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

Abstract

The invention provides a recombinant engineering bacterium for preparing recombinant canine 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 genes of PBN1, KAR2 and ERV2 related to protein folding are overexpressed and integrated into the strain while the recombinant canine albumin is expressed, so that the expression of the canine albumin is remarkably improved, the relative protein expression quantity can be up to 491% at most, the specific yield can be up to 32.83 g / L at most, and the recombinant canine albumin has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, specifically, to the fields of genetic engineering technology and microbiology, and particularly to a recombinant engineering bacterium for preparing recombinant canine albumin and its application. Background Art

[0002] Canine serum albumin (CSA) is the most abundant protein in canine blood, mainly synthesized by the liver, accounting for about 35 - 50% of the total plasma protein. It plays an important role in maintaining plasma colloid osmotic pressure, transporting endogenous and exogenous substances (such as fatty acids, hormones, drugs, etc.), and regulating acid-base balance. The liver parenchymal cells synthesize the CSA precursor containing 608 amino acids. The signal peptidase in the endoplasmic reticulum recognizes and cleaves the 18 signal peptide residues at the N-terminus, and the Golgi apparatus further cleaves 6 signal peptide residues, finally becoming a mature CSA molecule composed of 584 amino acids.

[0003] Hypoalbuminemia, usually defined as a serum albumin concentration below 30 g / L, is a common complication in critically ill dogs and cats, and is clearly regarded as a sign of disease severity. It is generally considered to be caused by certain diseases, and its adverse reactions will only occur when they become severe (below 20 g / L). Long-term malnutrition, liver diseases, glomerular dysfunction or gastrointestinal diseases, etc. will lead to a decrease in the albumin concentration in the body, which will further lead to edema, inflammatory bowel disease and familial glomerulopathy, and severe cases will cause damage to organs.

[0004] Currently, there is no stable CSA product on the market for treatment. When dogs need to be infused with albumin, the main choice is still HSA. However, heterologous proteins may stimulate the immune system, and the total dose and administration rate of HSA during disease treatment may also potentially stimulate immune responses, resulting in different degrees of hypersensitivity reactions, including angioedema, urticaria, pruritus or fever, leading to shortness of breath, collapse or hypotension. Some studies have shown that critically ill dogs still had immediate and delayed hypersensitivity reactions after receiving undiluted or diluted to 10% - 25% HSA for rapid infusion or slow constant-rate infusion. Therefore, there is an urgent need for a method to rapidly and efficiently produce stable canine albumin.

[0005] 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. From the perspective of foreign gene modification, it mainly includes optimization of foreign gene codons, increase in the integration copy number of foreign genes, and selection of signal peptides; from the perspective of strain modification, it mainly includes knocking out protein degrading enzyme genes, introducing folding promoting factors, introducing Vitreoscilla hemoglobin (Vgb) genes, and knocking out glycerol transporter genes; fermentation level optimization includes adjusting temperature and dissolved oxygen levels in environmental factors, adding auxiliary expression substances such as non-inhibitory carbon sources, vitamin C, certain amino acids, and oleic acid, and fermentation process optimization includes research on the initial induction cell mass and methanol feeding method. (Zhu Wen, Hu Youjia, Xie Liping. Related strategies and research progress of high-level 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 the high-level expression of canine albumin. Summary of the Invention The present invention improves the yield of canine albumin by increasing the copy number of foreign genes, introducing the α mating factor signal peptide, and strain modification. Further, the present invention selects to overexpress the protein folding genes of the endoplasmic reticulum to achieve the purpose of improving protein expression. 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 canine albumin. The recombinant engineering bacterium overexpresses and integrates one or more of the genes PBN1, KAR2, and ERV2 related to protein folding into the strain while expressing recombinant canine albumin, thereby achieving the high-level expression of recombinant canine albumin and laying a foundation for the industrial application of recombinant canine albumin.

[0006] The amino acid sequence encoded by the PBN1 gene is shown in SEQ ID NO.1. The nucleotide sequence of the PBN1 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1, including the sequences publicly available in the current database and the 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.

[0007] The amino acid sequence encoded by the KAR2 gene is as shown in SEQ ID NO.3. The nucleotide sequence of the KAR2 gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3, 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.4 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.4.

[0008] The amino acid sequence encoded by the ERV2 gene is as shown in SEQ ID NO.5. The nucleotide sequence of the ERV2 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.

[0009] Furthermore, the amino acid sequence of the canine albumin is as shown in SEQ ID NO.7. The canine albumin is synthesized and cloned into an expression vector after codon optimization according to the codon preference of Pichia pastoris to construct a recombinant plasmid.

[0010] Furthermore, the nucleotide sequence of the recombinant canine albumin is as shown in SEQ ID NO.8 or a nucleotide sequence having at least 95% identity with SEQ ID NO.8.

[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 feline albumin, recombinant bovine albumin, recombinant equine albumin, recombinant ovine albumin, and recombinant porcine albumin.

