Pichia pastoris engineering bacterium as well as method for preparing human serum albumin and application of pichia pastoris engineering bacterium

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to pichia pastoris engineering bacteria, a method for preparing human serum albumin by using the pichia pastoris engineering bacteria and application of the pichia pastoris engineering bacteria. The invention provides a gene for expressing human serum albumin. The nucleotide sequence of the gene is shown as SEQ ID NO: 1. The recombinant pichia pastoris engineering bacteria are obtained by inserting the nucleotide sequence into a carrier, constructing recombinant plasmids and carrying out electric shock fusion on the recombinant plasmids and pichia pastoris host bacteria. The invention also provides a recombinant human serum albumin obtained by recombinant expression of pichia pastoris and a method for producing the human serum albumin by fermentation of the recombinant human serum albumin. The recombinant human serum albumin provided by the invention is simple in production process and low in price, the protein expression quantity is as high as 6.567 g / L, and the recovery purity reaches 90% or above.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an engineered Pichia pastoris and a method and application thereof for preparing human serum albumin. Background Art

[0002] Human serum albumin (HSA) is a vital component of the blood system, accounting for 40% to 60% of total plasma protein. It plays a crucial physiological role in the animal circulatory system. HSA acts as a buffer to maintain blood pH stability and also has a coagulation effect by inhibiting platelet aggregation. HSA is widely used clinically, primarily for the treatment of hypoproteinemia, burns, hemorrhagic shock, acute liver injury, tumors, and cancer. Currently, HSA is isolated and purified from human plasma, but this method limits plasma sources and carries significant costs and safety risks. Furthermore, with the development of national immunization programs and the strengthening of public awareness of health prevention, the demand for albumin has been increasing annually. Recombinant human serum albumin (rHSA) effectively reduces the risk of viral contamination, has an unlimited source, and shares molecular structure, immunological, and physicochemical properties with human serum albumin. Therefore, rHSA holds great promise for research and market development.

[0003] rHSA refers to human serum albumin produced by genetic engineering. This technology requires the identification of an ideal exogenous gene expression system. Studies have found that bacteria are not suitable as chassis cells. HSA is expressed in high levels in E. coli, but the product contains a large number of disulfide bonds, which prevent the protein from effectively folding and thus has difficulty in possessing biological activity. In Bacillus subtilis, HSA expression levels are low and the signal peptide removal rate is low. Therefore, the above two expression systems are not suitable for the industrial production of HSA. Currently, HSA is a relatively ideal system in yeast expression systems, but related expression and fermentation parameters still need to be optimized. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a gene expressing human serum albumin, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0005] The present invention also provides an expression vector containing the nucleotide sequence.

[0006] The present invention also provides a recombinant Pichia pastoris engineered bacterium, which is obtained by inserting the nucleotide sequence into a vector to construct a recombinant plasmid, and then fusing the recombinant plasmid with the Pichia pastoris host bacterium by electroporation.

[0007] Furthermore, the vector is a pPIC9K vector.

[0008] Furthermore, the deposit number of the Pichia pastoris strain is CGMCC NO.33874.

[0009] The present invention also provides a method for preparing human serum albumin using recombinant Pichia pastoris engineered bacteria. The recombinant Pichia pastoris engineered bacteria seed liquid is introduced into a fermentation tank for fed-batch fermentation. During the fermentation process, methanol is used to induce fermentation to obtain fermentation broth, which is then purified to obtain human serum albumin.

[0010] Furthermore, the induction conditions are as follows: the induction medium is BMMY medium, the induction temperature is 28° C., the amount of methanol added is 0.5%, and the induction time is 120 h.

[0011] Furthermore, the fermentation broth is purified as follows:

[0012] (1) centrifuging the fermentation supernatant, discarding the precipitate, heating the supernatant in a metal bath to inactivate the protease, and centrifuging again to obtain the raw material solution;

[0013] (2) The raw material solution was subjected to UniHR Phenyl 30L hydrophobic chromatography to obtain an elution fraction;

[0014] (3) The eluted fractions were subjected to Capto DEAE anion exchange chromatography to further remove impurities;

[0015] (4) Purified human serum albumin is obtained after ultrafiltration and concentration.

[0016] The present invention also provides the use of the human serum albumin gene in preparing human serum albumin.

