Coding gene of dog serum albumin, expression vector, recombinant engineering bacterium, and preparation method and application of recombinant engineering bacterium of dog serum albumin

Abstract

The invention provides a coding gene of canine serum albumin, an expression vector, a recombinant engineering bacterium, and a preparation method and application of the recombinant engineering bacterium of canine serum albumin, the coding gene of canine serum albumin comprises a co-translation translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence, the co-translation translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region and an alpha-factor signal peptide pro-peptide region of saccharomyces cerevisiae, and the Ost1 signal peptide pre-peptide region and the alpha-factor signal peptide pro-peptide region are chimeric to obtain the co-translation translocation chimeric signal peptide sequence. According to the coding gene of the dog serum albumin provided by the invention, the efficient secretory expression of the dog serum albumin in pichia pastoris cells is realized by utilizing the synergistic effect of an Ost1 signal peptide pre-peptide region and an alpha-factor signal peptide pro-peptide region in a co-translation translocation chimeric signal peptide sequence.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a canine serum albumin encoding gene, an expression vector, a recombinant engineering bacterium, and a preparation method and application of the canine serum albumin recombinant engineering bacterium. Background Art

[0002] Canine serum albumin (CSA) is the most abundant protein in canine plasma, accounting for 40–60% of total plasma protein. CSA has multiple physiological functions, primarily maintaining stable plasma colloid osmotic pressure, accounting for 75–80% of this pressure. Other physiological functions include transporting various metabolites and drugs, maintaining acid-base balance, supporting the immune system, and helping maintain blood volume, making it of great value in veterinary clinical practice. CSA is a single peptide chain composed of 584 amino acids. The entire molecule consists of three structurally similar domains arranged to form a unique heart-shaped structure. CSA has 35 Cys residues, forming 17 disulfide bonds. Cys-34 is the only free sulfhydryl group not involved in disulfide bond formation and is the primary source of reduced sulfhydryl groups within blood vessels.

[0003] Canine serum albumin deficiency, also known as hypoproteinemia, is caused by reduced liver albumin synthesis, secondary diseases, inadequate protein intake, malabsorption, or excessive protein consumption (such as from massive blood loss, severe burns, or febrile illness). Symptoms include decreased plasma protein, decreased colloid osmotic pressure, and intravascular water leakage. In severe cases, systemic edema and decreased immunity may occur, making secondary infections more likely. With the rapid development of the Chinese pet market, the demand for pet dog medical care continues to grow. Recombinant canine serum albumin has potential applications in the clinical treatment of canine hypoproteinemia and canine hypovolemia, as well as in the production of veterinary excipients.

[0004] Canine serum albumin (CSA) for clinical use is typically extracted from healthy canine plasma. It is a protein biopharmaceutical used to treat conditions such as hypoalbuminemia, malnutrition, hypoalbuminemia-induced edema, and acute blood loss. Preparation methods, including salting-out, organic solvent precipitation, and heat shock extraction, often result in low albumin yield and purity. Furthermore, the shortage of canine plasma itself, the complex nature of serum sources, and the risk of viral transmission via drugs in the bloodstream severely limit its commercial production. Genetic engineering techniques for producing recombinant canine serum albumin (rCSA) can overcome these challenges. In particular, Pichia pastoris offers advantages as a host organism, including simple culture, low endotoxin levels, high expression levels, a post-translational modification system, and ease of large-scale cultivation. Furthermore, the target product can be directly secreted into the culture medium, making it an ideal host system for heterologous expression of CSA.

[0005] The recombinant canine serum albumin expressed by Pichia pastoris in the prior art has the same physical and chemical characteristics as natural canine serum albumin. In addition, the 97% pure canine serum albumin prepared by recombinant Pichia pastoris in the prior art has the same therapeutic effect as recombinant human serum albumin in the treatment of ascites in rats with liver cirrhosis. When facilitating the expression of recombinant canine serum albumin by Pichia pastoris, the signal peptide is an important factor affecting the expression of exogenous proteins in Pichia pastoris. The signal peptide commonly used in the prior art is the α-mating factor signal peptide (α-factor) derived from Saccharomyces cerevisiae, which promotes the post-translational translocation of proteins to the endoplasmic reticulum. However, it also has certain limitations. If the α-mating factor signal peptide is fused with a protein folded in the yeast cytoplasm, the protein may not be able to cross the endoplasmic reticulum membrane and enter the secretory pathway, thereby affecting the secretion and expression of canine serum albumin. Summary of the Invention

[0006] Aiming at the problems of low expression and severe degradation of canine serum albumin in Pichia pastoris in the prior art, a canine serum albumin encoding gene, an expression vector, a recombinant engineered bacterium, and a preparation method and application of a canine serum albumin recombinant engineered bacterium are provided.

