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 application relates to the technical field of genetic engineering, and particularly relates to a coding gene of canine serum albumin, an expression vector, a recombinant engineering bacterium, a preparation method and application of the recombinant engineering bacterium of canine serum albumin. BACKGROUND

[0002] Canine serum albumin (CSA) is the most abundant protein in canine plasma, accounting for 40-60% of the total plasma protein. CSA has multiple physiological functions, the main function is to maintain the stability of plasma colloid osmotic pressure, accounting for 75-80% of plasma colloid osmotic pressure, other physiological functions also include transporting various metabolites and drugs, maintaining acid-base balance, supporting the immune system, and helping to maintain blood volume, and has important application value in veterinary clinics. CSA is a single polypeptide chain composed of 584 amino acids, the whole molecule is arranged by three similar domains to form a unique heart-shaped structure. CSA has 35 Cys residues, forming 17 pairs of disulfide bonds, among which Cys-34 is the only free thiol that does not participate in the formation of disulfide bonds, and is also the main source of intravascular reductive thiol.

[0003] Canine serum albumin deficiency is also called hypoproteinemia, the causes of which include decreased ability of liver to synthesize albumin, secondary diseases, insufficient protein intake, absorption disorders or excessive protein consumption (such as massive blood loss, severe burns, heat disease, etc.). The main manifestations are decreased plasma protein and decreased colloid osmotic pressure, water leakage in blood vessels, and severe cases may appear systemic edema and decreased resistance, and secondary infection is easy to occur. With the rapid development of the pet market in China, the medical needs of pet dogs continue to grow, and recombinant canine serum albumin has potential application value in the aspects of clinical treatment of canine hypoproteinemia, canine hypovolemia, and veterinary drug production adjuvant.

[0004] Clinical canine serum albumin is generally a protein biological product extracted from healthy canine plasma, and is usually used for treating hypoproteinemia, malnutrition, edema caused by hypoproteinemia and acute blood loss, etc. The preparation methods mainly include salting-out method, organic solvent precipitation method and heat shock method, and have defects of low albumin yield and low purity. In addition, the supply gap of canine plasma itself, the extremely complex serum source and the risk of blood virus transmission through drugs all seriously limit the industrialized production. The use of genetic engineering technology to prepare recombinant canine serum albumin (rCSA) can overcome these difficulties. In particular, Pichia pastoris as a host fungus has the advantages of simple culture method, low endotoxin content, high expression level, post-translational modification system, easy large-scale culture and the like, and can directly secrete the target product into the culture medium, which is an ideal host system for heterologous expression of canine serum albumin.

[0005] The recombinant canine serum albumin expressed by Pichia pastoris in the prior art has the same physicochemical characteristics as natural canine serum albumin. In addition, the 97% purity canine serum albumin prepared by recombinant Pichia pastoris in the prior art has the same treatment effect as recombinant human blood albumin in treating rat cirrhosis ascites. In the expression of recombinant canine serum albumin by Pichia pastoris, the signal peptide is an important factor affecting the expression of foreign proteins in Pichia pastoris. The commonly used signal peptide in the prior art is the alpha mating factor signal peptide (alpha-factor) derived from Saccharomyces cerevisiae, which promotes the post-translational translocation of proteins to the endoplasmic reticulum, but also has certain limitations. If the alpha mating factor signal peptide is fused with the protein folded in the cytoplasm of the yeast cell, the protein may not be able to pass through the endoplasmic reticulum membrane to enter the secretion pathway, thereby affecting the secretion and expression of canine serum albumin. SUMMARY

[0006] In view of the problems of low expression amount and high degradation of canine serum albumin in the prior art, the present application provides a canine serum albumin coding gene, an expression vector, a recombinant engineering bacterium, a preparation method and application of the canine serum albumin recombinant engineering bacterium.

[0007] The technical solution adopted by the present application to solve the above technical problems is as follows: On the one hand, the present application provides a canine serum albumin coding gene, which comprises a co-translational translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence. The co-translational translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region of Saccharomyces cerevisiae and an alpha-factor signal peptide pro-peptide region. The Ost1 signal peptide pre-peptide region and the alpha-factor signal peptide pro-peptide region are chimeric to obtain the co-translational translocation chimeric signal peptide sequence.