[0017] The present invention also provides the use of the recombinant Pichia pastoris engineering bacteria in preparing human serum albumin.

[0018] The beneficial effects of the present invention compared to the prior art are:

[0019] The present invention provides a codon-optimized human serum albumin gene and screens suitable Pichia pastoris host bacteria based on the nucleotide sequence for efficient expression of human serum albumin. The recombinant Pichia pastoris engineered bacteria of the present invention can express human serum albumin at a level of up to 6.567 g / L with a recovery purity of over 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the plasmid map of the pPIC9K vector.

[0021] Figure 2The following is the restriction enzyme digestion map of the recombinant plasmid pPIC9K-hsa, where M is protein marker; 1: pPIC9K-hsa recombinant plasmid extracted from 1 mL of bacterial culture, 2: pPIC9K-hsa double-enzyme digestion product extracted from 1 mL of bacterial culture; 3: pPIC9K-hsa recombinant plasmid extracted from 1.5 mL of bacterial culture, 4: pPIC9K-hsa double-enzyme digestion product extracted from 1.5 mL of bacterial culture.

[0022] Figure 3 SDS-PAGE identification of the expression product. M: Marker 10-250 kDa; 1-3 are strains GS115, DBN JM58, and DBN JM111, respectively;

[0023] Figure 4 The expression products were identified by SDS-PAGE. 1-3, 2-6, 7-9 are GS115, DBN JM111 and DBN JM58, respectively, and each was repeated 3 times.

[0024] Figure 5 The expression products were identified by SDS-PAGE after different induction times of DBN JM111 and ammonium sulfate precipitation. M: Marker 10-250 kDa, 1: 24 h, 2: 48 h, 3: 72 h, 4: 96 h, 5: 120 h, 6: after precipitation and concentration.

[0025] Figure 6 Amino acid sequence alignment of human serum albumin induced by DBN JM111 and human serum albumin in NCBI

[0026] Figure 7 This is the analysis of human serum recombinant albumin purified from DBN JM111 fermentation broth, where M: protein marker; 1: stock solution; 2: purified human serum recombinant albumin. DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0028] Example 1 Method for eukaryotic expression of recombinant human serum albumin

[0029] A method for eukaryotic expression of recombinant human serum albumin, wherein the target protein is expressed in a Pichia pastoris expression system to obtain a secretory recombinant protein. The specific steps are as follows:

[0030] 1.1 Construction of recombinant vector pPICK-rHSA

[0031] According to the NCBI website, the nucleotide sequence of the human serum albumin gene is as follows:

[0032]

[0033] The amino acid sequence of human serum albumin is shown below:

[0034] (SEQ ID NO: 3).

[0035] With reference to the codon preference of the Pichia pastoris expression system, the extracted human serum albumin sequence was optimized and the synthesized sequence was inserted into the pPIC9K vector (e.g. Figure 1 ), a recombinant plasmid was constructed and transformed into Escherichia coli cells. The optimized nucleotide sequence of human serum albumin is shown below, and the optimized amino acid sequence of human serum albumin is consistent with SEQ ID NO: 3.

[0036]

[0037] The transformed cells were plated on a solid plate containing 50 μg / mL ampicillin resistance LB and cultured at 37°C overnight. Single clones were randomly selected and cultured in liquid medium containing ampicillin resistance LB at 37°C overnight. After plasmid extraction, positive clones were obtained by enzyme digestion identification (e.g. Figure 2 The positive plasmid was named pPIC9K-hsa. Sequencing of the recombinant plasmid revealed the correct insert sequence, absence of other mutations, and insertion orientation. Compared to the codon-optimized human serum albumin nucleotide sequence provided by the present invention, it exhibits improved compatibility with Pichia pastoris and is more suitable for subsequent experiments.

[0038] The length of the recombinant plasmid in this experiment is 11085 bp, which exceeds the applicable length of the plasmid purification kit (3000-10000 bp). Therefore, the alcohol precipitation method is used to extract the plasmid. The steps are as follows.

[0039] 1.1.1 Add 1 / 10 volume of sodium acetate (23 mol / L, pH 5.2) to 50 μL of plasmid and mix thoroughly to a final concentration of 0.3 mol / L.

[0040] 1.1.2 Add twice the volume of ice-cold anhydrous ethanol, mix thoroughly and place at -20℃ for more than 30 minutes.

