Recombinant engineering bacterium for preparing recombinant bovine albumin and application of recombinant engineering bacterium

Abstract

The invention provides a recombinant engineering bacterium for preparing recombinant bovine albumin and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. When the recombinant bovine albumin is expressed, one or more of protein degrading enzyme genes MKC7 and ULP1 are knocked out in a targeted manner, and / or one or more of protein folding related genes PDI-2 and SHR3 are overexpressed and integrated into the strain, so that the expression quantity of the recombinant bovine albumin is increased. According to the recombinant engineering bacterium disclosed by the invention, the yield of the recombinant bovine albumin can be obviously improved, the highest yield can reach 38.79 g / L, and compared with an unmodified recombinant engineering bacterium, the yield is improved by 4.85 times, and the recombinant engineering bacterium has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a recombinant engineering bacterium for preparing recombinant bovine albumin and its application. Background Art

[0002] Bovine serum albumin (BSA) is an abundant plasma protein and is widely used in the fields of biology and biomedical research. In cell culture, BSA is commonly used as one of the important components of cell culture media, providing nutrients and growth factors required by cells. In the pharmaceutical field, BSA can also be used to transport and stabilize bioactive molecules and as a carrier for drug delivery systems. By binding to drugs and promoting their transport and release, it can improve the stability and bioavailability of drugs. Many studies have shown that BSA can be structurally modified or combined with different biomaterials, and it can support cell attachment, tissue formation, and healing. Therefore, it has become a multifunctional drug carrier and scaffold. Related new therapeutic strategies will greatly benefit from more in-depth research. And BSA has a wide source and low price (the market price is only 1 / 10 - 1 / 5 of human serum albumin (HSA)), so it has a more extensive application prospect in the pharmaceutical field.

[0003] Currently, the BSA on the market mainly comes from bovine serum, which has the risk of potential pathogen contamination, and factors such as different cattle herds, feeding conditions, and seasons lead to fluctuations in serum components, affecting the repeatability of experiments and bringing certain inconveniences to the use of BSA. Currently, there is an urgent need for a method for quickly and efficiently producing stable bovine serum albumin.

[0004] The heterologous expression of proteins is of great significance in various biopharmaceutical industries and industrial applications. As one of the most successful eukaryotic protein expression systems, Pichia pastoris has experienced rapid development and received extensive attention in the past 20 years, generating huge economic and social value. Currently, the main methods for highly expressing foreign proteins mainly include foreign gene modification, strain modification, and fermentation level optimization. (Zhu Wen, Hu Youjia, Xie Liping. Related strategies and research progress of highly expressing foreign proteins in Pichia pastoris [J], Chinese Journal of Pharmaceuticals 2018, 49(4): 417 - 425). These optimizations of Pichia pastoris provide a basis for highly expressing bovine albumin. Summary of the Invention

[0005] The present invention aims to improve protein expression by selecting to introduce protein folding genes into the endoplasmic reticulum and simultaneously knocking out protease degradation genes in the target strain. The specific scheme of the present invention is as follows: The first invention of the present invention provides a recombinant engineering bacterium for preparing recombinant bovine albumin. While expressing recombinant bovine albumin, the recombinant engineering bacterium also includes the following operations: targeting and knocking out one or more of the protease degradation genes MKC7 and ULP1, and / or overexpressing and integrating one or more of the protein folding-related genes PDI-2 and SHR3 into the strain, thereby achieving the high-efficiency expression of recombinant bovine albumin.

[0006] Preferably, the operation at least includes any one of the following: (1) Knocking out the MKC7 gene; (2) Knocking out the MKC7 and ULP1 genes; (3) Knocking out the MKC7 and ULP1 genes, and overexpressing the PDI-2 gene; (4) Knocking out the MKC7 and ULP1 genes, and overexpressing the SHR3 gene; (5) Knocking out the MKC7 and ULP1 genes, and overexpressing the PDI-2 and SHR3 genes Further, the amino acid sequence encoded by the MKC7 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the MKC7 gene is as shown in SEQ ID NO.2; the amino acid sequence encoded by the ULP1 gene is as shown in SEQ ID NO.3, and the nucleotide sequence of the ULP1 gene is as shown in SEQ ID NO.4.

[0007] The amino acid sequence encoded by the PDI-2 gene is as shown in SEQ ID NO.5, and the nucleotide sequence of the PDI-2 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.5, including the sequences publicly available in the current database and the nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is as shown in SEQ ID NO.6 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.6.

