A recombinant engineering bacterium producing human albumin and its construction method and application

Abstract

The present application relates to a recombinant engineering bacterium producing human albumin and its construction method and application, belonging to the field of genetic engineering technology. In the present application scheme, the human albumin gene sequence is codon-optimized to construct a highly efficient recombinant human albumin Pichia yeast strain, further overexpress PDI and SSB2, and the gene sequence of the overexpressed protein in the recombinant plasmid is codon-optimized, and after transformation into the recombinant human albumin Pichia yeast strain, a Pichia yeast genetic engineering strain with high expression of human albumin is successfully obtained, and the strain achieves efficient expression of human albumin through high-density fermentation, with a yield of up to 38.6 g / L, laying the foundation for the industrial application of recombinant human albumin, and having important industrial application prospects and economic value.

Description

Technical Field

[0001] The present application relates to a recombinant engineering bacterium producing human albumin and a construction method and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Human albumin is a normal component of the human body. Its main physiological function is to maintain blood osmotic pressure and transport function. Human albumin injection is suitable for low protein caused by the following diseases: shock and cerebral edema caused by blood loss, trauma and burns, increased intracranial pressure caused by injury, edema or ascites caused by cirrhosis and kidney disease, prevention and treatment of hypoproteinemia, neonatal hyperbilirubinemia, and can also be used for cardiopulmonary bypass, adjuvant treatment of burns, adjuvant treatment of hemodialysis, adult respiratory distress syndrome, etc.

[0003] Currently, albumin-based pharmaceutical preparations are mainly derived from direct extraction of blood. However, due to the instability of blood sources and imperfect detection technology, it is very easy for users to be infected with HIV or other diseases that can be transmitted through blood. Moreover, extracting human albumin from blood for medical use cannot meet the huge market demand at all. Therefore, a new method of producing albumin safely and in large quantities is urgently needed.

[0004] Although the yeast expression system has the characteristics of fast growth and simple operation, when the exogenous protein is expressed at a high level, it is easy to cause protein misfolding and accumulation and even precipitation. Protein disulfide isomerase (PDI) is a multifunctional protein mainly present in the endoplasmic reticulum, which can oxidize, reduce and enzymatically catalyze the formation of disulfide bonds in nascent peptide chains, helping the nascent peptide chains to fold into the correct structure, thereby helping to reduce incorrect shearing in the protein secretion pathway, reduce protein degradation, and improve the expression quality of exogenous proteins.

[0005] Heat shock protein 70 (Hsp70) is a built-in component of the folding and signal transduction pathway. It has a housekeeping function in cells, which can help newly synthesized proteins fold and assemble, refold misfolded and aggregated proteins, regulate the membrane translocation of organelles and secretory proteins, and regulate protein activity. As a member of the Hsp70 family, SSB2 protein is involved in many processes, including translation termination, ribosome biogenesis, and metabolic regulation. SSB2 interacts with ribosomes and nascent polypeptides, mainly playing the role of a molecular chaperone, which can bind to misfolded proteins and assist them in folding correctly.

[0006] There is no report on overexpressing PDI and SSB2 in Pichia pastoris GS115 to increase the expression of human albumin. Therefore, it is necessary to provide an engineered bacterium that efficiently expresses human albumin. Summary of the invention

[0007] In order to solve the above problems, a recombinant engineered bacterium producing human albumin and a construction method and application thereof are provided. In the present application scheme, the human albumin gene sequence is codon-optimized to construct a highly efficient recombinant human albumin Pichia pastoris strain, further overexpressing PDI and SSB2, and codon optimization is performed on the gene sequence of the overexpressed protein in the recombinant plasmid. After transformation into the recombinant human albumin Pichia pastoris strain, a Pichia pastoris genetically engineered strain highly expressing human albumin is successfully obtained, and the strain achieves efficient expression of human albumin after high-density fermentation, with a yield of up to 38.6 g / L.

