A recombinant pepsinogen a and a fermentation method and application thereof

Abstract

This invention relates to a fermentation method for recombinant porcine pepsinogen A (rPGA). The method involves high-density fermentation of recombinant Pichia pastoris containing a porcine pepsinogen encoding gene. During the induction expression phase, the temperature is controlled at 19-23°C and the pH at 5.5-6.0, and a fed-batch solution containing methanol and sorbitol is used. The mass ratio of methanol to sorbitol in the fed-batch solution is 0.3-1.6:1. This fermentation method significantly improves protein expression levels and stability. The resulting recombinant porcine pepsinogen A (rPGA) exhibits stable quality, high enzyme activity, no active precursors, is not easily degraded, and poses no risk of pathogenic microorganism residue, demonstrating high safety and applicability in the biomedical field.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant porcine pepsinogen A, its fermentation method, and its application. Background Technology

[0002] This section is intended to provide background or context for embodiments of the present invention. The description herein is not intended to imply that it is prior art simply because it is included in this section.

[0003] Pepsin (enzyme classification number: EC 3.4.23.1) belongs to the aspartic protease family and is produced by the autocatalytic synthesis of its precursor pepsinogen under acidic conditions. Pepsin has a wide range of proteolytic applications and is widely used in various agricultural products, protein processing, and even biomedicine.

[0004] Currently, commercially available pepsin is mainly extracted from the gastric mucosa of mammals such as pigs, cattle, and sheep. However, this extraction method is heavily reliant on raw material supply from the slaughtering industry, and the extraction process is complex, resulting in low yields and unstable product purity. Most importantly, the products pose potential biosafety risks due to the presence of pathogens such as viruses and prions. With increasing global attention to animal welfare and the trend towards "animal-free" biopharmaceuticals, traditionally extracted animal-derived pepsin can no longer meet the stringent requirements of modern biopharmaceuticals and high-end industrial sectors for high purity, high activity, and high safety.

[0005] With the development of genetic engineering technology, Pichia pastoris ( Komagataella phaffii Recombinant expression systems for producing heterologous proteins have become an effective way to solve the above problems. Currently, high-density fermentation of recombinant Pichia pastoris for pepsinogen production commonly employs a methanol-induced culture process at pH 5.0 and 28°C. However, this process still suffers from insufficient target protein expression and low enzyme activity, limiting its large-scale application.

[0006] Therefore, developing a stable and efficient high-density fermentation and large-scale preparation process for recombinant porcine pepsinogen A is of great scientific value and application prospects for meeting the growing demand in the biopharmaceutical and related high-end industrial fields. Summary of the Invention

[0007] The purpose of this invention is to provide a fermentation method for recombinant porcine pepsinogen A with increased expression levels of the target protein and enzyme activity, and its application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a fermentation method for recombinant porcine pepsinogen A (rPGA), which involves high-density fermentation of recombinant Pichia pastoris containing a porcine pepsinogen encoding gene. During the induction expression stage, the temperature is controlled at 19-23℃ and the pH at 5.5-6.0, and a fed-batch solution containing methanol and sorbitol is used. The mass ratio of methanol to sorbitol in the fed-batch solution is 0.3-1.6:1.

[0009] In some embodiments, the temperature during the induction expression phase is maintained at 19°C, 20°C, 21°C, 22°C, or 23°C.

[0010] Furthermore, the temperature during the induction expression stage is maintained at 19-22°C, and even further, the temperature during the induction expression stage is maintained at 19-21°C.

[0011] In some embodiments, the pH during the induction expression phase is maintained at 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0012] Furthermore, the pH during the induction expression phase is maintained at 5.6-5.8.

[0013] In some embodiments, the mass ratio of methanol to sorbitol in the combined feed solution is 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, or 1.6:1.

[0014] Further, the mass ratio of methanol to sorbitol in the combined feed solution is 0.5-1.6:1; even further, the mass ratio of methanol to sorbitol in the combined feed solution is 0.8-1.6:1; still further, the mass ratio of methanol to sorbitol in the combined feed solution is 1-1.6:1; and still further, the mass ratio of methanol to sorbitol in the combined feed solution is 1-1.5:1.

[0015] In some embodiments, the sorbitol content in the combined feed solution is 24%-40% by mass, for example 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%.

[0016] Furthermore, the sorbitol content in the combined feed solution is 24%-30% by mass, and even further, the sorbitol content in the combined feed solution is 25%-28% by mass.

[0017] In some embodiments, the combined feed solution also contains 1-1.3 g / L of PTM1 and 0.05-0.15 g / L of sulfuric acid.

[0018] Furthermore, the concentration of PTM1 in the combined feeding solution is 1 g / L, 1.1 g / L, 1.2 g / L, or 1.3 g / L.

[0019] Further, the concentration of sulfuric acid in the combined feed solution is 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, or 0.15 g / L.

[0020] In some further embodiments, during the induction expression stage, the temperature is controlled at 19-21°C and the pH at 5.6-5.8, and the mass ratio of methanol to sorbitol in the combined feeding solution is 1-1.5:1.

[0021] Furthermore, the sorbitol content in the combined feed solution is 25%-28% by mass.

[0022] In some embodiments, 6-8 hours after the start of induction culture, ascorbic acid or mannitol is added to the reaction system until the final working concentration of ascorbic acid or mannitol is 3-8 mM, for example, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. Further, the final working concentration of ascorbic acid or mannitol is 4-6 mM.

[0023] Furthermore, 6-8 hours after the induction culture begins, ascorbic acid is added to the reaction system until the final working concentration of ascorbic acid is 4-6 mM.