[0008] The amino acid sequence encoded by the SHR3 gene is as shown in SEQ ID NO.7, and the nucleotide sequence of the SHR3 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.7, including the sequences publicly available in the current database and the nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is as shown in SEQ ID NO.8 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.8.

[0009] Further, the amino acid sequence of the bovine albumin is as shown in SEQ ID NO.9. After codon optimization, the bovine albumin is synthesized and cloned onto an expression vector to construct a recombinant plasmid.

[0010] Furthermore, the nucleotide sequence of the recombinant bovine albumin is as shown in SEQ ID NO.10 or a nucleotide sequence having at least 95% identity with SEQ ID NO.10.

[0011] Furthermore, the foreign gene expressed by the recombinant engineering bacteria can also be selected from one of other recombinant albumins. Preferably, the recombinant albumin includes one of recombinant human albumin, recombinant cat albumin, recombinant dog albumin, recombinant horse albumin, recombinant sheep albumin, and recombinant pig albumin.

[0012] Furthermore, the recombinant engineering bacteria are selected from one or more of Pichia pastoris, Hansenula anomala, Candida sp., and Saccharomyces cerevisiae. Preferably, the recombinant engineering bacteria are Pichia pastoris. Further, the Pichia pastoris strains include at least one of X-33, GS115, GS190, GS200, JC220, JC254, KM71, M-C100-3, SMDll63, SMDll65, and SMDll68. In a specific embodiment of the present invention, the donor strain is Pichia pastoris X-33.

[0013] Furthermore, the expression vector of the recombinant engineering bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα. Further, the expression vector is a multi-copy insertion type expression vector. Preferably, the multi-copy insertion type expression vector is selected from at least one of pPIC3.5K, pPIC9K, and pAO815.

[0014] Furthermore, the promoter of the recombinant engineering bacteria includes any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter, and AOX2 promoter. In a specific embodiment of the present invention, the promoter is AOX1 promoter.

[0015] Furthermore, the recombinant engineering bacteria use CRISPR-Cas9 gene editing technology and homologous recombination technology to targetedly knockout one or more genes of MKC7 and ULP1.

[0016] Furthermore, the recombinant engineering bacteria use CRISPR-Cas9 gene editing technology to targetedly knockout one or more genes of MKC7 and ULP1, including the following steps: 1-1: Construct a co-expression plasmid of Cas9 and gRNA; 1-2: Prepare donor DNA; 1-3: Prepare competent cells; 1-4: Construct a strain that knocks out one or more genes of MKC7 and ULP1.

[0017] Further, for the donor DNA, using the genomic DNA of Pichia pastoris as a template, one or more gene left and right homologous arm fragments of MKC7 and / or ULP1 are obtained by overlap extension PCR technology.

[0018] Further, the homologous recombination technology is EasyGeno seamless cloning technology. The recombinant engineering bacteria use EasyGeno seamless cloning technology to targetedly knockout one or more genes of MKC7 and ULP1, including the following steps: 2-1: Linearize the vector; 2-2: Prepare the insertion fragments, which respectively include the upstream and downstream fragments of the MKC7 and ULP1 genes; 2-3: Perform the recombination reaction, where the molar ratio of vector:fragment ≈ 1:2-3; 2-4: Directly transform the recombination reaction product into competent cells.

[0019] Further, the copy number of the nucleotide sequence encoding recombinant bovine albumin in the recombinant engineering bacteria is 4-7. In the examples of the present invention, the copy number of the nucleotide sequence encoding recombinant bovine albumin in the recombinant engineering bacteria is 6.

[0020] Further, the recombinant engineering bacteria of the present invention further include a drug resistance gene fragment. The drug resistance gene fragment is used for the screening of the recombinant engineering bacteria. In the examples of the present invention, the drug resistance fragment is one or more of a geneticin G418 resistance gene, a kanamycin resistance gene, an ampicillin resistance gene, a His4 gene, and a blasticidin resistance gene. Further, the recombinant engineering bacteria further include a signal peptide sequence, which is used for the secretion and expression of the foreign protein of the recombinant engineering bacteria. In the examples of the present invention, the signal peptide sequence in the recombinant engineering bacteria is the Saccharomyces cerevisiae α mating factor signal peptide; preferably, the nucleotide sequence encoding the Saccharomyces cerevisiae α mating factor (α-factor) signal peptide is as shown in SEQ ID NO.11.

