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

Abstract

The invention provides a recombinant engineering bacterium for preparing recombinant cat albumin and application of the recombinant engineering bacterium, and relates to the field of biochemistry, in particular to the technical field of genetic engineering. One or more of MIT1, EFT1 and SEC24 genes are overexpressed and integrated into the strain while the recombinant cat albumin is expressed, so that the expression of the cat albumin is remarkably improved, the relative protein expression quantity can be up to 443.7% at most, the specific yield can be up to 30.26 g / L at most, and the recombinant cat albumin has a wide application prospect.

Description

A recombinant engineered bacterium for preparing recombinant feline albumin and its application Technical Field

[0001] This invention relates to the field of biochemistry, specifically to the fields of genetic engineering and microbiology, and particularly to a recombinant engineered bacterium for preparing recombinant feline albumin and its application. Background Technology

[0002] Feline serum albumin (FSA) is the most abundant soluble protein in feline plasma, accounting for 35%–50% of total plasma protein. It is continuously synthesized by hepatocytes and secreted into the blood. Its mature molecule consists of 584 amino acids, and its precursor synthesis requires signal peptide cleavage modification by the endoplasmic reticulum and Golgi apparatus. The core physiological functions of FSA include: maintaining stable plasma colloid osmotic pressure and preventing extravasation of intravascular fluid that could lead to tissue edema; acting as an important "transporter" in the body, binding and transporting endogenous and exogenous substances such as fatty acids, bilirubin, hormones, and drugs; and participating in acid-base balance regulation by binding hydrogen ions, buffering fluctuations in blood pH. These functions make it one of the core molecules for maintaining metabolism and homeostasis in cats.

[0003] Clinically, it is mainly used to treat shock caused by blood loss or burns in cats, hypoalbuminemia, edema caused by cirrhosis and kidney disease, and other diseases. Currently, most serum albumin preparations used clinically to treat cats are feline serum albumin extracted from blood or human serum albumin (HSA). However, the insufficient supply of feline serum, the complex sources of serum, and the risk of viral transmission through blood severely limit the industrial production of FSA; while the use of HSA, the infusion of heterologous albumin may trigger specific allergic reactions, harming the cat's health and even endangering its life. Therefore, developing efficient, safe, and easily scalable FSA production technology has become a critical issue that urgently needs to be addressed in the field of veterinary medicine. Summary of the Invention

[0004] This invention improves the yield of feline albumin by increasing the copy number of exogenous genes, introducing α-mating factor signal peptides, and modifying bacterial strains. Furthermore, this invention achieves the goal of improving protein expression by simultaneously improving transcription efficiency, enhancing translational elongation, and opening secretion channels. The specific scheme of this invention is as follows: In the first aspect, this invention provides a recombinant engineered bacterium for preparing recombinant feline albumin. The recombinant engineered bacterium integrates one or more of MIT1, EFT1, and SEC24 into the strain while expressing recombinant feline albumin, thereby achieving high-efficiency expression of recombinant feline albumin and laying the foundation for the industrial application of recombinant feline albumin.

[0005] Optionally, the recombinant engineered bacteria, while expressing recombinant feline albumin, may also include any one of the following operations: A1) overexpressing and integrating the MIT1 gene into the strain; A2) overexpressing and integrating the EFT1 gene into the strain; A3) overexpressing and integrating the SEC24 gene into the strain; A4) simultaneously overexpressing and integrating the MIT1 and EFT1 genes into the strain; A5) simultaneously overexpressing and integrating the MIT1 and SEC24 genes into the strain; A6) simultaneously overexpressing and integrating the EFT1 and SEC24 genes into the strain; A7) simultaneously overexpressing and integrating the MIT1, EFT1, and SEC24 genes into the strain.

[0006] Among them, the MIT1 gene is a core transcription activator in the methanol-induced pathway, which directly binds to and activates the AOX1 promoter. Studies have shown that overexpression of the MIT1 gene in multi-copy strains can effectively improve transcriptional restriction caused by excessive gene dosage and restore cell growth and target protein yield.

[0007] The EFT1 gene is a crucial "transporter" in the process of cellular protein synthesis. During the translation phase, it is responsible for accurately transporting newly synthesized peptide chains from one location on the ribosome to the next, thereby ensuring that the protein chains can elongate normally.

[0008] The SEC24 gene acts as a "cargo adapter" for COPII-coated vesicles, responsible for recognizing and packaging secreted proteins at the endoplasmic reticulum exit.