[0024] Furthermore, the final working concentration of the ascorbic acid is 4.5-5.5 mM.

[0025] Furthermore, the ascorbic acid is fed in the form of an ascorbic acid aqueous solution with a concentration of 90-110 g / L, and the mannitol is fed in the form of a mannitol aqueous solution with a concentration of 80-90 g / L.

[0026] In some embodiments, during the induction expression stage, the temperature and pH are maintained at a set value, the stirring speed is 300-500 rpm, and the aeration rate is 1.5-3.0 vvm. The combined feed solution is added at a rate of 0.7-2 mL / L / h according to the volume of the fermentation system. Subsequently, the flow rate of the combined feed solution is gradually increased by 5-15% every 20-40 minutes, and the DO curve is monitored in real time to gradually stabilize the DO value between 20-30%. When the flow rate of the combined feed solution is increased to 2.4-4 mL / L / h, the DO-stat linked feed mode is activated. When the DO value is >30%, feed is automatically added, and when the DO value is below 20%, feed is paused, so that the DO value always fluctuates within the range of 20%-30%. The entire induction expression process lasts for 60-90 hours.

[0027] The induction culture begins after the glycerol feeding culture ends, and the entire induction expression process includes two parts: the induction adaptation period and the induction expression stage.

[0028] Furthermore, 6-8 hours after the induction begins, ascorbic acid solution or mannitol solution is added at a rate of 0.1-0.3 mL / L / h to ensure that the final working concentration of ascorbic acid or mannitol during the induction process is 3-8 mM, and the DO value fluctuation is monitored.

[0029] In some embodiments, prior to the induction expression stage, there is an induction adaptation period of 1-4 hours, during which anhydrous methanol is added at a rate of 0.4-1.0 mL / L / h, and the stirring speed and aeration rate are adjusted to gradually stabilize the DO value between 20-30%.

[0030] In some embodiments, the method for preparing the recombinant Pichia pastoris containing the porcine pepsinogen encoding gene is as follows: (1) Synthesize a plasmid containing a gene encoding porcine pepsinogen, wherein the gene encoding porcine pepsinogen is shown in SEQ ID NO.1; (2) The plasmid was linearized using restriction endonuclease, and then electroporated into competent Pichia pastoris. The recombinant Pichia pastoris was obtained by culturing and screening.

[0031] Furthermore, the Pichia pastoris is either strain GS115 or strain X33.

[0032] Furthermore, the plasmid is a plasmid containing the AOX1 promoter.

[0033] Furthermore, the plasmid is the pPIC9K plasmid.

[0034] In some embodiments, the fermentation method further includes seed culture of the recombinant Pichia pastoris to OD. 600 The initial concentration of DO was 20-50%. Then, the DO was inoculated into the fermentation medium. The initial culture temperature was set to 28-30℃, the stirring speed to 280-320 rpm, and the aeration rate to 1.0-2.0 vvm. The fermentation tank pressure was controlled at 0.03-0.05 MPa. Fermentation was carried out, and the DO value was monitored and maintained above 30%. The pH was adjusted to 5.0-5.5 using concentrated ammonia. Antifoaming agent was automatically added based on the foam signal. This stage of culture lasted 16-30 hours. When the DO value rose above 80%, glycerol feed solution was immediately added at a rate of 1-5 mL / L / h according to the volume of the culture medium in the tank. The stirring speed was set to 300-360 rpm, and the aeration rate to 1.5-2.5 vvm, maintaining the DO value between 20% and 30%. This stage of culture lasted 2-6 hours.

[0035] In some embodiments, the fermentation method further includes a purification method after high-density fermentation. The purification method involves: after fermentation, collecting the supernatant using a tubular centrifuge at 12,000-14,000 rpm, filtering to remove impurities, and then passing the supernatant through a tangential flow ultrafiltration device. The supernatant is concentrated to 10-20% of its original volume using a 5-10 kDa molecular weight membrane. The pH of the concentrate is adjusted to 7.2-7.6, imidazole is dissolved to a final concentration of 5-10 mM, and NaCl is dissolved to a final concentration of 0.4-0.6 M. The concentrate is then passed through a Ni... 2+ Affinity chromatography column was used to elute proteins with a buffer containing 20-50 mM PB, 100-500 mM imidazole, and pH 6.0-6.5, and the eluent was collected. The eluent was then passed into a tangential flow ultrafiltration device and concentrated to 10-20% of its original volume using a 5-10 kDa molecular weight filter membrane. The buffer system was then replaced with a 20-100 mM Tris-HCl buffer containing 100-200 mM NaCl, 400-600 g / L glycerol, and pH 6.0-7.0 to obtain purified recombinant porcine pepsinogen A.

[0036] In some embodiments, the expression level of the recombinant porcine pepsinogen A is above 700 mg / L.

[0037] Furthermore, the expression level of the recombinant porcine pepsinogen A is above 800 mg / L.

[0038] Furthermore, the expression level of the recombinant porcine pepsinogen A is above 1000 mg / L.

[0039] Furthermore, the expression level of the recombinant porcine pepsinogen A is above 1100 mg / L.

[0040] The present invention also provides a recombinant porcine pepsinogen A prepared by the fermentation method described in any of the preceding claims, wherein the enzyme activity of the recombinant porcine pepsinogen A is above 4000 U / mg.

[0041] Furthermore, the recombinant porcine pepsinogen A has an enzyme activity of over 5000 U / mg.

[0042] Furthermore, the recombinant porcine pepsinogen A has an enzyme activity of over 6000 U / mg.

