A recombinant engineering bacterium for preparing recombinant canine albumin and its application

Abstract

The present invention provides a recombinant engineered bacterium for producing recombinant canine albumin and its application, relating to the field of biochemistry, specifically, genetic engineering technology. By integrating overexpression of one or more of the protein-folding-related genes PBN1, KAR2, and ERV2 into the strain while simultaneously expressing recombinant canine albumin, the present invention significantly improves canine albumin expression, with relative protein expression reaching up to 491% and specific yields reaching up to 32.83 g / L, demonstrating broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, specifically, to the field of genetic engineering technology and microorganisms, and in particular to a recombinant engineering bacterium for preparing recombinant canine albumin and an application thereof. Background Art

[0002] Canine serum albumin (CSA) is the most abundant protein in canine blood, primarily synthesized in the liver and accounting for approximately 35-50% of total plasma protein. It plays an important role in maintaining plasma colloid osmotic pressure, transporting endogenous and exogenous substances (such as fatty acids, hormones, and drugs), and regulating acid-base homeostasis. Hepatic parenchymal cells synthesize a 608-amino acid CSA precursor, which is recognized by signal peptidases in the endoplasmic reticulum and cleaved off the 18 N-terminal signal peptide residues. The Golgi apparatus further cleaves off another 6 signal peptide residues, ultimately resulting in the mature CSA molecule consisting of 584 amino acids.

[0003] Hypoalbuminemia, typically defined as a serum albumin concentration below 30 g / L, is a common complication of critically ill dogs and cats. It is clearly considered a marker of disease severity and is generally considered to be caused by certain diseases, with adverse effects only occurring when the condition becomes severe (below 20 g / L). Conditions such as chronic malnutrition, liver disease, glomerular dysfunction, or gastrointestinal disease can lead to low albumin concentrations, which can lead to edema, inflammatory bowel disease, and familial glomerulopathy, which can cause organ damage in severe cases.

[0004] There are currently no stable CSA products available for therapeutic use on the market, and HSA remains the primary choice for canine albumin infusions. However, heterologous proteins may stimulate the immune system, and the total dose and rate of administration of HSA during disease treatment may also potentially stimulate immune stimulation, resulting in varying degrees of hypersensitivity reactions, including angioedema, urticaria, itching, or fever, leading to shortness of breath, collapse, or hypotension. Studies have shown that critically ill dogs still experience immediate and delayed hypersensitivity reactions after receiving undiluted or 10%-25% diluted HSA for rapid infusion or slow, constant-rate infusion. Therefore, a method for rapidly and efficiently producing stable canine albumin is urgently needed.

[0005] Heterologous protein expression is of great significance in various biopharmaceutical and industrial applications. As one of the most successful eukaryotic protein expression systems, Pichia pastoris has experienced rapid development and received widespread attention over the past 20 years, generating enormous economic and social value. Currently, methods for efficiently expressing exogenous proteins mainly include exogenous gene modification, strain modification, and fermentation optimization. From the perspective of exogenous gene modification, these methods mainly include optimizing the codons of the exogenous gene, increasing the number of copies of the integrated exogenous gene, and selecting the guide peptide. From the perspective of strain modification, these methods mainly include knocking out protein degrading enzyme genes, introducing folding-promoting factors, introducing the Vitreoscilla hemoglobin (Vgb) gene, and knocking out the glycerol transporter gene. Fermentation optimization includes adjusting environmental factors such as temperature and dissolved oxygen levels, adding auxiliary expression media such as non-inhibitory carbon sources, vitamin C, certain amino acids, and oleic acid. Fermentation process optimization includes studying the initial induction cell size and methanol feeding method. (Zhu Wen, Hu Youjia, Xie Liping. Strategies and research progress on efficient expression of foreign proteins in Pichia pastoris [J], Chinese Journal of Pharmaceuticals 2018, 49(4):417-425). These optimizations of Pichia pastoris provide a basis for efficient expression of canine albumin. Summary of the Invention