[0007] The technical solutions adopted by the present invention to solve the above technical problems are as follows: On the one hand, the present invention provides a gene encoding canine serum albumin, comprising a co-translational translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence, wherein the co-translational translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region and an α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae, and the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region are chimerized to obtain a co-translational translocation chimeric signal peptide sequence.

[0008] Optionally, the co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1; and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.3.

[0009] Optionally, the co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having more than 90% homology with SEQ ID NO.2, and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having more than 90% homology with SEQ ID NO.4.

[0010] Optionally, a Lys-Arg sequence, a recognition and cleavage site for endopeptidase Kex2, is inserted between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence.

[0011] On the other hand, the present application provides an expression vector of canine serum albumin, comprising an expression vector and a gene encoding the canine serum albumin.

[0012] Optionally, the expression vector comprises one or more of pGAPZB, pPICZB, pPIC3.5K and pAO815.

[0013] On the other hand, the present application provides a canine serum albumin recombinant engineering bacterium, comprising a host bacterium and an expression vector of the canine serum albumin; The host bacteria include one or more of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 and Pichia pastoris SMD1168.

[0014] Optionally, the method for preparing the canine serum albumin recombinant engineered bacteria comprises the following operations: The Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae were chimerized to obtain a co-translational translocation chimeric signal peptide sequence; The amino acid sequence of the co-translational translocation chimeric signal peptide and the amino acid sequence of the canine serum albumin mature peptide were codon-optimized to obtain the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide, respectively; Chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide to obtain a gene encoding canine serum albumin; The gene encoding canine serum albumin was connected to an expression vector to obtain a recombinant plasmid; The recombinant plasmid was digested with enzymes and then transformed into a host bacterium to obtain a canine serum albumin recombinant engineered bacterium.

[0015] Optionally, before “chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the canine serum albumin mature peptide nucleotide sequence”, the following operations are performed: A Lys-Arg sequence recognized and cleaved by endopeptidase Kex2 is inserted between the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide.

[0016] On the other hand, the canine serum albumin recombinant engineered bacteria of the present invention, or the canine serum albumin recombinant engineered bacteria prepared by the preparation method of the canine serum albumin recombinant engineered bacteria, are used in the preparation of canine serum albumin and canine serum albumin biological products.

[0017] The beneficial effects of this application are: The canine serum albumin coding gene provided by the present application includes a co-translational translocation chimeric signal peptide and a canine serum albumin mature peptide. The co-translational translocation chimeric signal peptide sequence is obtained by chimerizing the pre-peptide region of the Ost1 signal peptide of Saccharomyces cerevisiae and the pro-peptide region of the α-factor signal peptide. The canine serum albumin coding gene is formed by codon-optimizing the sequence of the co-translational translocation chimeric signal peptide and the canine serum albumin mature peptide sequence. In the coding gene, the pre-peptide region of the Ost1 signal peptide of Saccharomyces cerevisiae and the pro-peptide region of the α-factor signal peptide complement each other. The pre-peptide region of the Ost1 signal peptide has unique amino acid sequence and structural characteristics during the co-translational process. It is accurately recognized by the signal recognition particles in the cytoplasm, and after binding, translation is paused and the ribosome-polypeptide chain complex is pulled to be transported to the endoplasmic reticulum, guiding the recombinant canine serum albumin to efficiently pass through the endoplasmic reticulum channel, avoiding premature misfolding in the cytoplasm. The pro-peptide region of the α-factor signal peptide plays a role in the endoplasmic reticulum, ensuring that the recombinant canine serum albumin entering the endoplasmic reticulum is correctly folded and modified, guiding the canine serum albumin to be transported to the Golgi apparatus through the vesicles, and after further processing and modification, it is secreted outside the cell through the vesicles. That is, the synergistic effect of the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region in the co-translational translocation chimeric signal peptide sequence is utilized to achieve efficient secretory expression of canine serum albumin in Pichia pastoris cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is the enzyme electrophoresis diagram of the recombinant plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS provided by the present invention; (M stands for DL5000 DNA Marker, 1 stands for double digestion with BstBI and NotI, 2 stands for double digestion with XhoI and NotI, and 3 stands for single digestion with SacI) Figure 2 This is the PCR electrophoresis diagram of the canine serum albumin recombinant engineering bacteria pPICZB-pre-Ost1-pro-α-factor-rCBS / X-33 provided by the present invention; (M is DL5000 DNA Marker; 1-3 are PCR amplification products of engineered bacteria) Figure 3 This is the SDS-PAGE electrophoresis diagram of the comparative experiment of canine serum albumin recombinant engineering bacteria shake flask provided by the present invention; (M represents PageRuler™ Prestained Protein Ladder, 1-3 represent the expression of recombinant engineered bacteria pPICZαA-rCBS / X-33, and 4-6 represent the expression of recombinant engineered bacteria pPICZB-pre-Ost1-pro-α-factor- rCBS / X-33) Figure 4 This is the electrophoresis diagram of the results of the purified canine serum albumin recombinant engineering bacteria analysis provided by the present invention; (M is PageRuler™ Prestained Protein Ladder; 1-2: Purified recombinant canine serum albumin sample) Figure 5 The HPLC detection chart of canine serum albumin recombinant engineering bacteria provided by the present invention; Figure 6 A diagram showing the functional verification results of the canine serum albumin recombinant engineered bacteria provided by the present invention. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0020] The invention provides a canine serum albumin coding gene, comprising a co-translational translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence, wherein the co-translational translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region and an α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae, and the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region are chimerized to obtain a co-translational translocation chimeric signal peptide sequence.