[0041] 1.1.3 Centrifuge at 12000 rpm for 10 min. Carefully remove the supernatant and aspirate all droplets on the tube wall.

[0042] 1.1.4 Add 1 / 2 volume of 70% ethanol and centrifuge at 12000 rpm for 2 min. Carefully remove the supernatant and aspirate all droplets on the tube wall.

[0043] 1.1.5 Open the lid and place it in a vent in a clean bench until all the ethanol in the tube evaporates and becomes completely dry.

[0044] 1.1.6 Add ddH2O and mix thoroughly to dissolve the plasmid according to experimental requirements.

[0045] After purification, plasmid sequencing primers were used to perform PCR of the target sequence. The primer sequences are as follows:

[0046] 5′ sequencing primer: 5′AOX (5′-GACTGGTTCCAATTGACAAGC-3′);

[0047] 3' sequencing primer: 3'AOX (5'-GGCAAATGGCATTCTGACAT-3').

[0048] After PCR completion, agarose electrophoresis was performed for verification.

[0049] 1.2 Shake flask culture of human serum recombinant albumin

[0050] 1.2.1 Extraction and verification of pPIC9K-hsa recombinant plasmid

[0051] The positive plasmid pPIC9K-hsa, confirmed by PCR, was linearized using the endonuclease Sal I. The digestion system was as follows: 5 μL of 10× Qcut buffer, 1 μL of Qcut Sal I, 40 μL of extracted plasmid, and 4 μL of ddH2O, for a total of 50 μL. The digestion was performed at 30°C for 5 minutes.

[0052] 1.2.2 Preparation of electroporated yeast cells

[0053] Single colonies of Pichia pastoris GS115, DBN JM58 and DBN JM111 were picked and added to 5 mL of YPD medium, cultured at 250 rpm and 30°C overnight.

[0054] Among them, Pichia pastoris GS115 was purchased from Beijing Biobo Biotechnology Co., Ltd. in the form of a 2 mL glycerol tube with the platform number bio-82058.

[0055] The Pichia pastoris DBN JM58 was screened and obtained from the environment in a laboratory.

[0056] The Pichia yeast DBN JM111 strain was deposited at the General Microbiology Center of the China Culture Collection Administration on March 19, 2025, named BC-HSA, classified as: Pichia sp., with a collection number of CGMCC No. 33874, and the collection address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0057] Inoculate 1%-2% of the overnight culture of Pichia pastoris GS115, DBN JM58, and DBN JM111 into 50 mL (the specific amount can be adjusted as needed) YPD medium, and shake culture at 30°C and 250 rpm until the OD 600 It is 1.3-1.5.

[0058] Transfer the bacterial solution into a centrifuge tube, centrifuge at 4000 rpm, 4°C for 5 minutes, and discard the supernatant.

[0059] Resuspend the cells in 50 mL of ice-cold sterile water, centrifuge and discard the supernatant as above, and repeat twice.

[0060] Resuspend the cells in 4 mL of ice-cold 1 M sorbitol, transfer to a 5 mL centrifuge tube, and centrifuge as above.

[0061] When discarding the sorbitol supernatant for the last time during competent cell preparation, use a capillary pipette tip to aspirate as much of the supernatant as possible to ensure the purity of the competent cells.

[0062] Add 200 μL of 1 M sorbitol to resuspend the cells and store at 4°C until use.

[0063] 1.2.3 Construction of recombinant bacteria by electroporation

[0064] 7 μg of the linearized recombinant plasmid pPIC9K-hsa was electroporated into 80 μL of Pichia pastoris GS115, DBN JM58, and DBN JM111 competent cells. The steps are as follows:

[0065] 1) Mix 7 μg of recombinant plasmid pPIC9K-hsa with 80 μL of Pichia pastoris GS115, DBN JM58, and DBN JM111 competent cells respectively.

[0066] 2) Transfer the mixed liquid to an ice-cold electric rotating cup and place it on ice for 5 minutes.

[0067] 3) Perform electroporation with the parameters set to 25 μF, 200 Ω, and 1.0 kV.

[0068] 4) Immediately after electroporation, add 1 mL of ice-cold 1 M sorbitol, mix well, and transfer to a 2 mL centrifuge tube.

[0069] 5) Temperature 30℃, slow recovery for 2 hours.

