDS-PAGE electrophoresis to identify the recombinant DP1 production at various times of methanol induction (FIG. 4-a), indicating the OD of the fermentation broth 600 The absorption value, the variation trend of the wet weight of the thalli and the fermentation product are consistent with 3L of small-scale high-density fermentation. The applicant has thus determined that the fermentation process of the present invention is fully capable of industrial scale-up production.
The fermentation process described above for recombinant DP1 is equally applicable to recombinant DF 1.
EXAMPLE 3 recombinant DP1/DF1 Pilot (300L) high Density fermentation verification
Step 1: recombinant strain activation
And (3) streaking the glycerinum seeds frozen in a working seed library at-80 ℃ on YPD slant culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agarose 15 g/L), and culturing in a constant temperature and humidity box at 30 ℃ for 3-5 days.
Step 2: first-stage seed liquid culture
The monoclonal colony on the plate in the step 1 is picked up and cultured in YPD liquid culture medium (yeast extract 10g/L, peptone 20g/L, glucose 20 g/L) at 30 ℃ and 220rpm until OD 600 And (3) about 6.0, and observing the sterile condition under a microscope to obtain the primary seed liquid for fermentation.
Step 3: second-level seed liquid culture
Inoculating the primary seed solution obtained in step 2 into 30L seed tank of Di Wo Xin BVT-3000 type, culturing with 60% BSM, pH=5.5+ -0.2, and 27 deg.C, and 300rpm, and inoculating with aeration and rotation speed barThe member maintains dissolved oxygen at 25% and eventually to OD 600 And (4) obtaining the secondary seed liquid for fermentation, wherein the wet weight of the secondary seed liquid is about 80g/L, and the secondary seed liquid is obtained after observing the sterile under a microscope.
Step 4: fermentation process
Cleaning a Di Wo Xin BVT-3000 type 300L fermentation tank, respectively correcting pH meter probes of the fermentation tank by using standard solutions with pH=7.0 and pH=4.0, then determining that the fermentation medium is 40% BSM according to the design interval of key process parameters in the embodiment 1, preparing 100L of the fermentation medium, pouring the fermentation medium into the fermentation tank, sterilizing on line for 20min at 121 ℃, and regulating the pH=5.5+/-0.2 by using concentrated ammonia water after the temperature is reduced to 50 ℃.
And (3) inoculating the seed liquid obtained in the step (3) into a fermentation tank according to the proportion of 1:10 (V/V, seed liquid/fermentation medium). The initial recombinant Pichia pastoris is cultured at a fermentation temperature of 27 ℃ at pH=5.5+ -0.2 and a rotation speed=300 rpm at a DO value of 100% and PTM1 (2.4 ml/L) is added. About 20 hours at this stage, after the carbon source is consumed, the dissolved oxygen value is rapidly increased, the wet weight of the thalli reaches 100g/L, at this time, the thalli enters into the speed-limiting growth stage, 50% of glycerol is added at a rate of 3L/h for 2 hours at the beginning of the stage, and the feeding speed is up to 60ml/h after 2 hours -1 L -1 . And supplementing for 4 hours, stopping feeding when the wet weight of the thalli is about 200g/L, and increasing DO to indicate that the carbon source is exhausted and entering an induction stage. Acceleration of methanol flow is 60ml/h -1 L -1 Performing inducible expression recombinant DP1; maintaining DO not higher than 40%, inducing at 25-27deg.C and pH=6.5+ -0.2, fermenting, sampling at intervals after fermentation, and measuring OD of fermentation broth 600 Absorption values and cell wet weights, FIG. 5-b is a recombinant DP1 large scale fermentation broth OD 600 Absorption values and trend of bacterial cells during wet weight fermentation. After the end of the 26h induction fermentation, the supernatant was collected by centrifugation and SDS-PAGE electrophoresis to identify the recombinant DP1 production at various times of methanol induction (FIG. 5-a), indicating the OD of the fermentation broth 600 The absorption value, the variation trend of the wet weight of the thalli and the fermentation product are consistent with 30L large-scale high-density fermentation. The applicant has thus determined that the fermentation process of the present invention is fully capable of industrial scale-up production.