[0043] The present invention also provides the use of the recombinant porcine pepsinogen A in the preparation of antitoxin serum.

[0044] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The fermentation method of this invention significantly improves protein expression levels and stability. The resulting recombinant porcine pepsinogen A (rPGA) is of stable quality, has high enzyme activity, no active precursors, is not easily degraded, and has no risk of pathogenic microorganism residues, making it highly safe and applicable to the biomedical field. Attached Figure Description

[0045] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 SDS-PAGE gel electrophoresis images of the supernatant collected at different fermentation time periods in Example 1; Figure 2 This is a graph showing the comparison of yeast wet weight after fermentation in each embodiment and comparative example; Figure 3 The graph shows the comparison of expression levels of recombinant porcine pepsinogen A (rPGA) in each embodiment and comparative example. Figure 4 The graph shows the comparison of enzyme activities of recombinant porcine pepsinogen A (rPGA) in each embodiment and comparative example. Figure 5 A comparison of the results of digesting anti-viper venom serum with natural pepsin and recombinant porcine pepsinogen A prepared in Example 1. Detailed Implementation

[0047] To address the shortcomings of existing technologies, this invention optimizes the process parameters of the high-density fermentation method, particularly the pH and temperature during the induction culture stage. Supplementing with more ammonia to raise the pH provides a more abundant nitrogen source for Pichia pastoris growth and reduces the probability of rPGA self-activation, resulting in a more stable induction process. Low-temperature induction appropriately slows down the growth rate of Pichia pastoris, reducing the probability of misfolding of the target protein due to excessive growth and inhibiting protein degradation. This ultimately yields rPGA with lower misfolding, higher yield, and higher enzyme activity. Furthermore, during the induction culture stage, a combined feeding of methanol and sorbitol reduces the amount of flammable and explosive methanol used, improving process stability and safety. Sorbitol provides a non-inhibitory carbon source, alleviating methanol metabolic stress, resulting in higher cell activity and ultimately improving protein quality and yield. The recombinant porcine pepsinogen A prepared by this invention exhibits stable protein yield and batch quality, low product impurity content, stable enzyme activity, and is not limited by raw material supply. Moreover, the rPGA does not introduce exogenous viral factors, has no potential pathogens or chemical residues, and is highly safe, making it suitable for biomedical fields such as antitoxin serum production.

[0048] Furthermore, during the induction culture stage, the use of ascorbic acid as an auxiliary feed can reduce the large amount of H2O2 and reactive oxygen species produced by Pichia pastoris when metabolizing methanol, thereby reducing cellular oxidative stress damage, decreasing cell damage and protein misfolding, and improving the stability of protein expression; at the same time, it can reduce pigment impurities produced by yeast cells due to oxidative stress.

[0049] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0050] All features disclosed in this invention, or steps in all methods or processes disclosed, may be combined in any way, except for mutually exclusive features or steps.

[0051] The technical solutions of the present invention will be further described below with reference to specific embodiments. However, the present invention should not be limited to these embodiments. Unless specifically stated otherwise, all features can be replaced by other equivalent or similar alternative features. Unless specifically stated otherwise, each feature is only one example of a series of equivalent or similar features. The terminology used in the present invention, unless otherwise stated, generally has the meaning commonly understood by those skilled in the art. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use. Implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0052] In this invention, operations without specific instructions are performed at room temperature. The raw materials used in this invention are commercially available or can be prepared using conventional methods in the prior art. Unless otherwise specified, the methods described are those in the prior art.

[0053] In the fermentation process described in this invention, the set parameters such as temperature and pH are preferred or target values ​​determined based on process optimization. Those skilled in the art should understand that in actual fermentation systems, due to dynamic changes in cellular metabolic activity, these parameters may fluctuate slightly around the set values. Through conventional online monitoring and feedback control, these parameters can be stably maintained within a reasonably acceptable range of fluctuation recognized in the art (e.g., temperature ±1℃, pH ±0.1). This is an inherent characteristic of dynamic biological processes, and such slight fluctuations do not affect the technical effects of this invention.

[0054] Example 1: High-density fermentation method for recombinant porcine pepsinogen A

[0055] (2) The pPIC9K-PGA plasmid synthesized according to the aforementioned method was linearized using SacI restriction endonuclease (purchased from TaKaRa), and the plasmid was collected by ethanol precipitation. 10 μg of the linearized plasmid was added to competent Pichia pastoris strain GS115, and electroporation was performed at 1500V, 25μF, 200Ω, and 1 pulse. The electroporated Pichia pastoris was inoculated into RD medium for culture, and the recombinant strain GS115-pPIC9K-PGA with the highest relative expression level was finally screened.

[0056] (3) Preparation of culture medium and feed: YPD liquid culture medium: Weigh 10 g / L yeast extract, 20 g / L tryptone and 20 g / L glucose, add sterile water to dissolve and make up to volume; autoclave and cool to room temperature.

[0057] BSM medium: Measure 26.7 mL / L of 85% phosphate, weigh 18.2 g / L of potassium sulfate, 0.9 g / L of calcium sulfate, 4.1 g / L of potassium hydroxide, 14.9 g / L of magnesium sulfate heptahydrate, and 40 g / L of glycerol, mix them and add them directly to the fermenter. Add pure water and sterilize the fermenter in place.

[0058] PTM1 solution: Weigh 95 g / L PTM1 powder, dissolve it in pure water, then add 5 mL / L concentrated sulfuric acid, and make up to volume with pure water. Filter through a 0.22 μm filter to remove bacteria.