[0008] According to one aspect of the present application, a method for constructing a recombinant engineered bacterium producing human albumin is provided, the method comprising the following steps:

[0009] S1. Construction of albumin initial expression strain based on Pichia pastoris;

[0010] S2, constructing an expression vector for overexpressing PDI and SSB2;

[0011] S3, transforming the expression vector constructed in step S2 into the albumin initial expression strain constructed in step S1, and performing positive screening on the transformed strain to obtain the recombinant engineered bacteria producing human albumin;

[0012] The nucleotide sequence of PDI in the expression vector is shown as SEQ ID NO: 16 or SEQ ID NO: 18, and the nucleotide sequence of SSB2 in the expression vector is shown as SEQ ID NO: 30.

[0013] Optionally, the Pichia pastoris is X33, GS115, KM71 or SuperMan5-11.

[0014] Optionally, the Pichia pastoris is GS115.

[0015] Optionally, the albumin initial expression strain constructed in step S1 comprises the human albumin nucleotide sequence as shown in SEQ ID NO:9.

[0016] Optionally, in step S2, an expression vector for overexpressing PDI and an expression vector for overexpressing SSB2 are respectively constructed, and in step S3, the two vectors are respectively transformed into the albumin initial expression strain constructed in step S1. It should be noted that in the present application, PDI and SSB2 proteins are overexpressed by respectively transforming two vectors. Those skilled in the art can also use the same vector to overexpress PDI and SSB2 at the same time. In addition, in the present application, a plasmid vector is used as a vector for overexpressing PDI and SSB2. In addition, those skilled in the art can also select other available vectors, such as viral vectors, as long as the overexpression of PDI and SSB2 can be achieved.

[0017] Optionally, the expression vector for overexpressing PDI is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18 to a pPICZA vector; the expression vector for overexpressing SSB2 is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 30 to a pPICBSD vector.

[0018] Optionally, the expression vector for overexpressing PDI is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 16 to a pPICZA vector.

[0019] Optionally, the preparation method comprises the following steps:

[0020] 1) Optimize the coding sequence of human albumin and construct a recombinant expression vector of the optimized human albumin gene suitable for expression in Pichia pastoris;

[0021] 2) Construct human albumin expression strains and screen high-expression strains;

[0022] 3) Optimize the coding sequence of protein disulfide isomerase PDI and construct a recombinant expression vector of the PDI optimized gene suitable for expression in Pichia pastoris;

[0023] 4) The PDI recombinant expression vector is transformed into the human albumin high-expressing strain screened in step 2), and the transformed strain is subjected to positive screening.

[0024] 5) Optimize the coding sequence of SSB2 and construct a recombinant expression vector of the optimized SSB2 gene suitable for expression in Pichia pastoris;

[0025] 6) The SSB2 recombinant expression vector is transformed into the high-expression strain of PDI co-expressing human albumin screened in step 4), and the transformed strain is subjected to positive screening.

[0026] 7) Inducing the recombinant Pichia pastoris genetically engineered strain selected in step 6) to express human albumin.

[0027] According to one aspect of the present application, a recombinant bacterium that highly expresses human albumin is provided, wherein the recombinant bacterium is constructed by the above-mentioned method for constructing a recombinant engineered bacterium that produces human albumin.

[0028] According to one aspect of the present application, there is provided the use of the above-mentioned recombinant bacteria that highly express human albumin in the fermentation preparation of recombinant human albumin.

[0029] The beneficial effects of this application include but are not limited to:

[0030] 1. According to the recombinant engineered bacteria for producing human albumin of the present application, Pichia pastoris GS115 is used as the expression host, and by co-expressing the codon-optimized recombinant human albumin, the codon-optimized protein disulfide isomerase PDI and the codon-optimized SSB2, the degradation of albumin can be reduced, the correct folding of albumin can be promoted, and the expression amount of albumin produced by Pichia pastoris fermentation can be significantly increased, thereby achieving the efficient expression of recombinant human albumin.