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

[0009] Optionally, the co-translational translocation chimeric signal peptide sequence is selected from a nucleotide sequence as 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 a nucleotide sequence as 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 of endopeptidase Kex2, is inserted between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence.

[0011] In another aspect, the present application provides an expression vector of canine serum albumin, comprising an expression vector and the coding gene of the canine serum albumin.

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

[0013] In another aspect, the present application provides a recombinant engineering bacterium of canine serum albumin, comprising a host bacterium and the expression vector of the canine serum albumin. The host bacterium comprises one or more of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 and Pichia pastoris SMD1168.

[0014] Optionally, the preparation method of the recombinant engineering bacterium of canine serum albumin comprises the following operations: Chimerizing the pre-peptide region of Ost1 signal peptide and the pro-peptide region of α-factor signal peptide of Saccharomyces cerevisiae to obtain a co-translational translocation chimeric signal peptide sequence; Codon optimizing the amino acid sequence of the co-translational translocation chimeric signal peptide and the amino acid sequence of canine serum albumin mature peptide to respectively obtain a nucleotide sequence of the co-translational translocation chimeric signal peptide and a nucleotide sequence of canine serum albumin mature peptide; Chimerizing the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of canine serum albumin mature peptide to obtain a coding gene of canine serum albumin; The coding gene of canine serum albumin is connected to an expression vector to obtain a recombinant plasmid; The recombinant plasmid is subjected to enzyme digestion and then transformed into a host bacterium to obtain a canine serum albumin recombinant engineering bacterium.

[0015] Optionally, before the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide are chimerized, the following operation is performed: A Lys-Arg sequence 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 and is recognized and cut by an endopeptidase Kex2.

[0016] In another aspect, the canine serum albumin recombinant engineering bacterium or the canine serum albumin recombinant engineering bacterium prepared by the preparation method of the canine serum albumin recombinant engineering bacterium is used in the preparation of canine serum albumin and canine serum albumin biological products.

[0017] The present application has the following advantages: The coding gene of canine serum albumin provided by the present application comprises a co-translational translocation chimeric signal peptide and 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 alpha-factor signal peptide. The sequence of the co-translational translocation chimeric signal peptide and the sequence of the canine serum albumin mature peptide are subjected to codon optimization to form the coding gene of canine serum albumin. In the coding gene, the pre-peptide region of the Ost1 signal peptide and the pro-peptide region of the alpha-factor signal peptide are complementary to each other. The pre-peptide region of the Ost1 signal peptide is accurately recognized by the signal recognition particle in the cytoplasm due to its unique amino acid sequence and structural characteristics. After binding, the translation is paused and the ribosome-polypeptide chain complex is transported to the endoplasmic reticulum, guiding the recombinant canine serum albumin to efficiently pass through the endoplasmic reticulum channel and avoiding premature misfolding in the cytoplasm. The pro-peptide region of the alpha-factor signal peptide plays a role in the endoplasmic reticulum, ensuring the correct folding and modification of the recombinant canine serum albumin entering the endoplasmic reticulum, guiding the canine serum albumin to be transported to the Golgi body through vesicles, and then being secreted to the outside of the cell after further processing and modification. That is, the synergistic effect of the pre-peptide region of the Ost1 signal peptide and the pro-peptide region of the alpha-factor signal peptide in the co-translational translocation chimeric signal peptide sequence is utilized to realize the efficient secretion and expression of canine serum albumin in Pichia pastoris cells. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1is a restriction enzyme electrophoretogram of the recombinant plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS provided by the application; (M is DL5000 DNA Marker, 1 is BstBI and NotI double enzyme digestion, 2 is XhoI and NotI double enzyme digestion, and 3 is SacI single enzyme digestion) Figure 2 is a PCR electrophoretogram of the recombinant engineering bacteria pPICZB-pre-Ost1-pro-α-factor-rCBS / X-33 of canine serum albumin provided by the application; (M is DL5000 DNA Marker; 1-3 are PCR amplification products of the engineering bacteria) Figure 3 is a SDS-PAGE electrophoretogram of shake flask comparison experiments of the recombinant engineering bacteria of canine serum albumin provided by the application; (M is PageRuler™ Prestained Protein Ladder, 1-3 are expression conditions of the recombinant engineering bacteria pPICZαA-rCBS / X-33, and 4-6 are expression conditions of the recombinant engineering bacteria pPICZB-pre-Ost1-pro-α-factor-rCBS / X-33) Figure 4 is a result analysis electrophoretogram of the recombinant engineering bacteria of canine serum albumin after purification provided by the application; (M is PageRuler™ Prestained Protein Ladder; 1-2: purified recombinant canine serum albumin samples) Figure 5 The HPLC detection spectrum of the recombinant engineering bacteria of canine serum albumin provided by the application. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