[0009] Furthermore, the amino acid sequence encoded by the MIT1 gene is shown in SEQ ID NO.1. The nucleotide sequence of the MIT1 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1, including sequences currently published in the database and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown in SEQ ID NO.2 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0010] The amino acid sequence encoded by the EFT1 gene is shown in SEQ ID NO.3. The nucleotide sequence of the EFT1 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3, including sequences currently published in the database 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.

[0011] The amino acid sequence encoded by the SEC24 gene is shown in SEQ ID NO.5. The nucleotide sequence of the SEC24 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.5, including sequences currently published in the database and nucleotide sequences optimized according to actual needs. Preferably, the nucleotide sequence is shown in SEQ ID NO.6 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.6.

[0012] Furthermore, the amino acid sequence of the feline albumin is shown in SEQ ID NO.7. The feline albumin was synthesized and cloned into an expression vector after codon optimization according to the codon preference of Pichia pastoris to construct a recombinant plasmid.

[0013] Furthermore, the recombinant cat albumin nucleotide sequence is as shown in SEQ ID NO.8 or a nucleotide sequence having at least 95% identity with SEQ ID NO.8.

[0014] Furthermore, the exogenous gene expressed by the recombinant engineered bacteria can also be selected from other recombinant albumins. Preferably, the recombinant albumin includes one of recombinant human albumin, recombinant canine albumin, recombinant bovine albumin, recombinant equine albumin, recombinant sheep albumin, and recombinant porcine albumin.

[0015] Further, the recombinant engineered strain is selected from one or more of Pichia pastoris, Hansenula polymorpha, Candida albicans, and Saccharomyces cerevisiae. Preferably, the recombinant engineered strain is Pichia pastoris. Further, 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.

[0016] Further, 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. Further, 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.

[0017] 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.

[0018] Furthermore, the recombinant engineered bacteria of the present invention also include a drug resistance gene fragment. The drug resistance gene fragment is used for screening the recombinant engineered bacteria. In embodiments of the present invention, the drug resistance fragment is one or more of the following: genimycin G418 resistance gene, kanamycin resistance gene, ampicillin resistance gene, and 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 the embodiments of the present invention, the signal peptide sequence in the recombinant engineered bacteria is the Saccharomyces cerevisiae α-factor signal peptide; preferably, the nucleotide sequence encoding the Saccharomyces cerevisiae α-factor signal peptide is shown in SEQ ID NO.9.

[0020] In a second aspect, the present invention provides the use of the recombinant engineered bacteria in the preparation of recombinant feline albumin and / or in increasing the expression level of recombinant feline albumin.

[0021] In a third aspect, the present invention provides recombinant cat albumin prepared by fermentation using the aforementioned recombinant engineered bacteria.

[0022] In a fourth aspect, the present invention provides a method for culturing the recombinant engineered bacteria, wherein the culturing temperature is 22-30°C, the methanol concentration is 0.4-0.6%, and the induction time is 70-74 h.

[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 this invention include: the recombinant engineered bacteria of this invention, by overexpressing one or more of the MIT1, EFT1, and SEC24 genes, act on three key steps—transcriptional activation, translational elongation, and protein secretion—forming a complete gene-to-protein enhancement scheme. The recombinant engineered bacteria of this invention significantly increase the expression level of recombinant feline albumin, with a relative protein expression level reaching up to 443.7%, resulting in a recombinant feline albumin yield of 30.26 g / L. This recombinant engineered bacteria demonstrates strong potential in increasing the yield of high-economic-value proteins and has broad application prospects. Attached Figure Description

[0025] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 is a schematic diagram of the structure of the recombinant expression cassette encoding recombinant feline albumin; Figure 2 is a schematic diagram of the protein expression results of recombinant engineered bacteria 6-12 expressing recombinant feline albumin in the examples. Detailed Implementation

[0026] To more clearly illustrate the overall concept of the invention, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0027] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0028] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0029] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the kit and product instructions, or with reference to the methods described in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd edition).

[0030] The experimental materials and reagents involved in this invention are from the following sources: strain: Pichia pastoris X-33, purchased from Thermo Fisher Scientific. This strain itself has resistance to prokaryotic antibiotics, such as resistance to kanamycin and ampicillin. Moreover, X-33 is a His+ strain, which grows faster and can tolerate high copy numbers.

[0031] Expression vector and expression cassette: custom-made by Yunzhou Biotechnology Co., Ltd.