[0021] Specifically, the coding gene of canine serum albumin provided by the present application includes a co-translational translocation chimeric signal peptide and a canine serum albumin mature peptide. The co-translational translocation chimeric signal peptide sequence is obtained by chimerizing the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae. The sequence of the co-translational translocation chimeric signal peptide and the canine serum albumin mature peptide sequence are codon-optimized to form a canine serum albumin coding gene. In the coding gene, the pre-peptide region of the Saccharomyces cerevisiae Ost1 signal peptide and the pro-peptide region of the α-factor signal peptide complement each other. The pre-peptide region of the Ost1 signal peptide has a unique amino acid sequence and structural characteristics during the co-translational process. It is accurately recognized by the signal recognition particles in the cytoplasm. After binding, translation is paused and the ribosome-polypeptide chain complex is pulled to the endoplasmic reticulum, guiding the recombinant canine serum albumin to efficiently pass through the endoplasmic reticulum channel, avoiding premature misfolding in the cytoplasm. The pro-peptide region of the α-factor signal peptide plays a role in the endoplasmic reticulum, ensuring that the recombinant canine serum albumin entering the endoplasmic reticulum is correctly folded and modified, guiding the canine serum albumin to be transported to the Golgi apparatus through vesicles, and after further processing and modification, it is secreted into the extracellular space through the vesicles. That is, the synergistic effect of the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region in the co-translational translocation chimeric signal peptide sequence is utilized to achieve efficient secretory expression of canine serum albumin in Pichia pastoris cells.

[0022] In some embodiments, the co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1; and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.3.

[0023] Specifically, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is selected as a co-translational translocation chimeric signal peptide to guide canine serum albumin to quickly pass through the endoplasmic reticulum, avoid premature protein folding and degradation, and ensure efficient transport; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.3 is used as the mature peptide sequence of canine serum albumin, which can stably express the target protein with complete biological functions, which is conducive to ensuring the stable and efficient secretory expression of canine serum albumin in Pichia pastoris.

[0024] Specifically, the amino acid sequence of the co-translational translocation chimeric signal peptide shown in SEQ ID NO. 1 is: MRQVWFSWIVGLFLCFFNVSSAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR; The amino acid sequence of the mature peptide of canine serum albumin shown in SEQ ID NO.3 is:.

[0025] In some embodiments, the co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having more than 90% homology with SEQ ID NO.2, and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having more than 90% homology with SEQ ID NO.4.

[0026] Specifically, the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence with more than 90% homology to SEQ ID NO.2 is selected as a co-translational translocation chimeric signal peptide sequence, which can not only utilize its signal recognition and endoplasmic reticulum transport functions, but also enhance the stability of the gene through the homologous sequence, thereby avoiding the problem of decreased transport efficiency caused by gene mutations; the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence with more than 90% homology to SEQ ID NO.4 is used as the canine serum albumin mature peptide sequence, so that it can adapt to the Pichia pastoris translation system through codon degeneracy while keeping the amino acids in the core functional region of the protein unchanged. At the same time, the homologous sequence can tolerate base substitutions, reducing expression barriers caused by host replication preferences or synthesis processes, while achieving the secretion efficiency and sequence stability of the recombinant canine serum albumin.