The fermentation process described above for recombinant DP1 is equally applicable to recombinant DF 1.
EXAMPLE 4 recombinant DP1/DF1 (30L) purification Process
1. First step purification of recombinant DP1 fermentation supernatant
Step 1: pretreatment of fermentation broths
Centrifuging the fermentation broth obtained in the above example at high speed to obtain supernatant; adding diatomite for auxiliary filtration overnight to obtain a clarified fermentation broth sample; diluting with 10KD membrane package ultrafiltration, and reducing electric conductivity to below 5 mS/cm.
Step 2: cation exchange chromatography
The supernatant of the fermentation broth after the treatment is subjected to acetic acid adjustment to pH4.0, and is put on an SP FF chromatographic column, wherein the column volume is BPG140/100, the column bed volume is 2800mL, the balance buffer is 50mM NaAc, the pH is 4.0, the elution buffer is 50mM NaAc,1.0M NaCl,pH4.0, the DP1 target protein is mainly concentrated on the second elution peak according to 0-100% linear elution, the first-step purification chromatogram (pH 4.0) of the DP1 protein is shown in FIG. 6-a, and the first-step purification SDS-PAGE analysis chart (pH 4.0) of the DP1 protein is shown in FIG. 6-b.
The rest of steps 1, 2 are unchanged, and the pH of the sample and buffer is adjusted to 5.0. FIG. 7-a shows a first-step purification chromatogram (pH 5.0) of the DP1 protein, and FIG. 7-b shows a first-step purification SDS-PAGE analysis (pH 5.0) of the DP1 protein.
The remaining steps of steps 1, 2 were unchanged and the pH of the sample and buffer was adjusted to 6.5. FIG. 8-a shows a first-step purification chromatogram (pH 6.0) of the DP1 protein, and FIG. 8-b shows a first-step purification SDS-PAGE analysis (pH 6.0) of the DP1 protein.
As can be seen from the results of FIGS. 6 to 8, the purified DP1 protein of FIG. 7 has the highest purity, and the remaining conditions have impurities, wherein pH5.0 is the optimal condition.
2. Second step of purification of recombinant DP1 protein:
step 1: ultrafiltration
Combining the second elution peak in the first purification step 2, and carrying out ultrafiltration dilution by a10 KD membrane package until the electric conductance is less than 2.0mS/cm.
Step 2: anion exchange chromatography
Adding 20mM NaH to the sample obtained in step 1 2 PO 4 Adjusting pH to 6.0, loading onto Q FF anion exchange chromatography column with Hiscale 50/40, column bed volume of 500ml, and balancing buffer solution of 20mM NaH 2 PO 4 pH6.0, elution buffer 20mM NaH 2 PO 4 1.0M NaCl, pH6.0, collecting penetration, 0-100% linear elution, and concentrating the target protein mainly. FIG. 9-a shows the second step purification chromatogram of the DP1 protein, and FIG. 9-b shows the second step purification SDS-PAGE analysis of the DP1 protein.
The remaining steps of steps 1, 2 were unchanged, and the sample and buffer pH7.0 were adjusted to give a conductance of 10.0mS/cm. FIG. 10-a shows the second step purification chromatogram of the DP1 protein, and FIG. 10-b shows the second step purification SDS-PAGE analysis of the DP1 protein.
The remaining steps in steps 1, 2 were unchanged, and the sample and buffer pH was adjusted to 8.0 (phosphate buffer was changed to Tris buffer), and the conductance was 20.0mS/cm. FIG. 11-a shows the second step purification chromatogram of the DP1 protein, and FIG. 11-b shows the second step purification SDS-PAGE analysis of the DP1 protein.