[0031] 2. The recombinant engineered bacteria producing human albumin according to the present application achieves efficient expression of recombinant human albumin, with a maximum yield of 38.6 g / L, laying a foundation for the industrial application of recombinant human albumin and having important industrial application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0033] Figure 1 This is the plasmid map of the pPIC9K vector;

[0034] Figure 2 This is the plasmid map of the pPICZA vector;

[0035] Figure 3 This is the plasmid map of the pPICBSD vector;

[0036] Figure 4 The SDS-PAGE result diagram of human albumin expression induced by shake flasks of recombinant engineered bacteria in Example 2 (1 is GS115-HAS-1, 2 is GS115-HAS-2, 3 is GS115-HAS-3, 4 is GS115-HAS-4, 5 is GS115-HAS-5, 6 is GS115-HAS-6, 7 is GS115-HAS-7, 8 is GS115-HAS-8, 9 is GS115-HAS-9, 10 is GS115-HAS-10, 11 is human albumin sample, and 12 is protein Marker);

[0037] Figure 5 This is a graph showing the results of inducing recombinant human albumin expression in a 50 L bioreactor in Example 7. DETAILED DESCRIPTION

[0038] The present application is described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are purchased through commercial channels.

[0039] The molecular biology experimental methods not specifically described in the following examples are all carried out according to the kits and product instructions, or with reference to the methods described in the book "Molecular Cloning Laboratory Manual" (3rd edition) by J. Sambrook. The experimental materials and reagents involved in the present invention are as follows.

[0040] Experimental materials: Escherichia coli DH5α and Pichia pastoris GS115 were purchased from Fenghui Biotechnology; expression vectors pPIC9K and pPICZA were purchased from Thermo Fisher Scientific; restriction endonucleases EcoRI, NotI, SalI and PmeI were purchased from NEB; plasmid extraction kits were purchased from Tiangen Biotechnology Co., Ltd.; Zeocin and blasticidin screening reagents were purchased from Thermo Fisher Scientific, and colony PCR kits were purchased from Ruiyuan Biotechnology.

[0041] Culture medium: Escherichia coli culture medium LB (1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0); yeast culture medium YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose); yeast screening medium YPDZ (YPD + different concentrations of zeocin); yeast screening medium YPDB (YPD + different concentrations of blasticidin); yeast screening medium MD (1.34% (w / v) YNB, 0.00004% (w / v) B iotin, 2% (w / v) glucose); yeast medium BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (V / V)), yeast induction medium BMMY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 0.4% methanol (V / V)). Note: YNB is yeast nitrogen base (Yeast Nitrogen Base) and Biotin is biotin.

[0042] Example 1 Optimization of human albumin gene

[0043] The amino acid sequence of human albumin is shown in SEQ ID NO: 1, the original gene sequence is shown in SEQ ID NO: 2, and the coding sequence of human albumin is optimized. Through optimization design, 9 optimized sequences of human albumin HSA-2 to HSA-10 are obtained, and their gene sequences are shown in SEQ ID NO: 3 to SEQ ID NO: 11. The specific information is shown in Table 1.

[0044] Table 1

[0045]

[0046] The above sequences SEQ ID NO: 2 to SEQ ID NO: 11 were directly synthesized by a biological company and then ligated into the plasmid pPIC9K (eg, Figure 1 The expression vectors were formed and named pPIC9K-HSA-1 to pPIC9K-HSA-10, respectively.

[0047] The expression vector was linearized with DNA restriction enzyme SalI, and Pichia pastoris GS115 was transformed by electroporation. The cells were spread on MD plates and cultured at 30°C until colonies appeared. Positive transformants were identified by colony PCR. Positive transformants were stored in glycerol tubes and named GS115-HSA-1 to GS115-HSA-10. The specific information is shown in Table 2.

[0048] Table 2

[0049]

[0050] Example 2 Shake flask induced expression of recombinant human albumin strain

[0051] The positive transformants GS115-HSA-1 to GS115-HSA-10 obtained in Example 1 were inoculated into 100 ml of BMGY medium and cultured overnight to adjust the starting OD 600 The culture medium was changed to BMMY, and 0.4% methanol was added every 24 hours for induction, and the induction time was 72 hours. The fermentation liquid was collected after centrifugation, and the supernatant was used for SDS-PAGE electrophoresis detection of the target protein. The results are shown in Figure 4 .