[0027] Specifically, the nucleotide sequence of the co-translational translocation chimeric signal peptide shown in SEQ ID NO. 2 is: atgagacaggtgtggttctcttggattgtgggactgttcttgtgtttcttcaacgtcagttctgctgctcctgttaacactactaccgaagatgaaactgctcagataccagccgaagccgttataggttactcagaccttgaaggagactttgatgtcgcagtgttgccattctctaactccactaacaacggactgttgttcataaacacaactatcgcttccatcgctgctaaagaagagggagtatccctcgagaaaaga;

[0028] In some embodiments, a Lys-Arg sequence, a recognition and cleavage site for endopeptidase Kex2, is inserted between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence.

[0029] Specifically, a Lys-Arg cleavage site recognized by endopeptidase Kex2 is inserted between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence. The endopeptidase Kex2 in the endoplasmic reticulum of Pichia pastoris can be used to specifically recognize and cleave this site, thereby achieving efficient separation of the signal peptide and the mature peptide, thereby completely removing the signal peptide sequence from the secreted target protein, thereby ensuring that its amino acid sequence is 100% consistent with the natural canine serum albumin mature peptide, avoiding the uncut chimeric peptide chain from affecting the spatial folding and biological activity of canine serum albumin, and thus significantly improving the secretion efficiency and functional integrity of the recombinant canine serum albumin.

[0030] Another embodiment of the present application provides an expression vector of canine serum albumin, comprising an expression vector and a gene encoding the canine serum albumin.

[0031] Specifically, the canine serum albumin expression vector constructs a recombinant plasmid by combining the coding gene and the expression vector. The strong promoter of the expression vector drives efficient transcription of the gene, the ribosome binding site ensures translation efficiency, the terminator stabilizes the transcription product, and the signal peptide guide sequence is adapted to the endoplasmic reticulum and Golgi apparatus secretion pathway, which can promote the accurate secretion of the recombinant protein to the extracellular space, ultimately achieving efficient regulation of canine serum albumin in Pichia pastoris and providing a stable expression system.

[0032] In some embodiments, the expression vector comprises one or more of pGAPZB, pPICZB, pPIC3.5K, and pAO815.

[0033] Specifically, when preparing canine serum albumin expression vectors, pGAPZB, pPICZB, pPIC3.5K, and pAO815 can be selected as expression vectors; among the above expression vectors, their respective advantages can be used to improve the expression efficiency of canine serum albumin. Among them, the pPICZB and pPIC3.5K carry methanol-inducible AOX1 promoter, which can accurately control the expression amount by regulating the methanol concentration to avoid the accumulation of toxic proteins. pAO815 contains multiple copy integration sites, which can increase the copy number of the target gene and enhance the expression intensity.

[0034] Specifically, in a preferred embodiment, the expression vector for canine serum albumin is pPICZB.

[0035] Another embodiment of the present application provides a canine serum albumin recombinant engineered bacterium, comprising a host bacterium and an expression vector of the canine serum albumin; The host bacteria include one or more of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 and Pichia pastoris SMD1168.

[0036] Specifically, the canine serum albumin recombinant engineered bacteria achieves the combination of gene expression and protein production by introducing a canine serum albumin expression vector into a Pichia pastoris host bacterium. The expression vector carries elements such as a promoter, a screening marker, and a signal peptide, which can drive the efficient transcription and translation of the canine serum albumin encoding gene, and at the same time, quickly enrich positive transformants with the help of the screening marker; the Pichia pastoris host bacterium has protein folding, modification, and secretion mechanisms, which can correctly process and secrete the recombinant protein outside the cell, thereby helping to improve the expression efficiency of the recombinant protein.

[0037] Pichia pastoris X-33 has efficient homologous recombination capabilities, which can ensure the stable integration of expression vectors into the genome and reduce genetic drift; GS115 has a complete methanol utilization pathway and can achieve high expression of canine serum albumin with the help of the methanol-inducible promoter of vectors such as pPICZB; KM71 is a slow methanol utilization strain, suitable for proteins that are toxic to the host or require slow expression, reducing metabolic pressure; SMD1168, due to the lack of intracellular proteases, can significantly reduce the degradation of recombinant proteins and ensure the integrity and activity of canine serum albumin.

[0038] In some embodiments, the method for preparing the canine serum albumin recombinant engineered bacteria comprises the following operations: The Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae were chimerized to obtain a co-translational translocation chimeric signal peptide sequence; The amino acid sequence of the co-translational translocation chimeric signal peptide and the amino acid sequence of the canine serum albumin mature peptide were codon-optimized to obtain the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide, respectively; Chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide to obtain a gene encoding canine serum albumin; The gene encoding canine serum albumin was connected to an expression vector to obtain a recombinant plasmid; The recombinant plasmid was treated with enzymes and then transformed into a host bacterium to obtain a canine serum albumin recombinant engineered bacterium.