[0012] Furthermore, the recombinant engineering bacteria are selected from one or more of Pichia pastoris, Hansenula, Candida, 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] Further, the expression vectors of the recombinant engineered bacteria include 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] Further, the promoters of the recombinant engineered bacteria include 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] Further, the copy number of the nucleotide sequence encoding recombinant canine albumin in the recombinant engineered bacteria is 4 to 7. In an embodiment of the present invention, the copy number of the nucleotide sequence encoding recombinant canine albumin in the recombinant engineered bacteria is 6.

[0016] Further, the recombinant engineered 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 engineered bacteria. In an embodiment of the present invention, the drug resistance fragment is one or more of geneticin G418 resistance gene, kanamycin resistance gene, ampicillin resistance gene, and His4 gene. Further, the recombinant engineered bacteria further include a signal peptide sequence. The signal peptide sequence is used for the secretion expression of exogenous proteins of the recombinant engineered bacteria. In an embodiment of the present invention, the signal peptide sequence in the recombinant engineered bacteria is the α-mating factor signal peptide of Saccharomyces cerevisiae. Preferably, the nucleotide sequence encoding the α-mating factor signal peptide of Saccharomyces cerevisiae is as shown in SEQ ID NO.8.

[0017] In the second aspect of the present invention, there is provided the use of the recombinant engineered bacteria in the preparation of recombinant canine albumin and / or the increase of the expression level of recombinant canine albumin.

[0018] In the third aspect of the present invention, there is provided recombinant canine albumin prepared by fermenting the above-mentioned recombinant engineered bacteria.

[0019] In the fourth aspect of the present invention, there is provided a method for culturing the recombinant engineered bacteria. The culture temperature in the method is 26 to 30 °C, the methanol concentration is 0.4 to 0.6%, and the induction time is 70 to 74 h.

[0020] 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.

[0021] The beneficial effects of the present invention include: The recombinant engineering bacteria of the present invention overexpress one or more of the genes PBN1, KAR2, and ERV2 related to protein folding, solving the problem that when the copy number of the foreign protein is too high during expression, excessive protein accumulates in the endoplasmic reticulum or Golgi apparatus, triggering the unfolded protein response and blocking the secretion pathway. It significantly increases the expression level of recombinant canine albumin, with the relative protein expression level reaching up to 491% at most, and enables the production of recombinant canine albumin to reach 32.83 g / L. This recombinant engineering bacteria shows great potential in improving the production of proteins with high economic value and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute 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 of the present invention. In the drawings: Figure 1 It is a schematic structural diagram of a recombinant expression cassette encoding recombinant canine albumin; Figure 2 It is an SDS-PAGE gel electrophoresis diagram of the recombinant engineering bacteria 5-9 expressing recombinant canine albumin in the examples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Recombinant engineering bacteria: Bacterial cell lines in which foreign genes are highly expressed by genetic engineering methods.

[0024] 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.

[0025] For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0026] Unless otherwise specified, in the following embodiments, for reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0027] All the molecular biology experimental methods not specifically described in the following examples are carried out according to the kits and product manuals, or are carried out with reference to the methods described in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook.

[0028] The sources of the experimental materials and reagents involved in the present invention are as follows: 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.

[0029] Expression vector and expression cassette: Customized from Yunzhou Biotechnology Co., Ltd.

[0030] Enzymes and kits: Restriction endonucleases BgI II / Not I were purchased from Thermo Fisher Scientific; Plasmid extraction kit, Agarose gel recovery kit, DNA product purification kit were purchased from Tiangen Biotech Co., Ltd.; Kanamycin and Geneticin were purchased from Thermo Fisher Scientific; SYBR Green Master Mixture was purchased from Bio-Rad.

[0031] 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.

[0032] E. coli liquid medium LB: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0.

[0033] Yeast solid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar.

[0034] Yeast liquid medium YPD: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, PBS buffer, pH = 7.0.

[0035] 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).

[0036] 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.

[0037] Buffer: 0.283% (w / v) Na2HPO4, 0.4% (w / v) NaH2PO4, pH = 7.4. Example 1: Construction of recombinant Pichia pastoris strain expressing canine albumin The nucleic acid molecule encoding the mating factor signal peptide of Saccharomyces cerevisiae (sequence shown in SEQ ID NO.9) and the nucleic acid molecule encoding recombinant canine albumin (nucleotide sequence shown in SEQ ID NO.8) were entrusted to Cloud-Zhou 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 enzyme sites EcoRI and NotI to form an expression vector, named pPIC9K-rCHA (as Figure 1 shown).

[0038] 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, cultured at 28 °C for 48 - 72 h, added with a sterile water scraper, and spread on a YPD plate containing 250 μg / mL G418, and cultured at 28 °C for 48 - 72 h. Positive monoclonal colonies 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 rCSA, and the expression level of recombinant canine albumin was determined by SDS-PAGE after shake flask culture.

[0039] Shake flask culture steps: The recombinant engineering bacteria were inoculated into 100 mL of BMGY medium respectively, 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 10000 rpm for 5 min at room temperature, the supernatant was discarded aseptically, 100 mL of BMMY medium was added to fully suspend the cells, cultured at 28 °C and 220 rpm, and a certain amount of methanol was added every 24 h to keep the methanol concentration constant at 0.5%. After 72 h, centrifuged at 10000 rpm for 5 min, and the supernatant was collected.