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

[0021] Specifically, the application provides a canine serum albumin encoding gene, which comprises 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 a pre-peptide region of an Ost1 signal peptide and a pro-peptide region of an alpha-factor signal peptide of Saccharomyces cerevisiae. The canine serum albumin encoding gene is obtained by codon optimization of the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence. In the canine serum albumin encoding gene, the pre-peptide region of the Ost1 signal peptide and the pro-peptide region of the alpha-factor signal peptide are complementary to each other. The pre-peptide region of the Ost1 signal peptide is accurately recognized by a signal recognition particle in the cytoplasm by virtue of a unique amino acid sequence and structural characteristics, and after binding, translation is paused and the ribosome-polypeptide chain complex is transported to the endoplasmic reticulum, guiding the recombinant canine serum albumin to efficiently pass through the endoplasmic reticulum channel and avoiding premature misfolding in the cytoplasm. The pro-peptide region of the alpha-factor signal peptide functions in the endoplasmic reticulum, ensuring correct folding and modification of the recombinant canine serum albumin entering the endoplasmic reticulum, guiding the canine serum albumin to be transported to the Golgi apparatus by vesicles, and then secreted to the outside of the cell after further processing and modification. That is, by virtue of the synergistic effect of the pre-peptide region of the Ost1 signal peptide and the pro-peptide region of the alpha-factor signal peptide in the co-translational translocation chimeric signal peptide sequence, efficient secretion and expression of the canine serum albumin in Pichia pastoris cells are achieved.

[0022] In some embodiments, the co-translational translocation chimeric signal peptide sequence is selected from a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO. 1; and / or, The canine serum albumin mature peptide sequence is selected from a nucleotide sequence encoding an amino acid sequence as 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 the co-translational translocation chimeric signal peptide, which guides the canine serum albumin to quickly pass through the endoplasmic reticulum, avoids premature folding and degradation of the protein, and ensures efficient transport. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 3 is selected as the canine serum albumin mature peptide sequence, which can stably express the target protein with complete biological function, and is conducive to ensuring stable and efficient secretion and expression of the canine serum albumin in Pichia pastoris.

[0024] In particular, 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 canine serum albumin mature peptide shown in SEQ ID NO. 3 is: EAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACAAEESGANCDKSLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDDNPGFPPLVAPEPDALCAAFQDNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGVFAECCQAADKAACLGPKIEALREKVLLSSAKERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVHKECCHGDLLECADDRADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLYEYARRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKVGTKCCKKPESERMSCAEDFLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCFSGLEVDETYVPKEFNAETFTFHADLCTLPEAEKQVKKQTALVELLKHKPKATDEQLKTVMGDFGAFVEKCCAAENKEGCFSEEGPKLVAAAQAALV.

[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 90% or more 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 90% or more homology with SEQ ID NO. 4.

[0026] Specifically, the nucleotide sequence represented by SEQ ID NO. 2 or a nucleotide sequence having more than 90% homology with SEQ ID NO. 2 is selected as the co-translational translocation chimeric signal peptide sequence, which can utilize its signal recognition and endoplasmic reticulum transport function, and can enhance the stability of the gene through homologous sequences to avoid the problem of reduced transport efficiency caused by gene mutation; the nucleotide sequence represented by SEQ ID NO. 4 or a nucleotide sequence having more than 90% homology with SEQ ID NO. 4 is selected as the canine serum albumin mature peptide sequence, which can adapt to the Pichia pastoris translation system through codon degeneracy while keeping the amino acid of the protein core functional region unchanged, and the homologous sequence can tolerate base substitution to reduce the expression obstacles caused by host replication preference or synthesis process, and at the same time realize 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 represented by SEQ ID NO. 2 is: atgagacaggtgtggttctcttggattgtgggactgttcttgtgtttcttcaacgtcagttctgctgctcctgttaacactactaccgaagatgaaactgctcagataccagccgaagccgttataggttactcagaccttgaaggagactttgatgtcgcagtgttgccattctctaactccactaacaacggactgttgttcataaacacaactatcgcttccatcgctgctaaagaagagggagtatccctcgagaaaaga;