[0021] In the second aspect of the present invention, there is provided the use of the recombinant engineering bacteria in the preparation of recombinant bovine albumin and / or the increase of the expression level of recombinant bovine albumin.

[0022] In the third aspect of the present invention, there is provided recombinant bovine albumin prepared by fermenting the above-mentioned recombinant engineering bacteria.

[0023] In the fourth aspect of the present invention, there is provided a method for culturing the recombinant engineering bacteria. The culture temperature in the method is 26-30 °C, the methanol concentration is 0.4-0.6%, and the induction time is 70-74 h.

[0024] In a specific embodiment of the present invention, the culture temperature in the method is 28 °C, the methanol concentration is 0.5%, and the induction time is 72 h.

[0025] The beneficial effects of the present invention include: The present invention constructs a recombinant engineering bacterium for preparing recombinant bovine albumin. While expressing recombinant bovine albumin, one or more of the protease degradation genes MKC7 and ULP1 are targeted for knockout, and / or one or more of the protein folding-related genes PDI-2 and SHR3 are overexpressed and integrated into the strain to achieve the high-efficiency expression of recombinant bovine albumin, laying a foundation for the industrial application of recombinant bovine albumin. The present invention modifies the recombinant engineering bacterium from two aspects of promoting protein folding and inhibiting protease degradation, which can significantly increase the expression level of recombinant bovine albumin. The highest yield of recombinant bovine albumin can reach 38.79 g / L, which is 4.85 times higher. This recombinant engineering bacterium shows great potential in improving the yield of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 It is a schematic structural diagram of a recombinant expression cassette encoding recombinant bovine albumin; Figure 2 It is an SDS-PAGE gel electrophoresis diagram of recombinant engineering bacteria 4, recombinant engineering bacteria 6-10 expressing recombinant bovine albumin in the examples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Recombinant engineering bacterium: A strain of fungal cells in which foreign genes are highly expressed by genetic engineering methods.

[0028] In order to more clearly illustrate the overall concept of the present invention, the following will be described in detail by way of examples in conjunction with the drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0029] For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0030] Unless otherwise specified, in the following embodiments, reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0031] In the following examples, the molecular biology experimental methods not specifically described are carried out in accordance with the kits and product instructions, or are carried out with reference to the methods described in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook.

[0032] The sources of the experimental materials and reagents involved in the present invention are as follows.

[0033] Strain: Pichia pastoris X-33, purchased from Thermo Fisher Scientific. This strain itself has resistance to antibiotics of prokaryotes, such as resistance to kanamycin and ampicillin, and X-33 is a His+ strain, growing faster and being able to tolerate high copy numbers.

[0034] Expression vector and expression cassette: Customized from GenScript Biotech Corporation.

[0035] Enzymes and kits Restriction endonucleases BgI II, Not I, and BamH I were purchased from Thermo Fisher Scientific; plasmid extraction kit, agarose gel recovery kit, and DNA product purification kit were purchased from Tiangen Biotech Co., Ltd.; kanamycin, Geneticin, and Blasticidin were purchased from Thermo Fisher Scientific; SYBR Green Master Mixture was purchased from Bio-Rad; EasyGeno seamless cloning kit was purchased from Tiangen Biotech Co., Ltd.; alkaline phosphatase (CIP) was purchased from NEB; DH5α competent cells were purchased from Tiangen Biotech Co., Ltd.

[0036] Media E. coli solid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0037] E. coli liquid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0038] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar.

[0039] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH = 7.0.

[0040] Yeast medium BMGY: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) biotin, 0.23% (w / v) K2HPO4, 1.18% (w / v) KH2PO4 (where 0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0041] Yeast induction medium BMMY: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 0.5% methanol (V / V), 1.34% (w / v) YNB, 0.00004% (w / v) biotin, 0.23% (w / v) K2HPO4, 1.18% (w / v) KH2PO4 (where 0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0042] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH = 7.4. Example 1: Construction of recombinant bovine albumin Pichia pastoris strain The nucleic acid molecule encoding the mating factor signal peptide of Saccharomyces cerevisiae a (SEQ ID NO.11) and the nucleic acid molecule encoding recombinant bovine albumin (the nucleotide sequence is shown in SEQ ID NO.10) were directly synthesized by a biological company, and the kanamycin resistance gene was changed to the geneticin G418 resistance gene (the nucleotide sequence is shown in SEQ ID NO.12), and then ligated into the plasmid pPIC9K digested with the restriction enzyme sites EcoRI and NotI to form an expression vector, named pPIC9K-rBHA (as Figure 1 shown).