[0039] A Lys-Arg sequence recognized and cleaved by endopeptidase Kex2 is inserted between the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide.

[0040] In the specific operation, based on the codon preference of Pichia pastoris, the upstream restriction enzyme site was designed to be BstBⅠ, and the downstream restriction enzyme site was designed to be NotⅠ. The endopeptidase Kex2 recognition and cleavage site Lys-Arg was added between the co-translational translocation chimeric signal peptide nucleotide sequence and the canine serum albumin mature peptide nucleotide sequence to synthesize the nucleotide sequence of the canine serum albumin coding gene pre-Ost1-pro-α-factor-rCBS; The gene encoding the synthetic canine serum albumin was ligated into the vector pPICZαB through the restriction sites BstBI+NotⅠ to construct the recombinant plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS. The recombinant plasmid was linearized with endonuclease SacⅠ and then transformed into Pichia pastoris X-33 competent cells by electroporation. Canine serum albumin recombinant engineered bacteria were obtained after screening on YPDS medium plates containing 100 μg / mL Zeocin.

[0041] Specifically, in the preparation method of recombinant engineered bacteria of canine serum albumin, first, the chimera of the Ost1 signal peptide pre-peptide and the α-factor signal peptide pro-peptide not only utilizes the co-translational translocation ability of Ost1 to efficiently guide the protein into the endoplasmic reticulum, but also retains the transport guidance function of α-factor, and uses this transport function to secrete the protein extracellularly. Secondly, the codon optimization process adjusts the nucleotide sequence for Pichia pastoris. Furthermore, co-translational translocation avoids the misfolding and degradation of the protein in the cytoplasm, and the precise cleavage of the Kex2 cleavage site ensures that the mature peptide sequence is 100% consistent with the natural protein. The selection of an adapted expression vector and the transformation with the Pichia pastoris host bacteria achieves efficient gene integration, thereby promoting the controllable expression and stable secretion of canine serum albumin in the host bacteria after recombinant.

[0042] In some embodiments, before “chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the canine serum albumin mature peptide nucleotide sequence”, the following operations are performed: Specifically, a Lys-Arg sequence recognized by the endopeptidase Kex2 is inserted between the co-translational translocation chimeric signal peptide nucleotide sequence and the canine serum albumin mature peptide nucleotide sequence. During the Pichia pastoris expression process, the Kex2 enzyme can specifically recognize this site. When the recombinant protein enters the endoplasmic reticulum, the Kex2 enzyme can efficiently and accurately cut the connection between the signal peptide and the mature peptide (N-terminal signal peptide), ensuring that the secreted canine serum albumin mature peptide is completely consistent with the natural sequence, avoiding the influence of the signal peptide residue on the protein structure and function.

[0043] In some embodiments, the canine serum albumin recombinant engineered bacteria provided herein include a fermentation operation, and the specific fermentation operation includes the following process: Single colonies of recombinant engineered bacteria expressing canine serum albumin were screened in shake flasks to obtain highly expressing strains, which were then fermented at high density through four stages: seed culture medium cultivation, bacterial growth, and induction with glycerol and methanol. The seed culture medium was added to the fermentor, the pH was adjusted to 5.0 with ammonia water, and the temperature was controlled at 30°C. At the end of the glycerol addition stage, the pH was adjusted to 5.85, and the temperature was controlled at 25°C to enter the methanol induction stage. The dissolved oxygen was kept above 20% by adjusting the rotational speed, tank pressure, air flow, and methanol flow rate, and the fermentation was completed after 96 hours of induction.

[0044] In some embodiments, the present application provides a method for purifying canine serum albumin from a recombinant engineered canine serum albumin bacterium, comprising: subjecting the fermentation broth of the fermented canine serum albumin recombinant engineered bacteria to solid-liquid separation, and purifying the supernatant by hydrophobic chromatography and anion exchange chromatography to obtain canine serum albumin, wherein the purity of the recombinantly separated canine serum albumin is greater than 99.125%.

[0045] The canine serum albumin recombinant engineered bacteria provided in this application, or the canine serum albumin recombinant engineered bacteria prepared by the preparation method of the canine serum albumin recombinant engineered bacteria provided, are used in the preparation of canine serum albumin and canine serum albumin biological products.