[0059] Ascorbic acid supplementation solution: Weigh 100 g / L ascorbic acid, dissolve in pure water and bring to a final volume, filter through 0.22 μm for sterilization, and store in a sterile, light-proof bottle.

[0060] Glycerin feed solution: Weigh 500 g / L glycerin, dissolve it in pure water and make up to volume, then add 12 mL / L PTM1 solution.

[0061] Combined feed solution: Anhydrous methanol and 500 g / L sorbitol aqueous solution were mixed at a ratio of 1:1.2 (v / v), with the mass ratio of methanol to sorbitol being approximately 1.3:1. Then, 12 mL of PTM1 solution was added to each liter of feed solution.

[0062] (4) Primary seed culture: The frozen GS115-pPIC9-PGA working cell bank strain was taken out from -80℃ and inoculated into 100mL YPD medium at a ratio of 1:100 (v / v) in a clean bench. The culture was incubated at 30℃ for approximately 36 hours with a shaker at 220rpm. After the culture was completed, a small amount of the bacterial culture was diluted, and the final OD was measured. 600 It ranges from 6 to 10.

[0063] (5) Secondary seed culture: The BSM medium in the 10L seed tank was sterilized and cooled in advance. Sterile concentrated ammonia was introduced to adjust the pH to 5. All primary seeds were inoculated by flame. The culture was carried out at 28℃, 280rpm, and an aeration rate of 0.8~1.2vvm for 16h. After the culture was completed, a small amount of bacterial solution was taken, diluted, and the final OD was measured. 600 It ranges from 20 to 50.

[0064] (6) Glycerol batch culture: The BSM medium in the 100L fermenter was sterilized and cooled in advance. Sterile concentrated ammonia was introduced to adjust the pH to 5. The initial temperature was set to 28℃, the stirring speed to 300rpm, and the aeration rate to 1.0~2.0vvm; the pressure in the fermenter was controlled at 0.03~0.05MPa. Then, the secondary seed from the seed tank was introduced into the fermenter. DO was monitored and maintained at >30%, and the pH was adjusted to 5.0~5.5 using concentrated ammonia; the defoamer 204 was automatically added based on the foam signal. This stage was continued for 20h, and at the end, a portion of the culture medium was taken out to measure OD. 600 And the wet weight of yeast.

[0065] (7) Glycerol-feeded culture: When the DO rises sharply to above 80%, immediately start adding glycerol feed solution at a rate of 50-200 mL / h according to the culture medium in the tank. Set the stirring speed to 340 rpm and the aeration rate to 1.5-2.5 vvm to maintain the DO between 20% and 30%. Continue this stage of culture for 2 hours, then stop adding glycerol. At the end of the culture, take out a portion of the culture medium to measure the OD. 600 And the wet weight of yeast.

[0066] (8) Induction and adaptation period: After the glycerol feeding is completed, the DO level rises sharply to over 80%, and the induction phase begins. At this time, anhydrous methanol is added at a rate of 20-40 mL / h. The DO level will start to fluctuate at this point. Adjust the stirring speed and aeration rate to gradually stabilize the DO level between 20-30%. This phase is continued for 2 hours to ensure that the yeast adapts and consumes methanol in the early stage of induction, thus avoiding cell stress.

[0067] (9) rPGA Induction Expression: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, and the pH was maintained at 5.7, the temperature at 20℃, the stirring speed at 400 rpm, and the aeration rate at 1.5~3.0 vvm during the induction expression process. At this time, the combined feed solution was added at a rate of 40~80 mL / h, and then the flow rate of the combined feed solution was gradually increased by 10% every 30 min, and the DO curve was monitored in real time to gradually stabilize it between 20~30%. When the flow rate of the combined feed solution was gradually increased to 100~150 mL / h, the DO-stat linked feed mode was started. When DO>30%, feed was automatically added, and when DO was below 20%, feed was stopped, so that DO always fluctuated within the range of 20%~30%. Six hours after induction began, ascorbic acid solution was added at a rate of 8 mL / h to maintain a final working concentration of 5 mM during induction, and DO (dissolved oxygen) fluctuations were monitored. Freshly prepared ascorbic acid solution was used 48 hours after the start of induction, and induction continued. The entire induction process lasted 84 hours. From the start to the end of induction, 100-200 mL of culture medium was collected every 12 hours to measure OD (dissolved oxygen). 600 Yeast wet weight and protein expression.

[0068] (10) The fermentation supernatant collected at each stage was diluted 10-fold with 1×PBS, and the protein expression was identified by SDS-PAGE gel electrophoresis. Figure 1 ).like Figure 1 As shown, no obvious band was observed at the 43 kDa position in the lanes of the 0-hour (uninduced) sample. After induction with anhydrous methanol for 2 hours, a weak band began to appear at the same position. With the extension of induction time, the band signal at this position significantly increased and became clearly distinguishable in samples inducing for 6 hours and beyond. The size of this band is consistent with the expected molecular weight of recombinant porcine pepsinogen A, and its expression has a clear time dependence, indicating that the exogenous gene was successfully expressed under the induction of the combined feeding solution, and recombinant porcine pepsinogen A was effectively synthesized in the host.

[0069] (11) Take 300 mL of the culture medium after fermentation and divide it into 3 groups of 100 mL each. Centrifuge at 8,000 rpm, pour out the supernatant, weigh the remaining precipitate w1 in each group, weigh the blank container w2, calculate the wet weight w = (w1 - w2) × 10, and finally take the average of the three wet weights. ( Figure 2 ).