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

[0029] Specifically, the insertion of the Lys-Arg cleavage site, a recognition site of endopeptidase Kex2, between the co-translational translocation chimeric signal peptide sequence and the canine serum albumin mature peptide sequence allows the specific recognition and cleavage of the site by endopeptidase Kex2 in the endoplasmic reticulum of Pichia pastoris, achieving efficient separation of the signal peptide and the mature peptide, and thus completely removing the signal peptide sequence from the secreted target protein, ensuring that the amino acid sequence of the target protein is 100% identical to that of the native canine serum albumin mature peptide, and avoiding the influence of the uncut chimeric peptide chain on the spatial folding and biological activity of canine serum albumin, thereby 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 for canine serum albumin, comprising an expression vector and a coding gene for the canine serum albumin.

[0031] Specifically, the canine serum albumin expression vector constructs a recombinant plasmid by combining the coding gene with the expression vector, drives high-efficiency transcription of the gene by a strong promoter of the expression vector, ensures translation efficiency by a ribosome binding site, stabilizes the transcription product by a terminator, and adapts the signal peptide guide sequence to the endoplasmic reticulum and Golgi secretion pathway to 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, in the preparation of the canine serum albumin expression vector, pGAPZB, pPICZB, pPIC3.5K, and pAO815 can be selected as the expression vector. Among the above expression vectors, the expression efficiency of canine serum albumin can be improved by taking advantage of each of them. Specifically, the pPICZB and pPIC3.5K carry a methanol-inducible AOX1 promoter, which can be used to accurately control the expression amount by adjusting the methanol concentration, thereby avoiding the accumulation of toxic proteins. The pAO815 contains multiple copy integration sites, which can increase the copy number of the target gene and enhance the expression strength.

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

[0035] Another embodiment of the present application provides a recombinant engineering bacterium for canine serum albumin, comprising a host bacterium and the expression vector for canine serum albumin. The host fungus includes 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 engineering bacteria realize the combination of gene expression and protein production by introducing the canine serum albumin expression vector into the Pichia pastoris host fungus. The expression vector carries elements such as promoters, selection markers and signal peptides, which can drive efficient transcription and translation of the canine serum albumin coding gene, and at the same time, the selection marker can quickly enrich positive transformants. The Pichia pastoris host fungus has protein folding, modification and secretion mechanisms, which can correctly process and secrete recombinant proteins to the extracellular, thereby improving the expression efficiency of recombinant proteins.

[0037] Pichia pastoris X-33 has high homologous recombination ability, which can ensure the stable integration of the expression vector into the genome and reduce genetic drift; GS115 has a complete methanol utilization pathway, which can realize the high expression of canine serum albumin by using the methanol inducible promoter of pPICZB and other vectors; KM71 is a slow methanol utilization strain, which is suitable for proteins that are toxic to the host or need to be expressed slowly, thereby reducing metabolic pressure; SMD1168 lacks intracellular proteases, which can significantly reduce the degradation of recombinant proteins, thereby ensuring the integrity and activity of canine serum albumin.

[0038] In some embodiments, the preparation method of the canine serum albumin recombinant engineering bacteria comprises the following operations: The Ost1 signal peptide pre-peptide region and the alpha-factor signal peptide pro-peptide region of Saccharomyces cerevisiae are 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 are 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; The nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide are chimerized to obtain the coding gene of canine serum albumin; The coding gene of canine serum albumin is connected to an expression vector to obtain a recombinant plasmid; The recombinant plasmid is subjected to enzyme cutting treatment and then transformed into a host fungus to obtain canine serum albumin recombinant engineering bacteria.