[0006] The present invention improves canine albumin production by increasing the number of exogenous gene copies, introducing an α-mating factor signal peptide, and modifying the bacterial strain. Furthermore, the present invention selects overexpression of endoplasmic reticulum protein folding genes to achieve the purpose of increasing protein expression. The specific scheme of the present invention is as follows:

[0007] The first invention of the present invention provides a recombinant engineered bacterium for preparing recombinant canine albumin. The recombinant engineered bacterium achieves efficient expression of recombinant canine albumin by overexpressing and integrating one or more of the protein folding-related genes PBN1, KAR2, and ERV2 into the strain while expressing the recombinant canine albumin, thereby laying the foundation for the industrial application of recombinant canine albumin.

[0008] The amino acid sequence encoded by the PBN1 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the PBN1 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1, including sequences currently disclosed in databases and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown as SEQ ID NO.2 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0009] The amino acid sequence encoded by the KAR2 gene is shown in SEQ ID NO.3. The nucleotide sequence of the KAR2 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3, including sequences currently disclosed in databases and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown in SEQ ID NO.4 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.4.

[0010] The amino acid sequence encoded by the ERV2 gene is shown as SEQ ID NO.5. The nucleotide sequence of the ERV2 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.5, including sequences currently disclosed in databases and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown as SEQ ID NO.6 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.6.

[0011] Furthermore, the amino acid sequence of the canine albumin is shown in SEQ ID NO. 7. The canine albumin is synthesized after codon optimization according to the codon preference of Pichia pastoris and cloned into an expression vector to construct a recombinant plasmid.

[0012] Furthermore, the nucleotide sequence of the recombinant canine albumin is shown as SEQ ID NO.8 or a nucleotide sequence having at least 95% identity with SEQ ID NO.8.

[0013] Furthermore, the exogenous gene expressed by the recombinant engineered bacteria can also be selected from one of other recombinant albumins. Preferably, the recombinant albumin includes one of recombinant human albumin, recombinant feline albumin, recombinant bovine albumin, recombinant horse albumin, recombinant sheep albumin, and recombinant porcine albumin.

[0014] Furthermore, the recombinant engineered bacteria is selected from one or more of Pichia pastoris, Hansenula, Candida, and Saccharomyces cerevisiae. Preferably, the recombinant engineered bacteria is Pichia pastoris. Furthermore, the Pichia pastoris strain includes at least one of X-33, GS115, GS190, GS200, JC220, JC254, KM71, M-C100-3, SMD1163, SMD1165, and SMD1168. In a specific embodiment of the present invention, the donor strain is Pichia pastoris X-33.

[0015] Furthermore, the expression vector of the recombinant engineered bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα. Furthermore, the expression vector is a multi-copy insertion expression vector, preferably, the multi-copy insertion expression vector is selected from at least one of pPIC3.5K, pPIC9K, and pAO815.

[0016] Furthermore, the promoter of the recombinant engineered bacteria includes any one or more of the AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter and AOX2 promoter. In a specific embodiment of the present invention, the promoter is the AOX1 promoter.

[0017] Furthermore, the number of copies of the nucleotide sequence encoding the recombinant canine albumin in the recombinant engineered bacteria is 4 to 7. In an embodiment of the present invention, the number of copies of the nucleotide sequence encoding the recombinant canine albumin in the recombinant engineered bacteria is 6.

[0018] Furthermore, the recombinant engineered bacteria of the present invention also include a drug resistance gene segment. The drug resistance gene segment is used for screening recombinant engineered bacteria. In an embodiment of the present invention, the drug resistance segment is one or more of the following: the geneticin G418 resistance gene, the kanamycin resistance gene, the ampicillin resistance gene, and the His4 gene.

[0019] Furthermore, the recombinant engineered bacteria also includes a signal peptide sequence, which is used for the secretory expression of exogenous proteins in the recombinant engineered bacteria. In an embodiment of the present invention, the signal peptide sequence in the recombinant engineered 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 shown in SEQ ID NO.8.