[0070] (12) Take the supernatant after fermentation, determine the His tag concentration by ELISA, and calculate the rPGA protein expression level. Figure 3Purified rPGA protein with a 6×His tag (purity ≥95%) was used as a standard and diluted to 0 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 1500 ng / mL, and 2000 ng / mL, with three replicates for each concentration. The supernatant was diluted 1000-fold. Then, 100 μL of the standard solution at each concentration and the diluted supernatant were added to the wells of the ELISA plate pre-coated with Anti-His tag antibody. 1×PBS was added to three wells as a blank control. The plate was incubated at 37°C for 60 min. After incubation, the liquid in the wells was discarded, and each well was washed with 200 μL of PBST for 3 min. This process was repeated five times. Next, 100 μL of HRP-labeled Anti-His tag detection antibody was added to each well, and the mixture was incubated at 37°C for 60 min. The liquid in the wells was then discarded, and each well was washed with 200 μL of PBST for 3 min, repeated 5 times. Then, 100 μL of TMB chromogenic solution was added to each well, and the mixture was incubated at 37°C in the dark for 10 min. The reaction was stopped when a clear blue gradient appeared in the standard wells, and 50 μL of 2M sulfuric acid was added to each well to terminate the reaction. The absorbance of each well was read at 450 nm, and a standard curve was plotted based on the absorbance of the standards. The rPGA expression level was then calculated.

[0071] (13) After fermentation, the yeast was fed into a tubular centrifuge and the supernatant was collected at 14,000 rpm. Then, it was subjected to gradient filtration to remove impurities. The supernatant was then fed into a tangential flow ultrafiltration device and concentrated to 10% of its original volume using a 10 kDa molecular weight membrane.

[0072] (14) Adjust the pH of the concentrate to 7.4, dissolve imidazole to a final concentration of 10 mM, and dissolve NaCl to a final concentration of 0.5 M. Then pass the concentrate through Ni. 2+ Affinity chromatography column was used to linearly elute proteins with 20 mM PB + 500 mM imidazole buffer at pH 6.4, and the eluent of the A280 signal peak was collected.

[0073] (15) Pass the eluent into a tangential flow ultrafiltration device and concentrate it to 10% of its original volume using a 10 kDa molecular weight filter membrane. Replace the buffer system with a 50 mM Tris-HCl + 100 mM NaCl + 50% (v / v) glycerol, pH=7.0 buffer solution and store at -20°C.

[0074] (16) Take 1 mL of purified rPGA, calculate the concentration, and then measure the enzyme activity ( Figure 4Dilute rPGA 1000 times with 65mM HCl solution. Take 6 test tubes, add 1 mL of 0.5 mg / mL tyrosine reference solution to 3 tubes, and add 1 mL of diluted rPGA solution to the other 3 tubes. Incubate at 37 ℃ for 5 min. Then add 5 mL of hemoglobin solution preheated to 37 ℃. Shake well and react at 37 ℃ for 10 min. Immediately add 5 mL of 5% trichloroacetic acid. Shake well and filter 0.22 μm for later use. Take another 2 test tubes, add 5 mL of hemoglobin solution to each, react at 37 ℃ for 10 min, and immediately add 5 mL of 5% trichloroacetic acid to stop the reaction; add 1 mL of diluted rPGA solution to one tube and 1 mL of 65mM HCl solution to the other tube. Shake well and filter 0.22 μm for later use as blank controls for rPGA and the reference standard, respectively. Take all the filtrates and measure the absorbance at a wavelength of 275 nm, then calculate the average value. S and Calculate pepsin activity (U / g) using the following formula.

[0075] In the formula S The average absorbance of the reference standard; The average absorbance of rPGA; W S The amount of tyrosine in 1 mL of the reference standard, in μg; W represents the rPGA sample size, in grams. This represents the dilution factor of rPGA.

[0076] Enzyme activity is defined as the amount of enzyme required to hydrolyze hemoglobin to produce 1 μmol of tyrosine per minute at 37°C, which is one unit of protease activity.

[0077] Example 2: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that: in this example, competent Pichia pastoris strain X33 is used instead of competent Pichia pastoris strain GS115 in Example 1 in order to screen for the recombinant strain X33-pPIC9K-PGA with the highest relative expression level.

[0078] The results showed that the fermentation conditions of this embodiment could also produce a significant and effective rPGA induction effect on Pichia pastoris X33 strain, but the rPGA expression level and enzyme activity were not as good as in Example 1.

[0079] Example 3: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in embodiment 1, except that the temperature for rPGA-induced expression in step (9) is maintained at 22°C.

[0080] Example 4: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that the pH for rPGA-induced expression in step (9) is maintained at 5.5.

[0081] Example 5: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that the pH for rPGA-induced expression in step (9) is maintained at 6.0.

[0082] Example 6: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except for the formulation of the combined feeding solution.

[0083] The combined feed solution in this embodiment is as follows: anhydrous methanol and 500 g / L sorbitol aqueous solution are mixed at a ratio of 1:5 (v / v), with the mass ratio of methanol to sorbitol being approximately 0.3:1. Then, 12 mL of PTM1 solution is added to each liter of the combined feed solution.

[0084] Example 7: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except for the formulation of the combined feeding solution.

[0085] The combined feed solution in this embodiment is as follows: anhydrous methanol and 500 g / L sorbitol aqueous solution are mixed at a ratio of 1:1 (v / v), with the mass ratio of methanol to sorbitol being approximately 1.6:1. Then, 12 mL of PTM1 solution is added to each liter of the combined feed solution.

[0086] Example 8: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that in step (9) rPGA induction expression, the ascorbic acid aqueous solution is not fed in this process.