[0039] A Lys-Arg sequence recognized and cut 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 a specific operation, according to the codon bias of Pichia pastoris, the upstream enzyme cutting site is BstB I, the downstream enzyme cutting site is Not I, the endopeptidase Kex2 recognition and cutting site Lys-Arg is added between the co-translational translocation chimeric signal peptide nucleotide sequence and the mature peptide nucleotide sequence of canine serum albumin, and the nucleotide sequence of the coding gene pre-Ost1-pro-α-factor-rCBS of canine serum albumin is synthesized. The synthesized coding gene of canine serum albumin is directionally connected to the vector pPICZαB through the enzyme cutting site BstB I+Not I to construct the recombinant plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS. The recombinant plasmid is linearized by endonuclease Sac I, and then is electroporated into Pichia pastoris X-33 competent cells, and the recombinant engineering bacteria of canine serum albumin are obtained after screening on a YPDS medium containing 100 μg / mL Zeocin.

[0041] Specifically, in the preparation method of the recombinant engineering bacteria of canine serum albumin, firstly, the chimeric of Ost1 signal peptide pre-peptide and α-factor signal peptide pro-peptide utilizes the high co-translational translocation ability of Ost1 to efficiently guide the protein into the endoplasmic reticulum, and retains the transport guiding function of α-factor, and utilizes the transport function to secrete the protein to the extracellular, secondly, the codon optimization process adjusts the nucleotide sequence for Pichia pastoris, thirdly, the co-translational translocation avoids the incorrect folding and degradation of the protein in the cytoplasm, and the precise cutting of the Kex2 cutting site ensures that the mature peptide sequence is 100% consistent with the natural protein, the selection of the suitable expression vector and the transformation of the Pichia pastoris host bacteria realizes the efficient integration of the gene, and further promotes the controllable expression and stable secretion of the recombinant canine serum albumin in the host bacteria.

[0042] In some embodiments, before the "chimerization of the co-translational translocation chimeric signal peptide nucleotide sequence and the canine serum albumin mature peptide nucleotide sequence", the following operation is performed: Specifically, the 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, and in the expression process of Pichia pastoris, the Kex2 enzyme can specifically recognize the site, and when the recombinant protein enters the endoplasmic reticulum, the Kex2 enzyme can efficiently and accurately cut the connection site (N-terminal signal peptide) between the signal peptide and the mature peptide, ensuring that the secreted canine serum albumin mature peptide is completely consistent with the natural sequence, and avoiding the influence of the residual signal peptide on the structure and function of the protein.

[0043] In some embodiments, the recombinant engineering bacteria of canine serum albumin provided in the present application includes a fermentation operation, and the specific fermentation operation includes the following flow: The single colony of the recombinant engineering bacteria of canine serum albumin obtained by the shake flask screening is subjected to high-density fermentation through four stages of seed culture solution culture, cell growth, glycerol supplement and methanol induction; the seed culture solution is added into the fermentation tank, ammonia water is used to adjust the pH to 5.0, and the temperature is controlled at 30°C; at the end of the glycerol supplement, the pH is adjusted to 5.85, and the temperature is controlled at 25°C to enter the methanol induction stage; the dissolved oxygen is greater than 20% by adjusting the rotation speed, tank pressure, air flow and methanol flow rate, and the fermentation is completed after 96 hours of induction.

[0044] In some embodiments, the application provides a method for purifying canine serum albumin from the recombinant engineering bacteria of canine serum albumin, comprising: subjecting the fermentation liquor of the recombinant engineering bacteria of canine serum albumin after fermentation to solid-liquid separation, and purifying the canine serum albumin obtained by hydrophobic chromatography and anion exchange chromatography from the supernatant; and the purity of the canine serum albumin after separation is greater than 99.125%.

[0045] The application provides the use of the recombinant engineering bacteria of canine serum albumin or the recombinant engineering bacteria of canine serum albumin prepared by the preparation method of the recombinant engineering bacteria of canine serum albumin in the preparation of canine serum albumin and canine serum albumin biological products.

[0046] Specifically, the engineering bacteria obtained by the recombinant engineering bacteria of canine serum albumin and the preparation method thereof are used in the field of canine serum albumin and related biological product preparation. The engineering bacteria realize efficient and stable expression of canine serum albumin through codon optimization, chimeric signal peptide design and adaptation of vectors and host systems, which is conducive to the production of canine serum albumin with high purity and high activity, can meet the demand for standard canine serum albumin in scientific research, and can be used as a core raw material to prepare biological products such as canine plasma substitutes, drug carriers and biological diagnostic reagents.

[0047] In some embodiments, the application also provides a method for purifying the recombinant engineering bacteria of canine serum albumin The application is further described below by way of examples.