[0032] Enzymes and kits: Restriction endonucleases BgIII / NotI were purchased from Thermo Fisher Scientific; plasmid extraction kit, agarose gel extraction kit, and DNA product purification kit were purchased from Tiangen Biotech Co., Ltd.; kanamycin and genticin were purchased from Thermo Fisher Scientific; and SYBR Green MasterMixture was purchased from Bio-Rad.

[0033] Culture medium for Escherichia coli (LB): 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1.5% (w / v) agar, 1% (w / v) NaCl, pH 7.0.

[0034] Escherichia coli liquid culture medium LB: 10% (w / v) tryptone, 5% (w / v) yeast extract, 10% (w / v) NaCl, pH 7.0.

[0035] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar.

[0036] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH=7.0.

[0037] BMGY yeast culture medium: 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 with PBS solution).

[0038] 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.

[0039] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH=7.4.

[0040] Example 1: Construction of the recombinant cat albumin Pichia pastoris strain. The nucleic acid molecules encoding the Saccharomyces cerevisiae α-mating factor signal peptide (sequence shown in SEQ ID NO. 9) and the nucleic acid molecules encoding recombinant cat albumin (nucleotide sequence shown in SEQ ID NO. 8) were directly synthesized by Yunzhou Biotechnology. The kanamycin resistance gene was changed to the genimycin G418 resistance gene (nucleotide sequence shown in SEQ ID NO. 10), and then ligated into the plasmid pPIC9K digested with ECORI and NotI restriction sites to form an expression vector named pPIC9K-rFSA (as shown in Figure 1).

[0041] The expression vector was linearized using the DNA restriction endonuclease NotI (enzyme digestion system shown in Table 1), and transformed into Pichia pastoris X-33 by electroporation. After incubation at 30 ℃ for 2 h, the vector was plated onto G418 plates containing 0.25 mg / mL and cultured at 28 ℃ for 48–72 h. A sterile water scraper was then added, and the vector was plated onto YPD plates containing 2 mg / mL G418 and cultured at 28 ℃ for 48–72 h. Positive single clones were screened for further expression. X-33 strains selected from YPD plates were picked with a sterile 10 μL pipette tip for rFSA expression. After shake-flask culture, the expression level of recombinant feline albumin was determined by SDS-PAGE.

[0042] Shake flask culture procedure: Inoculate each recombinant engineered bacteria into 100 mL of BMGY medium and culture at 28 ℃ and 210 rpm until OD. 600 =1.0, take 1 mL of bacterial culture for subsequent SDS-PAGE analysis, collect all bacterial culture, centrifuge at 10000 rpm for 5 min at room temperature, aseptically discard the supernatant, add 100 mL of BMMY medium to fully resuspend the bacterial cells, and incubate at 28 ℃ and 220 rpm. After 48 h of induction, lower the temperature to 22-25 ℃. 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.

[0043] Table 1. Digestion system using the restriction endonuclease NotI.

[0044] Table 2 Expression levels of recombinant engineered bacteria 1-5

[0045] As shown in Table 2, the recombinant engineered bacteria 3 had the highest expression level, and therefore recombinant engineered bacteria 3 was selected for subsequent modification.

[0046] Example 2: Construction of MIT1, EFT1, and SEC24 co-expression strains. Yunzhou Biotechnology was commissioned to synthesize recombinant expression cassettes encoding the MIT1 gene (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1), the EFT1 gene (nucleotide sequence as shown in SEQ ID NO.4, amino acid sequence as shown in SEQ ID NO.3), and the SEC24 gene (nucleotide sequence as shown in SEQ ID NO.6, amino acid sequence as shown in SEQ ID NO.5), respectively. The recombinant expression cassettes were then ligated into the plasmid pGAPZA (containing the Zeocin resistance gene) to construct expression vectors, which were named pART1-1, pART1-2, and pART1-3, respectively.

[0047] Yunzhou Biotechnology was commissioned to ligate any two of the three genes encoding MIT1, EFT1, and SEC24 into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct expression vectors, named pART1-4, pART1-5, and pART1-6.

[0048] Yunzhou Biotechnology was commissioned to recombinantly express the three genes encoding MIT1, EFT1, and SEC24 into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector named pART1-7.