[0087] In this embodiment, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining it at 5.7, 20°C, 400 rpm, and 1.5–3.0 vvm during the induction process. A combined feed solution was then added at a rate of 40–80 mL / h, with the flow rate gradually increased by 10% every 30 min, while the DO curve was monitored in real time to stabilize it between 20% and 30%. When the flow rate reached 100–150 mL / h, a DO-stat-linked feeding mode was activated, automatically feeding when DO > 30% and pausing when DO falls below 20%, ensuring DO remained within the 20%–30% range. The entire induction process lasted 84 h. From the start to the end of induction, 100–200 mL of culture medium was collected every 12 h to measure OD. 600 Yeast wet weight and protein expression.

[0088] Example 9: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that in step (9), rPGA is induced to express, and the working concentration of ascorbic acid aqueous solution is 4 mM.

[0089] In this embodiment, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining the pH at 5.7 and the temperature at 20℃ during the induction expression process. The stirring speed was 400 rpm, and the aeration rate was 1.5–3.0 vvm. A combined feed solution was then added at a rate of 40–80 mL / h, and the flow rate was gradually increased by 10% every 30 min, while the DO curve was monitored in real time to gradually stabilize it between 20% and 30%. When the flow rate of the combined feed solution was gradually increased to 100–150 mL / h, the DO-stat linked feed mode was activated. Feeding was automatic when DO > 30% and paused when DO fell below 20%, ensuring that DO remained within the 20%–30% range. Six hours after induction began, ascorbic acid solution was added at a rate of 6 mL / h to maintain a final working concentration of 4 mM. Dissolved oxygen (DO) levels were monitored for fluctuations. Freshly prepared ascorbic acid solution was added 48 hours after the start of induction. The entire induction process lasted 84 hours. From the start to the end of induction, 100-200 mL of culture medium was collected every 12 hours to measure OD. 600 Yeast wet weight and protein expression.

[0090] Example 10: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that the rPGA induction expression in step (9) is performed at a working concentration of 6 mM for ascorbic acid aqueous solution.

[0091] In this embodiment, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining the pH at 5.7 and the temperature at 20℃ during the induction expression process. The stirring speed was 400 rpm, and the aeration rate was 1.5–3.0 vvm. A combined feed solution was then added at a rate of 40–80 mL / h, and the flow rate was gradually increased by 10% every 30 min, while the DO curve was monitored in real time to gradually stabilize it between 20% and 30%. When the flow rate of the combined feed solution was gradually increased to 100–150 mL / h, the DO-stat linked feed mode was activated. Feeding was automatic when DO > 30% and paused when DO fell below 20%, ensuring that DO remained within the 20%–30% range. Six hours after induction began, ascorbic acid solution was added at a rate of 10 mL / h to maintain a final working concentration of 6 mM. Dissolved oxygen (DO) levels were monitored for fluctuations. Freshly prepared ascorbic acid solution was used 48 hours after the start of induction. The entire induction process lasted 84 hours. From the start to the end of induction, 100-200 mL of culture medium was collected every 12 hours to measure OD. 600 Yeast wet weight and protein expression.

[0092] Example 11: High-density fermentation method for recombinant porcine pepsinogen A The method in this embodiment is basically the same as that in Example 1, except that in step (9) rPGA induction expression, the process is fed with mannitol aqueous solution.

[0093] In this embodiment, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining it at 5.7, 20°C, 400 rpm, and 1.5–3.0 vvm during the induction process. A combined feed solution was then added at a rate of 40–80 mL / h, with the flow rate gradually increased by 10% every 30 min, while the DO curve was monitored in real time to stabilize it between 20–30%. When the flow rate of the combined feed solution was gradually increased to 100–150 mL / h, the DO-stat-linked feeding mode was activated. Feeding was automatic when DO > 30% and paused when DO fell below 20%, ensuring DO remained within the 20–30% range. Six hours after induction began, mannitol aqueous solution was added at a rate of 10 mL / h, maintaining a final working concentration of 5 mM for DO fluctuations. The entire induction process lasted 84 hours. From the start to the end of induction, 100-200 mL of culture medium was taken every 12 hours to measure OD. 600 Yeast wet weight and protein expression.

[0094] The mannitol aqueous solution is a 90g / L mannitol aqueous solution that has been sterilized by 0.22μm filtration and stored in a sterile, light-proof bottle.

[0095] Comparative Example 1: High-density fermentation method for recombinant porcine pepsinogen A The method of this comparative example is basically the same as that of Example 1, except that the rPGA-induced expression in step (9) is different.

[0096] In this comparative example, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining the pH at 5.0, the temperature at 28℃, the stirring speed at 400 rpm, and the aeration rate at 1.5–3.0 vvm during the induction process. A methanol-only feed solution was then added at a rate of 40–80 mL / h, with the flow rate gradually increased by 10% every 30 min, while the DO curve was monitored in real time to stabilize it between 20% and 30%. When the flow rate of the methanol-only feed solution was gradually increased to 100–150 mL / h, the DO-stat-linked feeding mode was activated. Feeding was automatic when DO > 30% and paused when DO fell below 20%, ensuring that DO remained within the 20%–30% range. The entire induction process lasted 84 h. From the start to the end of induction, 100–200 mL of culture medium was collected every 12 h to measure OD. 600 Yeast wet weight and protein expression.

[0097] The single methanol feed solution is prepared by adding 12 mL of PTM1 solution to each liter of anhydrous methanol.

[0098] Comparative Example 2: High-density fermentation method for recombinant porcine pepsinogen A The method of this comparative example is basically the same as that of Example 1, except that the pH for rPGA-induced expression in step (9) is maintained at 5.0 and the temperature is maintained at 28°C.