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

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

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

[0051] BSM inorganic salt medium (1L): 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 6.0 g, MnSO4•H2O 3.0 g, Na2MoO4•2H2O 0.2 g, KI 0.088 g, H3BO3 0.02 g, CoCl2•6H2O 0.5 g, ZnCl2 0.0 g, FeSO4•7H2O 65.0 g, Biotin 0.2 g, concentrated H2SO4 5.0 ml.

[0053] Example 1 This example is used to illustrate the preparation of the coding gene of canine serum albumin and the expression vector disclosed in the present application, which comprises the following operation steps: Gene synthesis: According to the codon preference of Pichia pastoris, the factors of GC content, codon usage frequency, and RNA nuclease splicing sites, and RNA stability trans-acting elements are balanced, and the tandem gene pre-Ost1-pro-α-factor-rCBS of the co-translational translocation chimeric signal peptide nucleotide sequence SEQ ID NO. 2 and the canine serum albumin nucleotide sequence such as SEQ ID NO. 4 is artificially synthesized; Amplification of the target gene: The synthetic canine serum albumin coding gene pre-Ost1-pro-α-factor-rCBS is used as a template, and primers P1 and P2 are used for PCR amplification, and the target gene fragment is recovered by gel; The gene sequence of primer P1 is ttattcgaagccaccatgagacaggtgtggttc, and the gene sequence of primer P2 is ctggcggccgcttaaaccaaagcagcttg. Construction of recombinant plasmid: The gel recovery product and the vector plasmid pPICZB are digested with BstBI and NotI respectively, and connected by T4 DNA ligase at 16°C for 1h, and transformed into competent cells DH10b by heat shock, and coated on Zeocin-resistant LLB plates, and the plasmid was extracted and identified by enzyme digestion, and the electrophoresis detection result as shown in Figure 1 ​The BstBI+NotI double enzyme cutting product fragments are 2031 bp + 3268 bp, the XhoI+NotI double enzyme cutting product fragments are 1768 bp + 3531 bp, the SacI single enzyme cutting product fragment is 5299 bp, and the positive plasmid is named as pPICZB-pre-Ost1-pro-α-factor-rCBS. Figure 1 The electrophoresis test result of the control

[0054] Example 2 This example is used to illustrate the canine serum albumin recombinant engineering bacteria disclosed by the application, and includes the following operation steps. Electric shock transformation: The endonuclease SacI linearizes the plasmid pPICZB-pre-Ost1-pro-α-factor-rCBS, 1-10 μg of the linearized plasmid is added into 80 μl of Pichia pastoris X-33 competent cells, mixed uniformly, transferred into a 2 mm electric transformation cup, and then placed in an ice bath for 5 min; the electric shock instrument parameters are set as 1.5 KV, 200 Ω, and 25 μF; 1 ml of pre-cooled 1 M / L sorbitol solution is immediately added into the transformation cup after electric transformation, the mixture is transferred into a sterilized centrifuge tube, and then placed in a 30℃ incubator for 1 h; 200 μl of the bacterial solution is spread on a Zeocin-resistant (100 μg / ml) YPDS plate, and then cultured at 30℃ for 2-5 days until single colonies appear; PCR identification of the recombinant: The single colonies grown on the YPDS plate are picked up, and the genomic DNA is extracted therefrom to serve as a template for PCR identification; the PCR reaction system is 10×Taq buffer 5 μl, dNTP Mixture 4 μl, template 1 μl, 5'AOX1 and 3'AOX1 primers each 1 μl, Taq polymerase 1 μl, and sterilized double-distilled water 37 μl; the reaction parameters are 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and 72℃ re-extension for 10 min; the PCR product is then detected to obtain Figure 2 the detection result shown in the figure; It can be known from the agarose gel electrophoresis detection of Figure 2 that the PCR amplification band contains the target gene fragment, and the size thereof is about 2100 bp, which is consistent with the theoretically expected molecular weight.