[0043] The expression vector was linearized using the DNA restriction enzyme NotI (the digestion system is shown in Table 1), electrotransformed into Pichia pastoris X-33, incubated at 30 °C for 2 h, and then spread on a YPDK plate containing 50 μg / mL kanamycin. After culturing at 28 °C for 48 - 72 h, a sterile water scraper was added and spread on a YPD plate containing 250 μg / mL G418. After culturing at 28 °C for 48 - 72 h. Positive monoclonal clones were screened for subsequent expression. The X-33 strain screened on the YPD plate was picked with a sterilized 10 μL pipette tip for the expression of rBSA, and the expression level of recombinant bovine albumin was determined by SDS-PAGE after shake flask culture.

[0044] Flask culture procedure: Recombinant engineering bacteria were separately inoculated into 100 mL of BMGY medium and cultured at 28 °C and 210 rpm until OD 600 = 1.0. 1 mL of the bacterial solution was taken for subsequent SDS-PAGE analysis. All the bacterial solution was collected, centrifuged at 10,000 rpm for 5 min at room temperature, the supernatant was discarded aseptically, 100 mL of BMMY medium was added to fully suspend the bacterial cells, and the cells were cultured at 28 °C and 220 rpm. A certain amount of methanol was added every 24 h to keep the methanol concentration constant at 0.5%. After 72 h, the cells were centrifuged at 10,000 rpm for 5 min, and the supernatant was collected.

[0045] Table 1

[0046] Example 2: Determination of the copy number of recombinant bovine albumin The copy number of recombinant bovine albumin was calculated by qPCR (real-time quantitative PCR): Five recombinant engineering bacteria with high expression levels of recombinant bovine albumin prepared in Example 1 were selected, and the DNA of the test strains was prepared using a yeast genomic DNA extraction kit (TIANGEN DP325) to ensure the purity (A 260 / A 280 ≈ 1.8 - 2.0) and concentration (≥ 50 ng / μL). Primers were designed for the gene sequence of the target gene recombinant bovine albumin (rBSA) in the pPIC9K vector (SEQ ID NO.10) and the single-copy gene GAPDH gene of Pichia pastoris. The primer sequences are shown in Table 2. rBSA and GAPDH were cloned into the same vector to construct a plasmid containing the target gene and the internal reference gene, and were serially diluted (10 9 -10 6 copies / μL) as the standard. The qPCR amplification system is shown in Table 3.

[0047] Table 2

[0048] Table 3

[0049] Amplification program: Pre-denaturation at 95 °C for 5 min → 40 cycles (95 °C for 30 s → 60 °C for 30 s → 72 °C for 30 s) → Melting curve analysis. The copy number of rBSA was obtained by calculating the ratio of the copy numbers of the target gene and the internal reference gene according to the standard curve.

[0050] The corresponding results of the yields and copy numbers of the recombinant engineering bacteria with higher yields are shown in Table 4.

[0051] Table 4

[0052] As can be seen from Table 4, when the copy number is 4 - 7, the expression levels of the strains are not significantly different. When the copy number exceeds 5, the yield no longer increases significantly and even decreases. It is speculated that this may be due to the retention of excessive proteins in the endoplasmic reticulum or Golgi apparatus, triggering the unfolded protein response and resulting in blockage of the secretion pathway. To solve this problem, the present invention further modifies the recombinant engineering bacterium 4, overexpresses genes related to protein folding, and simultaneously knocks out protease-related genes to inhibit the degradation of recombinant bovine albumin.