[0070] 6) Take 200 μL of the transformation solution and plate the MM methanol nutrient plate: Take 90 mL of water and add 2 g of agar powder (20 g / L) and sterilize at 121°C for 20 min. After the temperature drops to 60°C, add 10 mL of 10× YNB (13.4 g / L), 0.2 mL of 500× biotin (4×10 -4 g / L). Multiple plates can be plated simultaneously to obtain as many positive clones as possible. Incubate at 30°C for 2-4 days until transformants emerge. For all positive strains, perform positive screening again using YPD medium containing a gradient of G418 and screen for polyclonal recombinant strains. The G418 concentrations are set at 0.25, 1, 2, 3, 4, and 5 mg / mL. After polyclonal screening, identify all recombinant strains and induce culture.

[0071] Plasmids were extracted from all positive clones according to the TaKaRa MiniBEST Plasmid Purification Kit protocol. PCR was performed on the extracted plasmids using the same primers used for recombinant plasmid verification. The primers were:

[0072] 5′ sequencing primer: 5′AOX (5′-GACTGGTTCCAATTGACAAGC-3′);

[0073] 3' sequencing primer: 3'AOX (5'-GGCAAATGGCATTCTGACAT-3').

[0074] After completion, perform electrophoresis to verify whether a single band of about 2000 bp appears (e.g. Figure 3 ), where 1, 2, and 3 correspond to GS115, DBN JM58, and DBN JM111, respectively, and all present a single band around 2000 bp. Positive strains showing the presence of the albumin gene were frozen at -80°C using glycerol at a final concentration of 25%.

[0075] 1.3 Induction culture conditions of recombinant bacteria

[0076] 1.3.1 Seed Medium BMGY: Prepare 1 L of culture medium with 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, and 11.8 g / L KH2PO4. Add water to 890 mL and sterilize at 121°C for 20 min. Then cool to 60°C and add 100 mL of 10× YNB (13.4 g / L), 1 mL of 500× biotin (4×10-4 g / L), and 10 mL of glycerol in a laminar flow hood.

[0077] 1.3.2 Induction Medium BMMY: Prepare 1 L of culture medium with 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, and 11.8 g / L KH2PO4. Add water to 890 mL and sterilize at 121°C for 20 min. Then cool to 60°C and add 100 mL of 10× YNB (13.4 g / L), 1 mL of 500× biotin (4 × 10-4 g / L), and 5 mL of methanol in a laminar flow hood.

[0078] 1.3.3 Prepare seed culture by inoculating an appropriate amount of cells into BMGY medium using shake flask culture. Culture conditions: 30 mL / 250 mL, 28°C, and 16 h. After completion, centrifuge the seed culture under aseptic conditions and re-inoculate into BMMY induction medium at a ratio of 1:5 between pre-centrifuged seed culture and post-centrifugation induction medium. Induction conditions: 30 mL / 250 mL, 28°C, 250 rpm. Add 0.5% methanol every 24 h (150 μL of methanol per 30 mL) for a total of 120 h. After induction, remove the culture and perform protein electrophoresis.

[0079] 1.3.4 Protein electrophoresis verification process: After the induction, the bacterial solution was centrifuged at 12000 rpm for 20 minutes. The supernatant was taken for protein electrophoresis verification. After electrophoresis, the protein results were observed according to the Maker to see if there was an albumin (66kD) band (such as Figure 4 shown).

[0080] 1.3.5 Protein Quantification: The protein content in the fermentation broth was quantitatively measured using a NanoPhotometer. The results are shown in Table 1. The results showed that the human serum albumin content of the fermentation broth of the DBN JM111 strain was the highest, at 6.567 g / L.

[0081] Table 1 Human serum albumin content in different Pichia pastoris fermentation broths

[0082]

[0083]

[0084] Example 2 Fermentation culture of human serum recombinant albumin

[0085] The DBN JM111 with the highest expression level in shake flasks was scaled up.

[0086] 2.1 Preparation of seed solution

[0087] Take DBN JM111 glycerol bacteria and streak it on MD plate. Incubate it at 28℃ for 2-3 days until a single colony grows. Pick a single colony and inoculate it into 10 mL YPD medium. Incubate it at 28℃ for 24 hours. Take 5 mL and inoculate it into 100 mL YPD medium and incubate it at 28℃ for 12 hours to prepare the seed solution.