As can be seen from the results of FIGS. 9 to 11, the optimal result of FIG. 11 shows that the separation degree of the target protein and the impurity is the highest, most of the target protein is remained in the penetration, the impurity is combined into the medium, the complete separation is realized, and the yield of the DP1 protein in the penetration is greatly improved.
3. Third step purification of recombinant DP1 protein
Adding ammonium sulfate to final concentration of 1.0M, adjusting pH to 6.5, loading onto Phenyl FF chromatographic column with Hiscale 50/40, column bed volume of 500ml, and balancing buffer solution of 20mM NaH 2 PO 4 ，1.0M(NH 4 ) 2 SO 4 pH6.0, elution buffer 20mM NaH 2 PO 4 pH6.0, eluting according to 25, 50%,70%,100% isocratic. FIG. 12-a shows the third step purification chromatogram of the DP1 protein, and FIG. 12-b shows the third step purification SDS-PAGE analysis of the DP1 protein.
The remaining steps were unchanged, and the sample and equilibration buffer ammonium sulfate concentrations were adjusted to 1.0M, pH7.5. FIG. 13-a shows the second step purification chromatogram of the DP1 protein, and FIG. 13-b shows the second step purification SDS-PAGE analysis of the DP1 protein.
The rest of the steps were unchanged, and the sample and equilibration buffer ammonium sulfate concentrations were adjusted to 2.0M, pH8.5 (phosphate buffer exchanged for Tris buffer). FIG. 14-a shows a second step purification chromatogram of the DP1 protein, and FIG. 14-b shows a second step purification SDS-PAGE analysis of the DP1 protein.
As can be seen from the results of FIGS. 12 to 14, the result of FIG. 14 is optimal, and the purity of the DP1 protein obtained after optimization is the highest, and impurities are contained
At least, the purity of the obtained protein can reach more than 95 percent.
The purification process shown in Table 2 was improved by about 30% in yield after optimization compared to that before optimization, and the activity (measured by ELISA) was also improved compared to that before process optimization.
TABLE 2 yield before and after optimization of DP1 protein purification process
The purification process described above for recombinant DP1 applies equally to recombinant DF 1.
EXAMPLE 5 identification of recombinant DP1/DF1 protein
1. Concentrating and measuring protein concentration
The gradient elution peak in example 3 was collected and then packaged with a Vivaflow50 tangential flow ultrafiltration membrane (VF 05P9-50 cm) 2 Sartorius) is concentrated, and the dialysis bag replacement buffer solution is a PBS solution with pH of 7.4; pierce BCA protein concentration kit determines protein concentration.
2. HPLC purity analysis of DP1/DF1 protein
And diluting the purified DP1/DF1 sample to 1mg/ml, and filtering with a 0.22um filter membrane to obtain the sample. The mobile phase was 20mM PB, pH7.5, the flow rate was 1ml/min, 10ul was injected, the purity was analyzed by RP column, FIG. 15-a, FIG. 15-b shows the purity detection result, and the purity of the three-step purified DP1/DF1 protein was more than 95%.
3. DP1/DF1 protein amino acid coverage analysis
The amino acid coverage analysis is one of very important indexes in the quality research of recombinant protein medicines, and the product can be ensured to have the same biological activity as the natural protein only if the primary structure of the amino acid is completely the same. The inventor entrusts the Shanghai middle-department of new life biotechnology limited company to carry out amino acid coverage analysis on the DP1/DF1 protein, and the results of the figure 16-a and the figure 16-b show that the amino acid coverage is more than 98 percent and accords with the requirements of related policy and regulation.
4. DP1/DF1 protein Activity assay
The biological activity of recombinant DP1, DF1 proteins (rDP 1, rDF 1) was determined using a competition inhibition ELISA method and compared to the native DP1 protein (nDP 1). The specific steps are (taking DP1 as an example):
1. coating: rDP1, nDP were diluted to 2ug/ml,100 ul/well with coating solution (pH 9.6.0.15M carbonate buffer), respectively, and incubated overnight at 4 ℃.