[0020] The second aspect of the present invention provides the use of the recombinant engineered bacteria in preparing recombinant canine albumin and / or increasing the expression level of recombinant canine albumin.

[0021] The third aspect of the present invention provides recombinant canine albumin prepared by fermentation using the above-mentioned recombinant engineered bacteria.

[0022] In a fourth aspect, the present invention provides a method for culturing the recombinant engineered bacteria, wherein the culture temperature is 26-30°C, the methanol concentration is 0.4-0.6%, and the induction time is 70-74 hours.

[0023] 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.

[0024] The beneficial effects of the present invention include:

[0025] By overexpressing one or more of the protein folding-related genes PBN1, KAR2, and ERV2, the recombinant engineered bacteria of the present invention address the problem of excessive protein retention in the endoplasmic reticulum or Golgi apparatus during exogenous protein expression, triggering an unfolded protein response and resulting in blockage of the secretory pathway. This significantly increases the expression of recombinant canine albumin, with relative protein expression reaching up to 491%, and enables a recombinant canine albumin yield of 32.83 g / L. This recombinant engineered bacteria demonstrates strong potential for increasing the production of highly economically valuable proteins 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 constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 is a schematic diagram of the structure of the recombinant expression cassette encoding recombinant canine albumin;

[0028] Figure 2 This is an SDS-PAGE gel electrophoresis diagram of the recombinant canine albumin expressed by the recombinant engineered bacteria 5-9 in the example. DETAILED DESCRIPTION

[0029] Recombinant engineered bacteria: a fungal cell line that uses genetic engineering methods to efficiently express exogenous genes.

[0030] In order to more clearly illustrate the overall concept of the present invention, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided 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 technical features well known in the art are not described.

[0031] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0032] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0033] Molecular biology experimental methods not specifically described in the following examples were performed 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.

[0034] The sources of experimental materials and reagents involved in the present invention are as follows:

[0035] Strain: Pichia pastoris X-33, purchased from Thermo Fisher Scientific. This strain inherently possesses resistance to prokaryotic antibiotics, such as resistance to kanamycin and ampicillin. X-33 is a His+ strain, grows faster, and can tolerate high copy numbers.

[0036] Expression vector and expression cassette: customized by Yunzhou Biotechnology Co., Ltd.

[0037] Enzymes and kits:

[0038] Restriction endonuclease BgIII / NotI was purchased from Thermo Fisher Scientific; plasmid extraction kit, agarose gel recovery kit, and DNA product purification kit were purchased from Tiangen Biotechnology Co., Ltd.; kanamycin and geneticin were purchased from Thermo Fisher Scientific; and SYBR Green Master Mixture was purchased from Bio-Rad.

[0039] culture medium

[0040] Escherichia coli solid culture medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0041] Escherichia coli liquid culture medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0042] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, and 2% (w / v) agar.

[0043] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH = 7.0.

[0044] 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 (0.23% (w / v) K2HPO4 and 1.18% (w / v) KH2PO4 can be directly replaced by PBS solution).

[0045] 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, PBS buffer.

[0046] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH=7.4.

[0047] Example 1: Construction of recombinant Pichia canis albumin strain

[0048] The nucleic acid molecule encoding the signal peptide of the mating factor of Saccharomyces cerevisiae a (sequence shown in SEQ ID NO.9) and the nucleic acid molecule encoding the recombinant canine albumin (nucleotide sequence shown in SEQ ID NO.8) were commissioned to Yunzhou Biotechnology for direct synthesis, and the kanamycin resistance gene was changed to the geneticin G418 resistance gene (nucleotide sequence shown in SEQ ID NO.10), and then ligated into the plasmid pPIC9K digested with the restriction sites EcoRI and NotI to form an expression vector named pPIC9K-rCHA (see Figure 1 shown).