[0040] Table 1

[0041] Example 2: Determination of the Copy Number of Recombinant Canine Albumin The copy number of recombinant canine albumin was calculated by qPCR (real-time quantitative PCR): Five recombinant engineering bacteria with high expression levels of recombinant canine albumin prepared in Example 1 were selected, and the DNA of the strains to be tested was prepared using a yeast genomic DNA extraction kit (TIANGEN DP325) to ensure 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 canine albumin (rCSA) (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, and gradient dilution (10 9 -10 6 copies / μL) was used as the standard. The qPCR amplification system is shown in Table 3.

[0042] Table 2

[0043] Table 3

[0044] 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 ratio of the target gene to the internal reference gene was calculated according to the standard curve to obtain the copy number of rCSA.

[0045] The corresponding results of the yield and copy number of the recombinant engineering bacteria with higher yields are shown in Table 4.

[0046] Table 4

[0047] 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 protein in the endoplasmic reticulum or Golgi apparatus, triggering the unfolded protein response and resulting in blockage of the secretion pathway. To solve this problem, considering factors such as the secretion ability of the strains, the present invention further modifies recombinant engineering bacterium 5 to overexpress genes related to protein folding.

[0048] Example 3: Construction of Strains Co-expressing PBN1, KAR2, and ERV2 Entrusted Yunzhou Biotechnology to synthesize recombinant expression cassettes encoding the PBN1 gene (nucleotide sequence as SEQ ID NO.2, amino acid sequence as SEQ ID NO.1), the KAR2 gene (nucleotide sequence as SEQ ID NO.4, amino acid sequence as SEQ ID NO.3), and the ERV2 gene (nucleotide sequence as SEQ ID NO.6, amino acid sequence as SEQ ID NO.5) respectively, and ligate 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. Ligate the recombinant expression cassettes of the three genes PBN1, KAR2, and ERV2 into the same plasmid pGAPZA (containing the Zeocin resistance gene) to construct an expression vector, named pART1-4.

[0049] Use the DNA restriction endonuclease BgI II to linearize the expression vectors pART1-1 to pART1-4, and electrotransform the recombinant engineering bacteria 5 respectively. Spread the electrotransformed cells on the selective plate YPDG containing Zeocin and culture at 28 °C until colonies appear. Identify the positive transformants as recombinant engineering bacteria 6-9 by colony PCR. The specific information is shown in Table 5.

[0050] Table 5

[0051] Example 5: Expression level of recombinant canine albumin in recombinant engineering bacteria Cultivation in a 5L fermenter: Inoculate recombinant engineering bacteria 5-9 into 100 mL of BMGY medium respectively, culture at 28 °C and 210 rpm for 16-24 h, then transfer to 500 mL of BMGY medium (inoculation ratio is 1:10) and culture 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. 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 cell wet weight reaches 200 g / L. Drain all the residual 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. Detect the supernatant by SDS-PAGE electrophoresis. The electrophoresis pattern is asFigure 2 As shown, the specific results are shown in Table 6.

[0052] The results show that when one or more of the genes PBN1, KAR2, and ERV2 related to protein folding are overexpressed, the expression level of recombinant canine albumin significantly increases. When all three genes are overexpressed simultaneously, the expression level of recombinant canine albumin reaches a maximum of 491%.

[0053] Table 6

[0054] 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 changes. Any modifications, equivalent replacements, improvements, 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 canine albumin, wherein the recombinant engineering bacterium overexpresses and integrates the PBN1, KAR2, and ERV2 genes into the strain while expressing recombinant canine albumin.

2. The recombinant engineering bacterium according to claim 1, characterized in that, The amino acid sequence encoded by the PBN1 gene is as shown in SEQ ID NO.1; the amino acid sequence encoded by the KAR2 gene is as shown in SEQ ID NO.3; the amino acid sequence encoded by the ERV2 gene is as shown in SEQ ID NO.

5.

3. The recombinant engineering bacterium according to claim 1, characterized in that, The nucleotide sequence of the PBN1 gene is as shown in SEQ ID NO.2 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.2; the nucleotide sequence of the KAR2 gene is as shown in SEQ ID NO.4 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.4; the nucleotide sequence of the ERV2 gene is as shown in SEQ ID NO.6 or has 95% homology with the nucleotide sequence shown in SEQ ID NO.

6.

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 spp., and Saccharomyces cerevisiae.

5. The recombinant engineering bacterium according to claim 1, wherein The expression vector of the recombinant engineering bacterium includes any one or more of pHIL-D2, pAO815, pPIC3K, pPICZ, pHWO10, pGAPZ, pPIC9K, pPICZα, and pGAPα.

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

7. The recombinant engineering bacteria according to claim 1, characterized in that, The copy number of recombinant canine 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 canine albumin and / or the increase of the expression level of recombinant canine albumin.

9. A method for preparing recombinant canine albumin, characterized in that, The method includes: fermenting to prepare recombinant canine 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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