[0099] Comparative Example 3: High-density fermentation method for recombinant porcine pepsinogen A The method of this comparative example is basically the same as that of Example 1, except that the inducing agent used for rPGA induction expression in step (9) is a methanol feed solution.

[0100] In this comparative example, step (9) is as follows: After the yeast adapted to the methanol environment, concentrated ammonia was used to adjust the pH, maintaining the pH at 5.7 and the temperature at 20℃ during the induction expression process. The stirring speed was 400 rpm, and the aeration rate was 1.5–3.0 vvm. A methanol-only feed solution was then added at a rate of 40–80 mL / h, with the flow rate gradually increased by 10% every 30 min, while the DO curve was monitored in real time to gradually stabilize it between 20% and 30%. When the flow rate of the methanol-only feed solution was gradually increased to 100–150 mL / h, the DO-stat linked feed mode was activated. Feeding was automatic when DO > 30% and paused when DO fell below 20%, ensuring that DO remained within the 20%–30% range. Six hours after induction began, ascorbic acid solution was added at a rate of 8 mL / h to maintain a final working concentration of 5 mM during induction, and DO (dissolved oxygen) fluctuations were monitored. Freshly prepared ascorbic acid solution was used 48 hours after the start of induction, and induction continued. The entire induction process lasted 84 hours. From the start to the end of induction, 100-200 mL of culture medium was collected every 12 hours to measure OD (dissolved oxygen). 600 Yeast wet weight and protein expression.

[0101] The single methanol feed solution is prepared by adding 12 mL of PTM1 solution to each liter of anhydrous methanol.

[0102] The percentage increase in wet weight, rPGA expression level, and enzyme activity of Pichia pastoris compared to Comparative Example 1 for each embodiment and comparative example is shown in Table 1 below.

[0103] Table 1 See Table 1 and Figure 2-4Compared to Comparative Example 1, Examples 1-11 showed significant improvements in Pichia pastoris wet weight, rPGA expression level, and enzyme activity. This indicates that a combined feed solution prepared with anhydrous methanol and 500 g / L sorbitol aqueous solution at a ratio of 1:1.1-1.5 (v / v) at pH 5.5-6.0 and 20-22℃ significantly improved rPGA expression and enzyme activity. Among these, Example 1, with its optimal induction parameters of pH 5.7, 20℃, a combined feed solution prepared with anhydrous methanol and 500 g / L sorbitol aqueous solution at a ratio of 1:1.2 (v / v), and the addition of 5 mM ascorbic acid, demonstrated the best results. A comparison of Examples 8 and 11 shows that adding mannitol during rPGA induction is detrimental to improving rPGA expression and enzyme activity. Comparisons of Examples 1 and 8-10 show that adding 4-6 mM ascorbic acid during rPGA induction expression is beneficial for increasing rPGA expression levels and enzyme activity. Comparisons of Example 1 and Comparative Example 2 show that at a reaction temperature of 28°C and pH 5.0, even with a combined feed solution and ascorbic acid addition, the increase in rPGA expression levels and enzyme activity is extremely limited. Comparisons of Example 1 and Comparative Example 3 show that even at the optimal reaction temperature, pH, and ascorbic acid addition, adding only methanol instead of a combined methanol and sorbitol feed solution results in extremely limited increases in rPGA expression levels and enzyme activity.

[0104] Example 12: Method for preparing purified rPGA-digested equine anti-viper venom serum as described in Example 1 (1) Take 200 mL of horse anti-viper venom serum and adjust the pH to 3.3 with 1 M HCl aqueous solution. Take out 100 mL and add 1 mL of 0.1 mg / mL rPGA. The digestion temperature is 30℃ and the digestion time is 60 min.

[0105] (2) Take another 100 mL of horse anti-viper venom serum (from the same batch), add 1 mL of 0.1 mg / mL porcine gastric mucosa extract pepsin (purchased from Shanghai Sangon Biotech), digest at 30℃ for 60 min.

[0106] (3) Take the sample after the reaction is complete, add SDS-PAGE loading buffer containing β-mercaptoethanol, heat in boiling water for 10 min, centrifuge at 12,000 rpm, and collect the supernatant for SDS-PAGE gel electrophoresis detection. Figure 5 The results showed that, for the same mass, rPGA was more effective than naturally extracted pepsin in digesting equine anti-viper venom serum, and rPGA had a higher relative enzyme activity.

[0107] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A fermentation method for recombinant porcine pepsinogen A, characterized in that: High-density fermentation was carried out on recombinant Pichia pastoris containing a porcine pepsinogen encoding gene. During the induction expression stage, the temperature was controlled at 19-23℃ and the pH at 5.5-6.0, and a fed-batch solution containing methanol and sorbitol was used. The mass ratio of methanol to sorbitol in the fed-batch solution was 0.3-1.6:

1.

2. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: The sorbitol content in the combined feed solution is 24%-40% by mass.

3. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: The combined feed solution also contains 1-1.3 g / L of PTM1 and 0.05-0.15 g / L of sulfuric acid.

4. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: During the induction expression stage, the temperature was controlled at 19-21℃ and the pH at 5.6-5.8, and the mass ratio of methanol to sorbitol in the combined feeding solution was 1-1.5:

1.

5. The fermentation method for recombinant porcine pepsinogen A according to claim 4, characterized in that: The sorbitol content in the combined feed solution is 25%-28% by mass.

6. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: Six to eight hours after the induction culture begins, ascorbic acid or mannitol is added to the reaction system until the final working concentration of ascorbic acid or mannitol is 3-8 mM.