[0049] The expression vector was linearized with the DNA restriction endonuclease NotI (enzyme digestion system is provided in Table 1) and transformed into Pichia pastoris X-33 by electroporation. After incubation at 30°C for 2 hours, the strain was plated onto YPDK plates containing 50 μg / mL kanamycin and cultured at 28°C for 48–72 hours. Sterile water was added to the plate and the strain was plated onto YPD plates containing 250 μg / mL G418. After culture at 28°C for 48–72 hours, positive single colonies were selected for subsequent expression. X-33 strains selected from the YPD plates were picked with a sterile 10 μL pipette tip for rCSA expression. After shake flask culture, the expression level of recombinant canine albumin was determined by SDS-PAGE.

[0050] Shake flask culture steps: take the recombinant engineered bacteria and inoculate them into 100 mL of BMGY medium, and culture them at 28 °C and 210 rpm until the OD 600 = 1.0, aspirate 1 mL of the bacterial suspension for subsequent SDS-PAGE analysis, collect the entire suspension, centrifuge at 10,000 rpm for 5 min at room temperature, discard the supernatant aseptically, add 100 mL of BMMY medium to fully resuspend the cells, and incubate at 28°C, 220 rpm, adding a certain amount of methanol every 24 h to maintain a constant methanol concentration of 0.5%. After 72 h, centrifuge at 10,000 rpm for 5 min, and collect the supernatant.

[0051] Table 1

[0052]

[0053] Example 2: Determination of recombinant canine albumin copy number

[0054] The copy number of recombinant canine albumin was calculated by qPCR (real-time quantitative PCR): 5 recombinant engineered bacteria expressing high amounts of recombinant canine albumin prepared in Example 1 were selected, and the yeast genomic DNA extraction kit (TIANGEN DP325) was used to prepare DNA of the strain to be tested to ensure the purity (A 260 / A 280 ≈1.8-2.0) and concentration (≥50 ng / μL). Primers were designed for the target gene recombinant canine albumin (rCSA) gene sequence (SEQ ID NO. 5) in the pPIC9K vector and the single-copy gene GAPDH gene of Pichia pastoris. The primer sequences are shown in Table 2. rCSA and GAPDH were cloned into the same vector to construct a plasmid containing the target gene and the internal reference gene. Serial dilution (10 9 -10 6 100 copies / μL) was used as a standard. The qPCR amplification system is shown in Table 3.

[0055] Table 2

[0056]

[0057] Table 3

[0058]

[0059] Amplification procedure: 95°C pre-denaturation 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 ratio of the target gene to the internal reference gene was calculated based on the standard curve to obtain the rCSA copy number.

[0060] The corresponding results of the yield and copy number of the recombinant engineering bacteria with higher yield are shown in Table 4.

[0061] Table 4

[0062]

[0063] As shown in Table 4, when the copy number is 4-7, the expression levels of the strains are not much 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 excessive protein retention in the endoplasmic reticulum or Golgi apparatus, triggering an unfolded protein response and resulting in blockage of the secretory pathway. To solve this problem, the present invention further modified the recombinant engineered strain 5 by overexpressing genes related to protein folding, taking into account factors such as the secretory capacity of the strain.

[0064] Example 3: Construction of PBN1, KAR2, and ERV2 co-expression strains

[0065] Yunzhou Bio was commissioned to synthesize recombinant expression cassettes encoding the PBN1 gene (nucleotide sequence such as SEQ ID NO.2, amino acid sequence such as SEQ ID NO.1), the KAR2 gene (nucleotide sequence such as SEQ ID NO.4, amino acid sequence such as SEQ ID NO.3), and the ERV2 gene (nucleotide sequence such as SEQ ID NO.6, amino acid sequence such as SEQ ID NO.5), and ligated the recombinant expression cassettes into the plasmid pGAPZA (containing the Zeocin resistance gene) to construct expression vectors, named pART1-1, pART1-2, and pART1-3, respectively. The recombinant expression cassettes encoding the PBN1, KAR2, and ERV2 genes were ligated into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector named pART1-4.