[0088] 2.2 Preparation of fermentation medium

[0089] Dissolve 20 g of tryptone, 10 g of yeast extract, 11.8 g of KH2PO4, and 2.3 g of K2HPO4 in 800 mL of water, place in a clean fermenter, add 300 μL of defoamer, and sterilize at 121°C for 15 min. After sterilization, add 100 mL of 40% glycerol (sterilized three times at 121°C for 15 min at 24-hour intervals), 100 mL of 10× YNB, and 2 mL of 500× biotin to the feed bottle in a laminar flow hood, bringing the volume to 1 L.

[0090] 2.3 Fermentation tank preparation

[0091] The fermentation tank temperature was stabilized at 28°C, the pH was adjusted to 5.75 with ammonia water, the ventilation volume was set to 2 L / min, the speed was 900 rpm, and the system was maintained in stable operation for 1 hour. After the dissolved oxygen electrode was stabilized, the dissolved oxygen electrode was calibrated. After the calibration was completed, the speed was reduced to 200 rpm.

[0092] 2.4 Inoculation of seed solution

[0093] The fermentation temperature of the engineered bacteria is 28°C. During the fermentation period, the engineered bacteria enter the acid production stage. Ammonia water is used to adjust to maintain the optimal pH. After the pH value of the thermometer stabilizes, 100 mL of seed liquid is slowly added to the fermenter through a peristaltic pump to start batch fermentation. Set the dissolved oxygen-speed coupling, the speed is set to 100-900 rpm, the solution is set to 20% (± 1%), and samples are taken at intervals for microscopic examination. Pay close attention to the fermentation tank parameters. When the dissolved oxygen suddenly increases and the speed suddenly drops, the batch addition stage begins.

[0094] 2.5 Fed-batch fermentation

[0095] Add 60 mL of 50% glycerol and 0.72 mL of PTM1 to a feeding bottle. Add the contents to the fermenter at a rate of 15 mL / h over 4 hours, while maintaining all other fermentation parameters. Samples were taken periodically after feeding and the fermenter was inspected. If the fermenter solution suddenly increased and the fermentation speed decreased, the methanol-induced fermentation phase was initiated.

[0096] 2.6 Induced fermentation

[0097] In a clean bench, 500 mL of methanol and 6 mL of PTM1 (12 mL / L) were added to the feeding bottle. Methanol was not added within 1 hour after the induction stage to ensure that the glycerol in the previous stage was completely consumed. After starvation treatment, the dissolved oxygen-speed coupling was exposed and switched to dissolved oxygen-methanol feeding coupling. The coupling was inversely correlated, and the dissolved oxygen critical value was 20%. When the dissolved oxygen was >20%, the feeding was started, and the methanol feeding cycle was 10 seconds. The fermentation temperature was kept constant at 28 ° C, the pH was changed to 5.85, and the speed was set to 900 rpm. The fermentation was run with these parameters until the end. Samples were taken at intervals during the fermentation process (24, 48, 72, 96, and 120 hours) for electrophoresis detection to estimate the expression of human serum albumin ( Figure 5 ), the results showed that the human serum albumin content gradually accumulated with the extension of fermentation time, and after ammonium sulfate precipitation, the initial purification effect was achieved. At this time, the protein content was quantitatively detected using NanoPhotometer, and the result was 6.567g / L. It was further sent to Qingke Bio for sequencing detection (the results are shown in Figure 6 ), and its similarity to the amino acid sequence of human serum albumin (SEQ ID NO: 3) is 88%.

[0098] Example 3 Purification process of human serum recombinant albumin

[0099] We then deeply purified the fermentation broth after DBN JM111 was induced to express as the host bacteria.

[0100] First, centrifuge the fermentation supernatant at 8000 rpm for 10 minutes and discard the precipitate. Heat the supernatant at 68°C in a metal bath for 30 minutes and then quickly cool it to 15°C to inactivate the protease, prevent degradation of the target protein during purification or storage, and effectively reduce the viscosity of the liquid. Centrifuge the supernatant at 12000 rpm for 10 minutes and discard the precipitate. Use this supernatant as the feedstock for subsequent purification.