[0049] The expression vectors pART1-1, pART1-2, pART1-3, pART1-4, pART1-5, pART1-6, and pART1-7 were linearized using the DNA restriction endonuclease BglII. These vectors were then electroporated to transform recombinant engineered bacteria 3. The transformed cells were plated on Zeocin-containing YPDG selective plates and cultured at 28°C until colonies appeared. Colony PCR identification confirmed that the positive transformants were recombinant engineered bacteria 6-9. Specific information is shown in Table 3.

[0050] Example 3: Expression level of recombinant feline albumin in recombinant engineered bacteria. 5L fermenter culture: Recombinant engineered bacteria 5-9 were inoculated into 100 mL of BMGY medium and cultured at 28 ℃ and 210 rpm for 16-24 h. The culture was then transferred to 500 mL of BMGY medium (inoculation ratio 1:10) and cultured until OD600 = 3.0. 2-3 L of BMGY (volume not exceeding 60%) was added to the 5L fermenter, and the pH was adjusted to 6.0. Autoclaving was performed at 121℃ for 20 minutes. Temperature: 28-30℃; Initial stirring: 300-500 rpm; Aeration rate: 0.5-1.0 vvm (volume ratio / min); pH: Automatically controlled (adjusted with 28% ammonia or 25% phosphoric acid, target pH 5.0). The seed culture was inoculated into a fermenter at a 10% inoculum and cultured for 18–24 h. Then, 50% glycerol was added at a rate of 90 mL / h to maintain DO ≥ 20% until the cell wet weight reached 200 g / L. Residual glycerol was drained, and BMMY medium was added at 28 °C. Methanol was added to a final concentration of 0.5%, and the culture was incubated at 220 rpm. After 48 h of induction, the temperature was lowered to 22–25 °C. A certain amount of methanol was added every 24 h to maintain a constant methanol concentration of 0.5%. After 72 h, the culture was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. The supernatant was analyzed by SDS-PAGE electrophoresis, and the protein expression levels of each recombinant engineered strain were calculated. The specific results are shown in Table 3 and Figure 2.

[0051] The results showed that when one or more of the genes related to protein folding, MIT1, EFT1, and SEC24, were overexpressed, the expression level of recombinant feline albumin increased significantly. When all three genes were overexpressed simultaneously, the expression level of recombinant feline albumin reached the highest level of 443.7%.

[0052] Table 3 Expression levels of recombinant engineered bacteria 6-12

[0053] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A recombinant engineered bacterium for preparing recombinant feline albumin, wherein the recombinant engineered bacterium, while expressing recombinant feline albumin, further includes any one of the following operations: A1) overexpressing and integrating the MIT1 gene into the strain; A2) simultaneously overexpressing and integrating the MIT1 and EFT1 genes into the strain; A3) simultaneously overexpressing and integrating the MIT1 and SEC24 genes into the strain; A4) simultaneously overexpressing and integrating the MIT1, EFT1, and SEC24 genes into the strain.

2. The recombinant engineered bacteria according to claim 1, characterized in that, The amino acid sequence encoded by the MIT1 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the EFT1 gene is shown in SEQ ID NO.3; and the amino acid sequence encoded by the SEC24 gene is shown in SEQ ID NO.

5.

3. The recombinant engineered bacteria according to claim 1, characterized in that, The nucleotide sequence of the MIT1 gene is shown in SEQ ID NO.2; the nucleotide sequence of the EFT1 gene is shown in SEQ ID NO.4; and the nucleotide sequence of the SEC24 gene is shown in SEQ ID NO.

6.

4. The recombinant engineered bacteria according to claim 1, characterized in that, The recombinant engineered bacteria are selected from one or more of Pichia pastoris, Hansenula polymorpha, Candida albicans, and Saccharomyces cerevisiae.

5. The recombinant engineered bacteria 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.

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

7. The recombinant engineered bacteria according to claim 1, characterized in that, The amino acid sequence of the recombinant feline albumin in the recombinant engineered bacteria is shown in SEQ ID NO.

7.

8. The use of the recombinant engineered bacteria as described in any one of claims 1-7 in the preparation of recombinant feline albumin and / or in increasing the expression level of recombinant feline albumin.

9. A method for preparing recombinant feline albumin, characterized in that, The method includes: preparing recombinant cat albumin by fermentation using recombinant engineered bacteria as described in any one of claims 1-7.

10. A method for culturing the recombinant engineered bacteria according to any one of claims 1-7, characterized in that, The culture temperature in the method is 22~30℃, and the methanol concentration is 0.4~0.6%.

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