[0053] Example 3: Construction of vectors lacking protease degradation genes MKC7 and ULP1 Using the EasyGeno seamless cloning kit, the Pichia pastoris MKC7 gene deletion vector pPICZαA-ΔMKC7 was constructed by amplifying the Pichia pastoris genomic DNA. In this process, the Zeocin resistance gene was replaced with the blasticidin resistance gene (the nucleotide sequence is shown in SEQ ID NO.13). The steps for constructing pPICZαA-ΔMKC7 were entrusted to a biological company. The primer sequences are shown in Table 5. After the amplified pPICZαA-ΔMKC7 vector was digested with BamH I single restriction enzyme and then treated with alkaline phosphatase to purify the linear vector, the purified pPICZαA-ΔMKC7 linear vector was obtained. The linear vector pPICZαA-ΔMKC7 was ligated by seamless cloning according to the EasyGeno kit instructions and then heat-shock transformed into DH5α, and the transformed cells were spread on LB plates containing blasticidin (25 μg / mL) to screen for positive transformants. The obtained positive transformants were used to transform pPICZαA-ΔMKC7 into the recombinant engineering bacterium 4 by electroporation, and clones resistant to blasticidin (500 μg / mL) were screened to obtain the recombinant engineering bacterium 6.

[0054] The PCR amplification system is the same as shown in Table 5. The specific nucleotide sequences of the upstream and downstream primers are shown in Table 5. The EasyGeno seamless cloning system is shown in Table 6.

[0055] Table 5

[0056] Table 6

[0057] The EasyGeno seamless cloning kit was used to amplify the genomic DNA of Pichia pastoris to construct the Pichia pastoris ULP1 gene deletion vector pPICZαA-ΔULP1, in which the Zeocin resistance gene was replaced with the blasticidin resistance gene (the nucleotide sequence is shown in SEQ ID NO.13). The steps of constructing pPICZαA-ΔULP1 were entrusted to a biological company. The amplified pPICZαA-ΔULP1 vector was digested with the single restriction enzyme BamH I and then alkaline phosphatase was added to purify the linear vector to obtain the pPICZαA-ΔULP1 linear vector. The linear vector pPICZαA-ΔULP1 was ligated by seamless cloning according to the EasyGeno kit instructions and then heat-shock transformed into DH5α, and the cells were spread on an LB plate containing blasticidin (25 μg / mL) to screen for positive transformants. The obtained positive transformants were used to transform pPICZαA-ΔULP1 into the recombinant engineering strain 6 by electroporation, and blasticidin (500 μg / mL) resistant clones were screened to obtain the recombinant engineering strain 7.

[0058] Example 4: Construction of Protein Folding Gene PDI-2 and SHR3 Expression Strains A biological company was entrusted to synthesize the recombinant expression cassettes encoding the PDI-2 gene (the nucleotide sequence is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.5) and the SHR3 gene (the nucleotide sequence is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO.7) respectively, and the recombinant expression cassettes were ligated into the plasmid pGAPZA (containing the Zeocin resistance gene) to construct expression vectors, named pART1-1 and pART1-2 respectively. The recombinant expression cassettes encoding the two genes PDI-2 and SHR3 were ligated into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector, named pART1-3.

[0059] The expression vectors pART1-1, pART1-2, and pART1-3 were linearized using the DNA restriction enzyme BgI II, and then electrotransformed into the recombinant engineering strain 7 respectively. The cells were spread on the selective plate YPDG containing Zeocin and cultured at 28 °C until colonies appeared. The positive transformants were identified by colony PCR as the recombinant engineering strains 8-10, and the specific information is shown in Table 7.

[0060] Table 7

[0061] Example 5: Expression Level of Recombinant Bovine Albumin in Recombinant Engineering Strains 5L Fermenter Culture: Recombinant engineering bacteria 4 and recombinant engineering bacteria 6 - 10 were separately inoculated into 100 mL of BMGY medium and cultured at 28 °C and 210 rpm for 16 - 24 h, then transferred to 500 mL of BMGY medium (inoculation ratio 1:10) and cultured until OD 600 = 3.0. Add 2 - 3 L of BMGY (volume not exceeding 60%) to the 5L tank and adjust the pH to 6.0. Autoclave at 121 °C for 20 minutes. After cooling, connect the pH and DO electrodes and calibrate (pH with 4.0 and 6.86 standard buffer solutions; DO electrode calibrated 100% with saturated air). Temperature: 28 - 30 °C, initial stirring: 300 - 500 rpm, ventilation rate: 0.5 - 1.0 vvm (volume ratio / minute), pH: automatically controlled (adjusted with 28% ammonia water or 25% phosphoric acid, target pH 5.0). Inoculate the seed liquid into the fermenter at an inoculation amount of 10% and culture for 18 - 24 h, then supplement 50% glycerol at a rate of 90 mL / h to maintain DO ≥ 20% until the wet cell weight reaches 200 g / L. Drain the remaining glycerol, add BMMY medium and culture at 28 °C, add methanol to a final concentration of 0.5%, and culture at 220 rpm. Add a certain amount of methanol every 24 h to keep the methanol concentration constant at 0.5%. After 72 h, centrifuge at 10000 rpm for 5 min and collect the supernatant. Perform SDS-PAGE electrophoresis on the supernatant, and the electrophoresis pattern is as Figure 2 shown, and the specific results are shown in Table 8.