2. Closing: PBST (pH 7.4. 7.4 0.15M PBS+0.05%Tween20) was washed 3 times, patted dry, 200ul of blocking solution (1% BSA/PBST) was added to each well and incubated at 37℃for 2h.
3. Sample dilution: samples rDP, nDP1 were 30-fold diluted and then 3-fold gradient diluted for a total of 7 dilutions. The samples of each dilution were mixed with serum pool serum of the appropriate dilution (15 parts above phadia100 detection d1/d2 specific IgE > 100Kua/L patient serum mix pool) in equal volumes, i.e. 125ul sample +125ul serum, and the mixed samples were incubated overnight at 4 ℃. Positive control: 125ul of diluent+125 ul of serum.
4. Sample adding: and adding the mixed incubated samples into corresponding coated holes, and incubating at 37 ℃ for 90min.
5. Secondary antibody incubation: PBST was washed 4 times, patted dry, 100ul of secondary antibody diluent (murine anti-human IgE-HRP 1:1500 dilution) was added to each well and incubated for 1h at 37 ℃.
6. Color development: PBST is washed for 4 times, the mixture is patted dry, 100ul TMBI color developing solution is added into each hole, and color development is carried out for 10min at 37 ℃.
7. Termination and reading: 50ul of 2M H was added per well 2 SO 4 Termination of the reactionReadings at a wavelength of 450 nm.
8. And (3) result processing: using EXCEL software, performing quadratic curve fitting with inhibition ratio (positive value-sample value/positive value) as abscissa and Log10 dilution as ordinate. The 50% inhibition was substituted into the curve equation and the dilution factor X100 was calculated as the biological activity value (BU/ml). Specific activity (BU/mg) =activity value/protein concentration.
The experimental results are shown in Table 3, and the calculated rDP1 activity value is 79616.0BU/ml; the specific activity was 9.95E+04BU/mg. The result of the experiment using nDF1 as a control and showing that yeast expression rDP, rDF1 had similar biological activity as compared to the native protein is shown in table 2.
Table 3: comparison of Activity values of recombinant proteins with Natural proteins
5.DP 1/DF1 host protein content detection
Host residual protein (Host Cell Protein, HCP for short) refers to host protein that remains in the biological product. The protein has complex components and various kinds, and can change according to different production processes and purification processes. The residual HCP in the genetic engineering product is an important factor influencing the purity of the product, and repeated use of the genetic engineering product containing the HCP can cause anaphylactic reaction of the organism, has potential adjuvant effect, and can also cause the organism to generate antibodies to the medicine so as to influence the curative effect of the medicine. The residual amount of HCP in a biological product reflects not only the consistency of the product lot to lot, but also an important indicator of the quality of the biological product. The inventors detected the prepared samples using the pichia pastoris HCP detection kit (F140, CYGNUS), and table 4 shows that the three-step purified DP1, DF1 protein HCP content was far below the recombinant biological product (yeast) HCP ceiling specified in the 2015 edition pharmacopoeia.
TABLE 4 HCP detection results of DP1 and DF1 proteins
6. DP1/DF1 residual DNA content detection
Although the recombinant protein medicines are purified in multiple steps, DNA fragments of host cells still possibly remain in the preparation, and the residual DNA may bring about infectious or tumorigenic risks, and may cause insertion mutation, cause the inactivation of cancer suppressor genes, the activation of cancer genes and the like; in addition, the genome DNA of microorganism source is rich in CpG and unmethylated sequences, which increases the immunogenicity risk of recombinant protein drugs in vivo, so that the limit requirement of WHO and national drug registration authorities on residual DNA is very strict. The current method for detecting the DNA residual quantity mainly comprises a DNA probe hybridization method, a fluorescent dye method and a qPCR method, wherein the two methods have technical defects, the sensitivity of impurity limit detection is difficult to reach, and the FDA prescribes qPCR in the latest version of USP as the only recommended residual DNA detection method, so the inventor adopts qPCR to detect the residual DNA in a sample (SK 030205P100, huzhou Shen Ke biotechnology Co., ltd.), and the results in Table 5 show that the residual DNA content of the sample purified by three steps is far lower than the maximum limit of the residual DNA content of a recombinant biological product (yeast) prescribed in the 2015 version of pharmacopoeia.