[0046] Specifically, the canine serum albumin recombinant engineered bacteria and the engineered bacteria obtained by the preparation method thereof of the present application, in the application in the field of canine serum albumin and related biological product preparation, the engineered bacteria achieve efficient and stable expression of canine serum albumin through codon optimization, chimeric signal peptide design and adaptation vector and host system, which is conducive to the production of high-purity and high-activity canine serum albumin, which can not only meet the demand for standard canine serum albumin in scientific research, but also can be used as a core raw material for the preparation of canine plasma substitutes, drug carriers, biological diagnostic reagents and other biological products.

[0047] In some embodiments, the present application also provides purification of canine serum albumin recombinant engineering bacteria The present invention is further described below with reference to the following examples.

[0048] LLB medium (1 L): yeast extract 5 g, peptone 10 g, sodium chloride 10 g (solid medium contains 2% agar powder).

[0049] YPD medium (1 L): yeast extract 10 g, peptone 20 g, glucose 20 g.

[0050] YPDS medium (1 L): yeast extract 10 g, peptone 20 g, glucose 20 g, 1 M sorbitol (solid medium containing 2% agar powder).

[0051] BSM inorganic salt medium (1 L): 85% H3PO4 26.7 ml, CaSO4•2H2O 0.93 g, K2SO4 18.2 g, MgSO4•7H2O 14.9 g, KOH 4.13 g, Glycerol 40 g, PMT1 4.35 ml.

[0052] PMT1 formula (1L): CuSO4•5H2O 6.0g, MnSO4•H2O 3.0g, Na2MoO4•2H2O 0.2g, KI 0.088g, H3BO3 0.02g, CoCl2•6H2O 0.5g, ZnCl2 20.0g, FeSO4•7H2O 65.0g, Biotin 0.2g, concentrated H2SO4 5.0ml.

[0053] Example 1 This example is used to illustrate the preparation of the canine serum albumin encoding gene and expression vector disclosed in the present invention, including the following steps: Gene synthesis: Based on the codon bias of Pichia pastoris, and by weighing factors such as GC content, codon usage frequency, RNase splicing sites, and RNA stabilizing trans-acting elements, a tandem gene pre-Ost1-pro-α-factor-rCBS consisting of a co-translational translocation chimeric signal peptide nucleotide sequence SEQ ID NO.2 and a canine serum albumin nucleotide sequence such as SEQ ID NO.4 was artificially synthesized; Amplify target gene: Using the gene encoding canine serum albumin, pre-Ost1-pro-α-factor-rCBS, as a template, primers P1 and P2 were used for PCR amplification, and the target gene fragment was recovered from the gel; The gene sequence of primer P1 is ttattcgaagccaccatgagacaggtgtggttc, and the gene sequence of primer P2 is ctggcggccgcttaaaccaaagcagcttg; Construction of recombinant plasmid: The product and the vector plasmid pPICZB were recovered by double enzyme digestion with BstBI and NotI, respectively, and ligated with T4 DNA ligase at 16℃ for 1h, heat-shocked and transformed into competent cells DH10b, and spread on Zeocin-resistant LLB plates. The plasmid was extracted and identified by enzyme digestion, and the following was obtained: Figure 1 The electrophoresis test results are shown; The fragments of the double digestion products of BstBI+NotI were 2031 bp + 3268 bp, the fragments of the double digestion products of XhoI+NotI were 1768 bp + 3531 bp, and the fragment of the single digestion product of SacI was 5299 bp. The positive plasmid was named pPICZB-pre-Ost1-pro-α-factor-rCBS. Figure 1 The electrophoresis test results showed that the inserted fragment sequence was correct, there was no mutation, and the insertion direction was correct, proving that the target recombinant plasmid had been successfully constructed.

[0054] Example 2 This example is used to illustrate the canine serum albumin recombinant engineering bacteria disclosed in the present invention, and includes the following steps: Electric shock conversion: Linearize the plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS with the endonuclease SacI. Add 1-10 μg of the linearized plasmid to 80 μl of Pichia pastoris X-33 competent cells, mix well, and transfer to a 2 mm electroporation cuvette. Incubate on ice for 5 minutes. Set the electroporator parameters to 1.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, add 1 ml of pre-chilled 1 M / L sorbitol solution to the cuvette and mix thoroughly. Transfer the mixture to a sterile centrifuge tube and incubate at 30°C for 1 hour. Spread 200 μl of the bacterial solution on a YPDS plate containing Zeocin resistance (100 μg / ml) and incubate at 30°C for 2-5 days until single colonies appear. PCR identification of recombinants: Single colonies grown on YPDS plates were picked to extract genomic DNA, and PCR identification was performed using the template. The PCR reaction system was 10× Taq buffer 5μl, dNTP Mixture 4μl, template 1μl, 5'AOX1 and 3'AOX1 primers 1μl each, Taq polymerase 1μl, sterile double-distilled water 37μl, reaction parameters were 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 2min, 30 cycles, and 72℃ extension for another 10min. After that, the PCR product was taken for detection. Figure 2 The test results shown; Depend on Figure 2 Agarose gel electrophoresis showed that the PCR amplified band contained the target gene fragment, which was about 2100 bp in size, consistent with the theoretical expected molecular weight.