7. The fermentation method for recombinant porcine pepsinogen A according to claim 6, characterized in that: Six to eight hours after the induction culture begins, ascorbic acid is added to the reaction system until the final working concentration of ascorbic acid is 4-6 mM.

8. The fermentation method for recombinant porcine pepsinogen A according to claim 7, characterized in that: The final working concentration of the ascorbic acid is 4.5-5.5 mM.

9. The fermentation method for recombinant porcine pepsinogen A according to claim 6, characterized in that: The ascorbic acid is added in the form of an aqueous solution of ascorbic acid with a concentration of 90-110 g / L, and the mannitol is added in the form of an aqueous solution of mannitol with a concentration of 80-90 g / L.

10. The fermentation method for recombinant porcine pepsinogen A according to any one of claims 1 to 9, characterized in that: During the induction expression phase, the temperature and pH were maintained at the set values, the stirring speed was 300-500 rpm, and the aeration rate was 1.5-3.0 vvm. The combined feed solution was added at a rate of 0.7-2 mL / L / h according to the volume of the fermentation system. Subsequently, the flow rate of the combined feed solution was gradually increased by 5-15% every 20-40 min, and the DO curve was monitored in real time to gradually stabilize the DO value between 20-30%. When the flow rate of the combined feed solution was increased to 2.4-4 mL / L / h, the DO-stat linked feed mode was activated. Feeding was automatic when the DO value was >30% and stopped when the DO value was below 20%, so that the DO value always fluctuated within the range of 20%-30%. The entire induction expression process lasted 60-90 h.

11. The fermentation method for recombinant porcine pepsinogen A according to claim 10, characterized in that: Six to eight hours after the induction begins, ascorbic acid or mannitol solution is added at a rate of 0.1-0.3 mL / L / h to ensure that the final working concentration of ascorbic acid or mannitol during the induction process is 3-8 mM. The DO value fluctuation is then monitored.

12. The fermentation method for recombinant porcine pepsinogen A according to any one of claims 1 to 9, characterized in that: Before the induction expression stage, there is also an induction adaptation period of 1-4 hours. During this stage, anhydrous methanol is added at a rate of 0.4-1.0 mL / L / h, and the stirring speed and aeration rate are adjusted to gradually stabilize the DO value between 20-30%.

13. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: The method for preparing the recombinant Pichia pastoris containing the porcine pepsinogen encoding gene is as follows: (1) Synthesize a plasmid containing a gene encoding porcine pepsinogen, wherein the gene encoding porcine pepsinogen is shown in SEQ ID NO.1; (2) The plasmid was linearized using restriction endonuclease, and then electroporated into competent Pichia pastoris. The recombinant Pichia pastoris was obtained by culturing and screening.

14. The fermentation method for recombinant porcine pepsinogen A according to claim 1 or 13, characterized in that: The Pichia pastoris is either strain GS115 or strain X33.

15. The fermentation method for recombinant porcine pepsinogen A according to claim 13, characterized in that: The plasmid is a plasmid containing the AOX1 promoter.

16. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: The fermentation method further includes seed culture of the recombinant Pichia pastoris to OD. 600 The initial concentration of DO was 20-50%. Then, the DO was inoculated into the fermentation medium. The initial culture temperature was set to 28-30℃, the stirring speed to 280-320 rpm, and the aeration rate to 1.0-2.0 vvm. The fermentation tank pressure was controlled at 0.03-0.05 MPa. Fermentation was carried out, and the DO value was monitored and maintained above 30%. The pH was adjusted to 5.0-5.5 using concentrated ammonia. Antifoaming agent was automatically added based on the foam signal. This stage of culture lasted 16-30 hours. When the DO value rose above 80%, glycerol feed solution was immediately added at a rate of 1-5 mL / L / h according to the volume of the culture medium in the tank. The stirring speed was set to 300-360 rpm, and the aeration rate to 1.5-2.5 vvm, maintaining the DO value between 20% and 30%. This stage of culture lasted 2-6 hours.

17. The fermentation method for recombinant porcine pepsinogen A according to claim 1, characterized in that: The fermentation method also includes a purification method after high-density fermentation. The purification method is as follows: after fermentation, the supernatant is collected using a tubular centrifuge at 12,000-14,000 rpm, then filtered to remove impurities, and passed through a tangential flow ultrafiltration device. The supernatant is concentrated to 10-20% of its original volume using a 5-10 kDa molecular weight membrane. The pH of the concentrate is adjusted to 7.2-7.6, imidazole is dissolved to a final concentration of 5-10 mM, and NaCl is dissolved to a final concentration of 0.4-0.6 M. The concentrate is then passed through a Ni... 2+ Affinity chromatography column was used to elute proteins with a buffer containing 20-50 mM PB, 100-500 mM imidazole, and pH 6.0-6.5, and the eluent was collected. The eluent was then passed into a tangential flow ultrafiltration device and concentrated to 10-20% of its original volume using a 5-10 kDa molecular weight filter membrane. The buffer system was then replaced with a 20-100 mM Tris-HCl buffer containing 100-200 mM NaCl, 400-600 g / L glycerol, and pH 6.0-7.0 to obtain purified recombinant porcine pepsinogen A.

18. A recombinant porcine pepsinogen A prepared by the fermentation method according to any one of claims 1 to 17, characterized in that: The recombinant porcine pepsinogen A has an enzyme activity of over 4000 U / mg.

19. The use of recombinant porcine pepsinogen A prepared by the fermentation method according to any one of claims 1 to 17 or the recombinant porcine pepsinogen A according to claim 18 in the preparation of antitoxin serum.

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