TABLE 5 detection results of residual DNA of DP1 and DF1 proteins
EXAMPLE 6 recombinant DP1/DF1 (300L) purification Process validation
1. First step purification of recombinant DP1 fermentation supernatant
Step 1: pretreatment of fermentation broths
Centrifuging the fermentation broth obtained in the above example at high speed to obtain supernatant; adding diatomite for auxiliary filtration overnight to obtain a clarified fermentation broth sample; diluting with 10KD membrane package ultrafiltration, and reducing electric conductivity to below 5 mS/cm.
Step 2: cation exchange chromatography
Regulating pH of the treated fermentation broth supernatant with acetic acid to 5.0, loading the fermentation broth supernatant onto an SP FF chromatographic column with BPG140/100, a column bed volume of 2800ml, an equilibrium buffer of 50mM NaAc, pH of 5.0, an elution buffer of 50mM NaAc,1.0M NaCl,pH5.0, and linearly eluting according to 0-100%, wherein the DP1 target protein is mainly concentrated in a second elution peak, wherein FIG. 17-a is a first step purification chromatogram of the DP1 protein, and FIG. 17-b is a first step purification SDS-PAGE analysis of the DP1 protein; it is known that the first purification process of 300L of DP1 fermentation broth has better amplification effect.
2. Second step of purification of recombinant DP1 protein:
step 1: ultrafiltration
Combining the second elution peak in the first purification step 2, and carrying out ultrafiltration dilution by a10 KD membrane package until the electric conductance is less than 2.0mS/cm.
Step 2: anion exchange chromatography
Adding 20mM Tris into the sample obtained in the step 1, adjusting the pH value to 8.0, loading the sample onto a Q FF anion exchange chromatographic column, wherein the column volume is Hiscale 50/40, the column bed volume is 500ml, the balance buffer solution is 20mM Tris, the pH value is 8.0, the elution buffer solution is 20mM Tris,1.0M NaCl,pH8.0, collecting penetration, linearly eluting at 0-100%, and the target protein is mainly concentrated in the penetration. FIG. 18-a shows a second step purification chromatogram of the DP1 protein, and FIG. 18-b shows a second step purification SDS-PAGE analysis of the DP1 protein; it is known that the second purification process of DP1 protein has good amplification effect, and the purification process is well amplified.
3. Third step purification of recombinant DP1 protein
Adding ammonium sulfate to final concentration of 1.0M, adjusting pH to 8.5, and loading onto Phenyl FF chromatographic column with Hiscale 50/40, column bed volume of 500ml, balancing buffer of 20mM Tris,1.0M (NH) 4 ) 2 SO 4 The elution buffer was 20mM Tris, pH8.5, eluting at 25, 50%,70%,100% isocratic. FIG. 19-a is a third step purification chromatogram of the DP1 protein, and FIG. 19-b is a third step purification SDS-PAGE analysis of the DP1 protein; it can be seen that the third purification process of the DP1 protein has good amplification effect, and the purity of the DP1 protein obtained by three steps of purification>95％。
The purification process described above for recombinant DP1 applies equally to recombinant DF 1.
EXAMPLE 7 recombinant DP1/DF1 (300L broth purified sample) protein Activity assay
The activity detection method in step 4 of example 5 was used to detect the activity of the DP1 and DF1 proteins obtained by 300L fermentation broth, and the sample obtained by 30L fermentation broth and the natural protein were compared, and table 6 shows that the activity of the 300L fermentation and purification sample is slightly higher than that of the 30L and natural protein, which indicates that the fermentation and purification process is well amplified.