[0055] Example 3 This example is used to illustrate the induced expression of canine serum albumin recombinant engineered bacteria disclosed in the present invention, and includes the following steps: Transfer a 1% inoculum of canine serum albumin recombinant engineered bacteria stored in glycerol to 50 ml of YPD medium, culture at 30°C, 200 rpm to an OD of 2-6, collect the cells by centrifugation at 1500 g for 5 min, resuspend them in 200 ml of BSM inorganic medium, culture at 30°C, 200 rpm, and add methanol to a final concentration of 1.0% (500 ml of methanol plus 6 ml of PMT1) every 24 hours to induce expression. Analyze the fermentation supernatant by SDS-PAGE electrophoresis to obtain highly expressing engineered bacteria. Effects of different signal peptides on target protein expression: A high-expression canine serum albumin recombinant engineering bacterium pPICZB-pre-Ost1-pro-α-factor-rCBS / X-33 and a control group high-expression engineering bacterium pPICZαA- rCBS / X-33 were selected for induced expression under the same conditions. Three parallel experiments were set up. The SDS-PAGE electrophoresis detection results were analyzed using ImageJ software. Figure 3 ; like Figure 3 As shown, compared with the signal peptide α-factor, the chimeric signal peptide increased the canine serum albumin target band by 2.63 times and reduced the degree of degradation by 29.5%, further demonstrating that the canine serum albumin recombinant engineered bacteria provided by the present application can be efficiently secreted and expressed in its host.

[0056] Example 4 This example is used to illustrate the high-density fermentation of canine serum albumin recombinant engineering bacteria disclosed in the present invention, and includes the following steps: Seed cultivation stage: The recombinant engineered bacteria were inoculated into YPD medium, cultured at 30°C and 220 rpm for 20 h to obtain seed solution, and the wet weight of the bacteria was measured and examined under a microscope; Bacteria growth stage: Transfer the seed liquid to a fermenter containing 20L of BSM inorganic salt medium, set the temperature to 30°C, the rotation speed to 500-700rpm, the ventilation to 20L / min, the dissolved oxygen to be higher than 20%, and the pH to be adjusted and maintained at 5.0 mainly by ammonia water; Glycerol fed-batch stage: When the dissolved oxygen level suddenly increased, 50% glycerol containing 12 ml / L PTM1 was added at a rate of 18 ml / h / L, and all parameters were kept unchanged until the wet weight of the cells reached 240 g / L. Methanol induction stage: After the fermentation dissolved oxygen rapidly rose again, the temperature was adjusted to 25° C. and the pH was adjusted to 6.0. No carbon source was added to the tank within 1 hour after entering the induction stage to ensure complete consumption of glycerol. Methanol containing 12 ml / L PTM1 was added for induction, and the dissolved oxygen was controlled above 30%. The fermentation was terminated after 72 hours of induction. The protein content in the fermentation broth was determined by Coomassie Brilliant Blue staining to be 10.12 g / L.

[0057] Example 5 This example is used to illustrate the purification of canine serum albumin from the canine serum albumin recombinant engineering bacteria disclosed in the present invention, and includes the following steps: Heat treatment: The fermentation broth was centrifuged (6000 rpm, 20 minutes) to collect the supernatant, which was then heated in a metal bath at 72°C for 30 minutes to inactivate the protease to prevent degradation of the target protein. Hydrophobic chromatography: After heating, the sample was filtered through a 0.45 μm membrane and equilibrated with 5 column volumes of a Butyl hydrophobic column using equilibration buffer (50 mM / L sodium phosphate, 0.1 M / L sodium chloride, pH 7.0). The sample was loaded and the flow-through peak was collected and concentrated using a 30,000 molecular weight cut-off membrane pack to a rCSA concentration of 100 mg / ml. Anion chromatography: equilibrate DEAE6FF anion exchange chromatography column with equilibration buffer (20 mmol / L Tris-HCl, 0.1 mol / L sodium chloride, pH 7.4) for 5 column volumes, load the sample, and elute with elution buffer (20 mmol / L Tris-HCl, 0.5 mol / L sodium chloride, pH 7.4). Collect the eluted fractions containing rCSA and perform SDS-PAGE electrophoresis to obtain Figure 4 ; like Figure 4 As shown in FIG, lane 1 and lane 2 are both single bands, indicating that the canine serum albumin in the recombinant engineering bacteria is highly purified, and the purity of the target protein is further detected by HPLC. Figure 5 After two steps of purification, the protein purity can reach 99.12%.