[0101] UniHR Phenyl 30L hydrophobic chromatography: The supernatant was diluted four-fold with PB buffer, pH 7.0, 1.5 mM (NH₄)₂SO₄, and filtered through a 0.22 μm filter to remove impurities. The sample was then loaded onto a UniHR Phenyl 30L hydrophobic chromatography column. Procedure: Buffer A (50 mM PB buffer, pH 7.0) and Buffer B (50 mM PB buffer, pH 7.0, 1.5 M (NH₄)₂SO₄) were used. The column was first washed with Buffer B for 5-10 column volumes, then equilibrated with Buffer A. The target protein solution, which had been desalted in the previous step, was loaded onto a 250 mL UniHR Phenyl 30L hydrophobic chromatography column. After loading, the sample was washed with Buffer A for another 5 column volumes. Elution was performed using a linear gradient, increasing the concentration of Buffer B from 0% to 100% over 10 column volumes. The eluted fractions were collected.

[0102] Capto DEAE Anion Exchange Chromatography: After capturing and purifying the target protein via hydrophobic chromatography, if significant impurities remain, further purification can be performed using a DEAE anion exchange column. Procedure: Desalt the sample from the previous hydrophobic chromatography step into 20 mM Tris-HCl, pH 7.0. Equilibrate the Capto DEAE column with 20 mM Tris-HCl, pH 7.0. Load the sample at a flow rate of 4 mL / min and elute the target protein in steps using 20 mM Tris-HCl, pH 7.0, 1 M NaCl. Assess the purity of the target protein in the flow-through by SDS-PAGE.

[0103] Ultrafiltration concentration / liquid exchange: Operation steps: Use Amicon Ultra 15 (10kDa) ultrafiltration tube, centrifuge at 4000rpm for 30min at 4℃. Discard the solution in the collection tube and continue to add 5-10mL PBS buffer. Centrifuge at 4000rpm for 30min at 4℃. Discard the solution in the collection tube and repeat 2 times. Determine the concentration of the target protein. The buffer is 1×PBS, pH 7.4, without adding 50% glycerol. Store in a -20℃ refrigerator for a long time. It can be divided into small tubes to avoid repeated freezing and thawing. Electrophoresis diagram before and after purification (such as Figure 7The gel was scanned by Image Quant 800, and the ratio of the optical density of the target protein band (79.60) to the total grayscale of all bands (88.45) was calculated, which was the sample purity (90%).

Claims

1. A gene expressing human serum albumin, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

1.

2. An expression vector containing the gene according to claim 1.

3. A recombinant Pichia pastoris engineered bacterium, characterized in that: The recombinant Pichia pastoris engineered bacteria is obtained by inserting the nucleotide sequence of claim 1 into a vector to construct a recombinant plasmid, and then fusing the recombinant plasmid with the Pichia pastoris host bacteria by electroporation.

4. The recombinant Pichia pastoris engineered bacterium according to claim 3, characterized in that The vector is a pPIC9K vector.

5. The recombinant Pichia pastoris engineered bacterium according to claim 3, characterized in that The deposit number of the Pichia pastoris host bacterial strain is CGMCC NO.33874.

6. The method for preparing human serum albumin by using the recombinant Pichia pastoris engineered bacteria according to claim 3, characterized in that: The recombinant Pichia pastoris engineered bacteria seed liquid is introduced into a fermentation tank for fed-batch fermentation. During the fermentation process, methanol is used to induce fermentation to obtain a fermentation liquid. The fermentation liquid is purified to obtain the product.

7. The method according to claim 6, characterized in that The induction conditions are as follows: the induction medium is BMMY medium, the induction temperature is 28° C., the amount of methanol added is 0.5%, and the induction time is 120 h.

8. The method according to claim 6, characterized in that The purification method of the fermentation broth is as follows: (1) Centrifuge the fermentation supernatant, discard the precipitate, heat the supernatant in a metal bath to inactivate the protease, and centrifuge again to obtain the raw material solution; (2) The raw material solution was subjected to UniHR Phenyl 30L hydrophobic chromatography to obtain an elution fraction; (3) The eluted fractions were subjected to Capto DEAE anion exchange chromatography to further remove impurities; (4) Purified human serum albumin is obtained after ultrafiltration and concentration.

9. Use of the human serum albumin gene according to claim 1 in the preparation of human serum albumin.

10. Use of the recombinant Pichia pastoris engineered bacteria according to claim 3 in the preparation of human serum albumin.

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