Table 6: comparison of 300L fermentation purified sample with 30L fermentation purified and Natural protein Activity value
Claims (8)
1. The preparation method of recombinant dust mite I type allergen DP1 and DF1 proteins comprises a fermentation process and a purification process, wherein the fermentation process comprises recombinant yeast strain activation, seed liquid culture and high-density fermentation, and is characterized in that a fermentation medium is 40% BSM medium, and the induction temperature in the induction stage of the high-density fermentation is as follows: the temperature is 25-27 ℃, the pH value is 6.5+/-0.2, and the components and the content of the BSM culture medium are K 2 SO 4 18.2g/L，MgSO 4 14.9g/L，KOH 4.13g/L，CaSO 4 0.93g/L，H 3 PO 4 26.7mL/L, glycerol 40g/L.
2. The method according to claim 1, wherein the methanol flow acceleration during the induction phase of the high-density fermentation is 30mL/h -1 L -1 And maintains DO not higher than 40%.
3. The method according to claim 1, wherein the fermentation temperature in the cell proliferation stage of the high-density fermentation is 27 ℃, ph=5.5±0.2, rotation speed 300rpm, do value 100%, and PTM 1.4 ml/L is added.
4. The method of claim 1, wherein the rate-limiting growth phase of the high-density fermentation is started at 30ml/h -1 L -1 Is added with 50 percent of glycerol, and the feeding speed is adjusted to be 60ml/h -1 L -1 。
5. The preparation method according to claim 1, wherein the purification process of the preparation method is three-step purification including cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography.
6. The process of claim 5, wherein the purified filler of cation exchange chromatography is SP FF and has a pH in the range of 4.0 to 6.5.
7. The process according to claim 5, wherein the purified filler of anion exchange chromatography is QFF, has a pH of 6.0-8.0 and a conductivity of 2.0mS/cm-20.0mS/cm.
8. The method according to claim 5, wherein the purified filler for hydrophobic chromatography is phenyl FF, and the pH is in the range of 6.5-8.5; the concentration of ammonium sulfate is 1.0-2.0M.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1780852A (en) * | 2003-04-25 | 2006-05-31 | 诺和酶股份有限公司 | Group 1 mite polypeptide variants |
| CN101821286A (en) * | 2007-10-08 | 2010-09-01 | 阿尔克-阿贝洛有限公司 | Produce the method for sophisticated recombinant mite group i allergen |
| CN101977929A (en) * | 2008-03-25 | 2011-02-16 | 拜尔工业制药有限公司 | Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies |
| CN102676568A (en) * | 2012-05-08 | 2012-09-19 | 崔玉宝 | Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein |
| CN103923894A (en) * | 2014-04-14 | 2014-07-16 | 崔玉宝 | Protein encoded by idiotoxin Der f4 gene of dermatophagoides farinae and application of protein |
| CN106929496A (en) * | 2015-12-31 | 2017-07-07 | 江苏众红生物工程创药研究院有限公司 | A kind of pharmaceutical grade recombined human kininogenase industrialization production method |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1780852A (en) * | 2003-04-25 | 2006-05-31 | 诺和酶股份有限公司 | Group 1 mite polypeptide variants |
| CN101821286A (en) * | 2007-10-08 | 2010-09-01 | 阿尔克-阿贝洛有限公司 | Produce the method for sophisticated recombinant mite group i allergen |
| CN101977929A (en) * | 2008-03-25 | 2011-02-16 | 拜尔工业制药有限公司 | Hypoallergenic hybrid proteins of major group 1 and 2 mite allergens for use in the treatment of allergies |
| CN102676568A (en) * | 2012-05-08 | 2012-09-19 | 崔玉宝 | Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein |
| CN103923894A (en) * | 2014-04-14 | 2014-07-16 | 崔玉宝 | Protein encoded by idiotoxin Der f4 gene of dermatophagoides farinae and application of protein |
| CN106929496A (en) * | 2015-12-31 | 2017-07-07 | 江苏众红生物工程创药研究院有限公司 | A kind of pharmaceutical grade recombined human kininogenase industrialization production method |
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