[0058] Example 6 The functional verification of the canine serum albumin recombinant engineered bacteria disclosed in this application includes the following steps: The canine serum albumin purified from the above-mentioned recombinant engineering bacteria was used to treat rats with hypoproteinemia due to liver fibrosis. 30 rats with CC14-induced hypoproteinemia model of liver fibrosis were selected and divided into a negative control group, a positive control group, and an experimental group, with 10 rats in each group. The negative control group was given normal saline, the positive control group was given 500 mg / ml canine serum albumin injection (CSA, Taizhou Bolideli Biotechnology Co., Ltd.), and the experimental group was given 100 mg / ml recombinant canine serum albumin rCSA purified from the recombinant engineering bacteria. The dosage of each group was 200 mg / kg, and the drug was administered by tail vein push injection once a day for a total of seven days. After the last dose, the rats were deprived of food and water. 18 hours later, blood was collected from the retinal venous plexus to detect the serum albumin (ALB) value. The results are as follows: Figure 6 As shown (*P < 0.05, **P < 0.01, ***P < 0.001); Depend on Figure 6 It can be seen that the albumin levels in the rats of the experimental group and the positive control group returned to normal levels, indicating that rCSA has the same therapeutic effect as the commercially available canine blood albumin injection in treating hypoalbuminemia.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A gene encoding canine serum albumin, characterized in that The invention comprises a co-translational translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence, wherein the co-translational translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region and an α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae, and the Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region are chimerized to obtain a co-translational translocation chimeric signal peptide sequence.

2. The gene encoding canine serum albumin according to claim 1, characterized in that The co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1; and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

3.

3. The gene encoding canine serum albumin according to claim 1, characterized in that The co-translational translocation chimeric signal peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having more than 90% homology with SEQ ID NO.2, and / or, The canine serum albumin mature peptide sequence is selected from the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having more than 90% homology with SEQ ID NO.

4.

4. The gene encoding canine serum albumin according to claim 1, characterized in that A Lys-Arg sequence, a recognition and cleavage site for endopeptidase Kex2, is inserted between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence.

5. An expression vector for canine serum albumin, characterized in that: The invention comprises an expression vector and a gene encoding the canine serum albumin according to any one of claims 1 to 4.

6. The expression vector of canine serum albumin according to claim 5, characterized in that The expression vector includes one or more of pGAPZB, pPICZB, pPIC3.5K and pAO815.

7. A canine serum albumin recombinant engineering bacterium, characterized in that: Comprising a host bacterium and the expression vector of canine serum albumin according to claim 5; The host bacteria include one or more of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 and Pichia pastoris SMD1168.

8. The method for preparing canine serum albumin recombinant engineering bacteria according to claim 7, characterized in that: The following operations are included: The Ost1 signal peptide pre-peptide region and the α-factor signal peptide pro-peptide region of Saccharomyces cerevisiae were chimerized to obtain a co-translational translocation chimeric signal peptide sequence; The amino acid sequence of the co-translational translocation chimeric signal peptide and the amino acid sequence of the canine serum albumin mature peptide were codon-optimized to obtain the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide, respectively; Chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide to obtain a gene encoding canine serum albumin; The gene encoding canine serum albumin was connected to an expression vector to obtain a recombinant plasmid; The recombinant plasmid was treated with enzymes and then transformed into a host bacterium to obtain a canine serum albumin recombinant engineered bacterium.

9. The method for preparing canine serum albumin recombinant engineering bacteria according to claim 8, characterized in that: Before "chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide", the following operations are performed: A Lys-Arg sequence recognized and cleaved by endopeptidase Kex2 is inserted between the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide.

10. Use of the canine serum albumin recombinant engineered bacterium according to claim 7, or the canine serum albumin recombinant engineered bacterium prepared by the preparation method of the canine serum albumin recombinant engineered bacterium according to any one of claims 8 to 9, in the preparation of canine serum albumin and canine serum albumin biological products.

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