[0052] The results showed that the 9 optimized genes HSA-2 to HSA-10 obtained after human albumin gene modification by various strategies were successfully recombinantly expressed in Pichia pastoris. Among them, HSA-8 (the corresponding HSA nucleotide sequence is shown in SEQ ID NO: 9) had the highest secretory expression level in Pichia pastoris.

[0053] This is because although the final encoded amino acid sequence is the same, after gene sequence optimization, due to consideration of factors such as codon usage preference of the expression host, DNA repeat sequence, mRNA secondary structure, GC content and SD sequence in the gene sequence, the expression level of human albumin is different when different amino acid sequences are transferred. In this application scheme, the HSA-8 gene is transferred into Pichia pastoris to obtain a recombinant human albumin strain. When the recombinant human albumin strain (GS115-HSA-8) is expressed, the secretory expression level of human albumin is significantly increased compared with other strains.

[0054] Although the GS115 yeast expression system grows fast and is easy to operate, when human albumin is expressed at high levels, it is easy to cause protein misfolding and accumulation and even precipitation. To address the above problems, the experimenters overexpressed protein disulfide isomerase (PDI). PDI is a multifunctional protein mainly found in the endoplasmic reticulum. It can oxidize, reduce and enzymatically catalyze the formation of disulfide bonds in nascent peptide chains, helping the nascent peptide chains fold into the correct structure, thereby helping to reduce incorrect shearing in the protein secretion pathway, reducing protein degradation, and increasing the expression of human albumin.

[0055] Example 3 Optimization of PDI gene

[0056] The amino acid sequence of protein disulfide isomerase (PDI) is shown in SEQ ID NO: 12, and the original gene sequence is shown in SEQ ID NO: 13. The coding sequence of PDI is optimized, and through optimization design, 9 optimized sequences of PDI, PDI-2 to PDI-10, are obtained, and their gene sequences are shown in SEQ ID NO: 14 to SEQ ID NO: 22. The specific information is shown in Table 3.

[0057] Table 3

[0058]

[0059] The above sequences SEQ ID NO: 13 to SEQ ID NO: 22 were directly synthesized by a biological company and then ligated into the plasmid pPICZA (eg, Figure 2 To form expression vectors, named pPICZA-PDI-1 to pPICZA-PDI-10.

[0060] The expression vector was linearized using the DNA restriction endonuclease PmeI, and electroporated into the GS115-HSA-8 strain. The cells were plated on the selection plate YPDZ containing zeocin antibiotics and cultured at 30°C until colonies appeared. Positive transformants were identified by colony PCR. Positive transformants were stored in glycerol tubes and named GS115-HSA-8-PDI-1 to GS115-HSA-8-PDI-10. The specific information is shown in Table 4.

[0061] Table 4

[0062]

[0063] Example 4 PDI co-expression recombinant human albumin strain shake flask induced expression

[0064] The positive transformants GS115-HSA-8-PDI-1 to GS115-HSA-8-PDI-10 obtained in Example 3 were inoculated into 100 ml of BMGY medium. The high-expression recombinant human albumin strain (GS115-HSA-8) obtained in Example 2 was used as a control strain and was also inoculated into 100 ml of BMGY medium. After overnight culture, the starting OD 600 6, the culture medium was replaced with BMMY, and 0.4% methanol was added every 24 hours for induction, and the induction was performed for a total of 72 hours. The fermentation broth was collected after centrifugation, and the supernatant was taken for SDS-PAGE electrophoresis detection of the target protein. The specific results are shown in Table 5.

[0065] Table 5

[0066]

[0067] After PDI was transformed by various strategies, the nine optimized genes PDI-2 to PDI-10 were successfully recombinantly expressed in yeast strains expressing human albumin. Among them, PDI-4 (nucleotide sequence as shown in SEQ ID NO: 16) and PDI-6 (nucleotide sequence as shown in SEQ ID: 18) can significantly increase the expression of human albumin.