[0066] The expression vectors pART1-1 to pART1-4 were linearized using the DNA restriction endonuclease BgIII and transformed into the recombinant engineered bacteria 5, respectively. The transformed cells were spread on a YPDG selective plate containing Zeocin and cultured at 28°C until colonies appeared. The positive transformants were identified by colony PCR as recombinant engineered bacteria 6-9. The specific information is shown in Table 5.

[0067] Table 5

[0068]

[0069] Example 5: Expression level of recombinant canine albumin in recombinant engineered bacteria

[0070] 5L fermenter culture: Take 5-9 recombinant engineered bacteria and inoculate them into 100 mL of BMGY medium, culture at 28°C, 210 rpm for 16-24 hours, transfer to 500 mL of BMGY medium (inoculation ratio of 1:10) and culture until OD 600=3.0, add 2-3 L of BMGY (no more than 60% by volume) to a 5 L flask and adjust the pH to 6.0. Autoclave at 121°C for 20 minutes. Temperature: 28-30°C, initial agitation: 300-500 rpm, aeration: 0.5-1.0 vvm (volume ratio / minute), and pH: automatically controlled (adjusted with 28% ammonia or 25% phosphoric acid, target pH 5.0). Inoculate the fermentor with 10% seed solution and incubate for 18-24 hours. Then, supplement with 50% glycerol at a rate of 90 mL / h, maintaining a DO ≥ 20% until the cell wet weight reaches 200 g / L. Drain any remaining glycerol, add BMMY medium, and incubate at 28°C with methanol to a final concentration of 0.5%. Incubate at 220 rpm, adding a constant amount of methanol every 24 hours to maintain a constant methanol concentration of 0.5%. After 72 hours, centrifuge at 10,000 rpm for 5 minutes, and collect the supernatant. The supernatant was subjected to SDS-PAGE electrophoresis detection, and the electrophoresis pattern was as follows Figure 2 The specific results are shown in Table 6.

[0071] The results showed that when one or more of the protein folding-related genes PBN1, KAR2, and ERV2 were overexpressed, the expression level of recombinant canine albumin increased significantly. When the three genes were overexpressed at the same time, the expression level of recombinant canine albumin reached a maximum of 491%.

[0072] Table 6

[0073]

[0074] The foregoing is merely an embodiment of the present invention and is not intended to limit the present invention. It will be apparent to those skilled in the art that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. A recombinant engineered bacterium for preparing recombinant canine albumin, wherein the recombinant engineered bacterium overexpresses PBN1, KAR2, and ERV2 genes into the strain while expressing recombinant canine albumin, wherein the recombinant engineered bacterium is Pichia pastoris, the amino acid sequence encoded by the PBN1 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the KAR2 gene is shown in SEQ ID NO.3; and the amino acid sequence encoded by the ERV2 gene is shown in SEQ ID NO.5, and the number of copies of recombinant canine albumin in the recombinant engineered bacterium is 4 to 7.

2. The recombinant engineered bacterium according to claim 1, characterized in that The nucleotide sequence of the PBN1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the KAR2 gene is shown in SEQ ID NO.4; and the nucleotide sequence of the ERV2 gene is shown in SEQ ID NO.

6.

3. The recombinant engineered bacterium according to claim 1, characterized in that The expression vector of the recombinant engineered bacteria includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα.

4. The recombinant engineered bacterium according to claim 1, characterized in that The promoter of the recombinant engineering bacteria includes any one or more of the AOX1 promoter, GAP promoter, TEF1 promoter, GCW14 promoter and AOX2 promoter.

5. Use of the recombinant engineered bacteria according to any one of claims 1 to 4 in preparing recombinant canine albumin and / or increasing the expression level of recombinant canine albumin.

6. A method for preparing recombinant canine albumin, characterized in that: The method comprises: preparing recombinant canine albumin by fermentation using the recombinant engineered bacteria according to any one of claims 1 to 4.

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