[0062] The results showed that when knocking out one or more genes of MKC7 and ULP1, and / or overexpressing one or more genes of PDI-2 and SHR3, the expression level of recombinant bovine albumin increased significantly. Among them, when knocking out the genes of MKC7 and ULP1 and overexpressing the genes of PDI-2 and SHR3 simultaneously, the expression level of recombinant bovine albumin reached a maximum of 38.79 g / L, which was increased by 4.85 times.

[0063] Table 8 also showed that when overexpressing the genes of PDI-2 and SHR3 simultaneously, the expression level of recombinant bovine albumin increased significantly. It was speculated that there might be a synergistic effect between the PDI-2 gene that promotes disulfide bond formation and the SHR3 gene that promotes protein folding.

[0064] It was also found that after knocking out the protease degradation genes MKC7 and ULP1, the other proteins with different molecular weights, especially the impurity proteins with molecular weights lower than the target protein, decreased significantly. Figure 2 Table 8

[0065] Table 8

[0066] The above are only embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A recombinant engineering bacterium for preparing recombinant bovine albumin, wherein the recombinant engineering bacterium, while expressing recombinant bovine albumin, further comprises at least any one of the following operations: (1) Knocking out the MKC7 gene; (2) Knocking out the MKC7 and ULP1 genes; (3) Knocking out the MKC7 and ULP1 genes and overexpressing the PDI-2 gene; (4) Knocking out the MKC7 and ULP1 genes and overexpressing the SHR3 gene; (5) Knocking out the MKC7 and ULP1 genes and overexpressing the PDI-2 and SHR3 genes.

2. The recombinant engineering bacterium according to claim 1, wherein The amino acid sequence encoded by the MKC7 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the ULP1 gene is shown in SEQ ID NO.3; the amino acid sequence encoded by the PDI-2 gene is shown in SEQ ID NO.5; the amino acid sequence encoded by the SHR3 gene is shown in SEQ ID NO.

7.

3. The recombinant engineering bacterium according to claim 1, wherein The nucleotide sequence of the MKC7 gene is shown in SEQ ID NO.2; the nucleotide sequence of the ULP1 gene is shown in SEQ ID NO.4; the nucleotide sequence of the PDI-2 gene is shown in SEQ ID NO.6; the nucleotide sequence of the SHR3 gene is shown in SEQ ID NO.

8.

4. The recombinant engineering bacterium according to claim 1, wherein The recombinant engineering bacterium is selected from one or more of Pichia pastoris, Hansenula anomala, Candida sp., and Saccharomyces cerevisiae.

5. The recombinant engineering bacteria according to claim 1, characterized in that, The expression vector of the recombinant engineering bacterium includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, pGAPα; the promoter of the recombinant engineering bacterium includes any one or more of AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter, and AOX2 promoter.

6. The recombinant engineering bacterium according to claim 1, wherein The recombinant engineering bacterium uses the CRISPR-Cas9 gene editing technology and homologous recombination technology to target and knock out one or more genes of MKC7 and ULP1.

7. The recombinant engineering bacterium according to claim 1, characterized in that, The copy number of recombinant bovine albumin in the recombinant engineering bacterium is 4 to 7.

8. Use of the recombinant engineering bacterium according to any one of claims 1-7 in the preparation of recombinant bovine albumin and / or increasing the expression level of recombinant bovine albumin.

9. A method for preparing recombinant bovine albumin, characterized in that, The method includes: fermenting and preparing recombinant bovine albumin using the recombinant engineering bacterium according to any one of claims 1-7.

10. A method for culturing the recombinant engineering bacteria according to any one of claims 1-7, characterized in that, The culture temperature in the method is 26 to 30 °C, and the methanol concentration is 0.4 to 0.6%.

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