[0068] Among them, the PDI-4 gene was transferred into the recombinant engineered bacteria GS115-HSA-8 to obtain the PDI co-expressing recombinant human albumin strain. When the PDI co-expressing recombinant human albumin strain (GS115-HSA-8-PDI-4) was expressed, the secretion expression level of human albumin was the highest, which was significantly increased by about 1.5 times compared with GS115-HSA-8. Therefore, GS115-HSA-8-PDI-4 was selected for further transformation.

[0069] Example 5 Optimization of SSB2 gene

[0070] The amino acid sequence of SSB2 is shown in SEQ ID NO: 23, the original gene sequence is shown in SEQ ID NO: 24, the coding sequence of SSB2 is optimized, and through optimization design, 9 optimized sequences of SSB2 from SSB2-2 to SSB2-10 are obtained, and their gene sequences are shown in SEQ ID NO: 25 to SEQ ID NO: 33. The specific information is shown in Table 6.

[0071] Table 6

[0072]

[0073] Using gene homologous recombination technology, the biological company was commissioned to replace the zeocin resistance gene in pPICZA with the blasticidin resistance gene (nucleotide sequence as shown in SEQ ID NO: 34) to form an expression vector named pPICBSD (as shown in Figure 3 as shown).

[0074] The above sequences SEQ ID NO: 24 to SEQ ID NO: 33 were commissioned to a biological company for direct synthesis, and then ligated into the plasmid pPICBSD digested with EcoRI and NotI to form expression vectors, which were named pPICBSD-SSB2-1 to pPICBSD-SSB2-10.

[0075] The expression vector was linearized using the DNA restriction endonuclease PmeI, and electroporated into the GS115-HSA-8-PDI-4 strain. The cells were plated on the selection plate YPDB containing the blasticidin antibiotic and cultured at 30°C until colonies appeared. The positive transformants were identified by colony PCR. The positive transformants were stored in glycerol tubes and named GS115-HSA-8-PDI-4-SSB2-1 to GS115-HSA-8-PDI-4-SSB2-10. The specific information is shown in Table 7.

[0076] Table 7

[0077]

[0078] Example 6 SSB2 co-expression of recombinant human albumin strain induced expression in shake flasks

[0079] The positive transformants GS115-HSA-8-PDI-4-SSB2-1 to GS115-HSA-8-PDI-4-SSB2-10 obtained in Example 5 were inoculated into 100 ml of BMGY medium. The PDI co-expressing recombinant human albumin strain (GS115-HSA-8-PDI-4) obtained in Example 4 was used as a control strain and was also inoculated into 100 ml of BMGY medium. After overnight culture, the starting OD 600 6, the culture medium was replaced with BMMY, and 0.4% methanol was added every 24 hours for induction, and the induction was performed for a total of 72 hours. The fermentation broth was collected after centrifugation, and the supernatant was taken for SDS-PAGE electrophoresis detection of the target protein. The specific results are shown in Table 8.

[0080] Table 8

[0081]

[0082] After SSB2 was transformed by various strategies, the 9 optimized genes SSB2-2 to SSB2-10 were successfully expressed in the PDI co-expression recombinant human albumin strain. Among them, SSB2-7 (nucleotide sequence as shown in SEQ ID NO: 30) can significantly increase the expression of human albumin. This is because SSB2, as a member of the Hsp70 family, is involved in translation termination, ribosome biogenesis and metabolic regulation. SSB2 interacts with ribosomes and nascent polypeptides, mainly playing the role of a molecular chaperone, can bind to misfolded proteins and assist them in folding correctly, thereby increasing the expression of human albumin.

[0083] In addition, just as the optimization of the human albumin nucleotide sequence affects the expression level of human albumin, the optimization of the SSB2 nucleotide sequence here will also affect the expression level of SSB2 in GS115 Pichia pastoris. Among them, the SSB2-7 gene was transferred into the recombinant engineered bacteria GS115-HSA-8-PDI-4 to obtain the SSB2 co-expression recombinant human albumin strain. When the SSB2 co-expression recombinant human albumin strain (GS115-HSA-8-PDI-4-SSB2-7) was expressed, the secretory expression level of human albumin was significantly increased by about 1.8 times.

[0084] The experimenters continued to scale up the culture of the obtained GS115-HSA-8-PDI-4-SSB2-7 in a 50L fermenter.

[0085] Example 7 Induction of expression of codon-optimized recombinant human albumin gene engineering strain in a 50 L fermenter

[0086] The experimenters selected the transformant with the highest expression level of human albumin in Example 6 (GS115-HSA-8-PDI-4-SSB2-7) to study its expression level in the fermentation device. Specifically, in addition to fermenting GS115-HSA-8-PDI-4-SSB2-7, which has the highest expression level in the shake flask culture medium, in a 50L fermenter, GS115-HSA-8-PDI-4, GS115-HSA-8 and GS115-HSA-1 were also used as controls to test their improvement effects. The 50L fermenter culture operation is as follows: Inoculate the corresponding strain into YPD liquid culture medium at a ratio of 1%, culture at 30°C and 220rpm, and wait until the shake flask seed OD 600 When the fermentation temperature reached 10, it was inoculated into 20L fermentation medium and fermented at 28°C and 500 rpm. It was induced with methanol for 120 h and then put into the tank. The fermentation product was centrifuged at 8000rpm for 20 minutes, the supernatant was collected, and the protein level was detected by HPLC. The specific results are as follows: Figure 5 shown.

[0087] It was calculated from the liquid phase detection results that the expression level of the strain with the original gene sequence (GS115-HSA-1) after 50L fermentation was 8.7g / L, and the expression level of the strain with the codon-optimized recombinant human albumin gene sequence (GS115-HSA-8) after 50L fermentation was 13.4g / L, which was significantly increased to 1.5 times that before optimization; the expression level of the codon-optimized strain co-expressing the recombinant human albumin gene sequence (GS115-HSA-8-PDI-4) after 50L fermentation was 21.7g / L, which was significantly increased to 2.5 times that before optimization; the expression level of the codon-optimized SSB2 co-expressing recombinant human albumin gene sequence (GS115-HSA-8-PDI-4-SSB2-7) after 50L fermentation was 38.6g / L, which was significantly increased to 4.4 times that before optimization.

[0088] It can be seen that after a series of codon optimization, the present invention achieves the purpose of further improving the expression level of recombinant human albumin, provides a practical and effective strategy for the industrial production of recombinant human albumin, and has important economic value and industrial application scenarios.

[0089] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a recombinant engineering bacterium producing human albumin, characterized in that: The construction method comprises the following steps: S1. Construction of albumin initial expression strain based on Pichia pastoris; S2, constructing an expression vector for overexpressing PDI and SSB2; S3, transforming the expression vector constructed in step S2 into the albumin initial expression strain constructed in step S1, and performing positive screening on the transformed strain to obtain the recombinant engineered bacteria producing human albumin; The nucleotide sequence of PDI in the expression vector is shown as SEQ ID NO: 16 or SEQ ID NO: 18, and the nucleotide sequence of SSB2 in the expression vector is shown as SEQ ID NO:

30.

2. The method for constructing a recombinant engineered bacterium producing human albumin according to claim 1, characterized in that: The Pichia pastoris is GS115.

3. The method for constructing a recombinant engineered bacterium producing human albumin according to claim 2, characterized in that: The albumin initial expression strain constructed in step S1 comprises the human albumin nucleotide sequence shown in SEQ ID NO:

9.

4. The method for constructing a recombinant engineered bacterium producing human albumin according to claim 1, characterized in that: The expression vector for overexpressing PDI is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18 to the pPICZA vector; the expression vector for overexpressing SSB2 is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 30 to the pPICBSD vector.

5. The method for constructing a recombinant engineered bacterium producing human albumin according to claim 4, characterized in that: The expression vector for overexpressing PDI is obtained by connecting the nucleotide sequence shown in SEQ ID NO: 16 to the pPICZA vector.

6. A recombinant bacterium that highly expresses human albumin, characterized in that: The recombinant bacteria are constructed by the method for constructing a recombinant engineering bacteria producing human albumin as described in any one of claims 1 to 5.

7. Use of the recombinant bacteria highly expressing human albumin as claimed in claim 6 in the fermentation preparation of recombinant human albumin.

Images