[0055] Example 3 This example is used to illustrate the induction expression of the canine serum albumin recombinant engineering bacteria disclosed by the application, and includes the following operation steps. The recombinant engineering bacteria of canine serum albumin preserved in glycerol were inoculated into 50 ml YPD medium at an inoculation amount of 1%, and cultured at 30°C and 200 rpm until OD=2-6. The bacteria were collected by centrifugation at 1500 g for 5 min, and then suspended in 200 ml BSM inorganic medium, and cultured at 30°C and 200 rpm. The final concentration of methanol (500 ml of methanol and 6 ml of PMT1) was added every 24 h for induction of expression. The fermentation supernatant was detected by SDS-PAGE electrophoresis to obtain the high-expression engineering bacteria; Influence of different signal peptides on expression of target proteins: One high-expression recombinant engineering bacteria pPICZB-pre-Ost1-pro-α-factor-rCBS / X-33 of canine serum albumin and one control high-expression engineering bacteria pPICZαA-rCBS / X-33 were selected for induction expression under the same conditions. Three parallel experiments were set up. The results of SDS-PAGE electrophoresis detection were analyzed by using ImageJ software, and the results are shown in Table 1. Figure 3 As shown in Table 1 and Figure 3 compared with the signal peptide α-factor, the chimeric signal peptide increased the target band of canine serum albumin by 2.63 times and reduced the degradation degree by 29.5%, which further proved that the recombinant engineering bacteria of canine serum albumin provided by the application can be efficiently secreted and expressed in the host.

[0056] Example 4 This example is used to illustrate the high-density fermentation of the recombinant engineering bacteria of canine serum albumin disclosed in the application, which comprises the following operation steps: Seed culture stage: The recombinant engineering bacteria were inoculated into YPD medium, and cultured at 30°C and 220 rpm for 20 h to obtain seed liquid. The wet weight of the bacteria was measured and microscopic examination was performed. Bacterial growth stage: The seed liquid was inoculated into a fermentation tank containing 20 L of BSM inorganic salt medium. The temperature was set to 30°C, the rotation speed was 500-700 rpm, the aeration was 20 L / min, and the dissolved oxygen was higher than 20%. The pH was maintained at 5.0 mainly by adjusting ammonia water. Glycerol feeding stage: When the dissolved oxygen suddenly rose, 50% glycerol containing 12 ml / L PTM1 was added at a rate of 18 ml / h / L. The parameters were maintained unchanged until the wet weight of the bacteria reached 240 g / L. Methanol induction stage: ​When the dissolved oxygen of the fermentation again rapidly rises, the temperature is adjusted to 25℃, the pH is adjusted to 6.0, and within 1 hour after entering the induction stage, no carbon source is added in the tank to ensure complete consumption of glycerol. Methanol containing 12 ml / L PTM1 is added for induction, and the dissolved oxygen is controlled to be higher than 30%. After 72 h of induction, the fermentation is ended. The protein content in the fermentation broth is 10.12 g / L, which is determined by the Coomassie brilliant blue staining method.

[0057] Example 5 This example is used to illustrate the purification of canine serum albumin in the recombinant engineering bacteria of canine serum albumin disclosed by the present application, which comprises the following operation steps: Heat treatment: the fermentation broth cultured above is centrifuged (6000 rpm, centrifugation for 20 minutes) to collect the supernatant, and the protease is inactivated by heating at 72℃ for 30 minutes in a metal bath to avoid degradation of the target protein; Hydrophobic chromatography: after heating, the sample is filtered through a 0.45 μm membrane, equilibrated with Butyl hydrophobic column for 5 column volumes, loaded, and the flow-through peak is collected and concentrated to rCSA concentration of 100 mg / ml using a molecular cut-off membrane of 30000; Anion chromatography: the DEAE6FF anion exchange chromatography column is equilibrated with equilibration buffer (20 mmol / L Tris-HCI, 0.1 mol / L sodium chloride, pH 7.4) for 5 column volumes, loaded, eluted using elution buffer (20 mmol / L Tris-HCl, 0.5 mol / L, sodium chloride, pH 7.4), and the elution fraction containing rCSA is collected and detected by SDS-PAGE electrophoresis to obtain Figure 4 ; As shown in Figure 4 , lanes 1 and 2 are single bands, indicating that the purification degree of canine serum albumin in the recombinant engineering bacteria is high, and further HPLC detection of the target protein is carried out, as shown in Figure 5 , the protein purity after two-step purification can reach 99.12%.

[0058] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gene encoding canine serum albumin, characterized in that, The co-translational 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 is chimeric with the alpha-factor signal peptide pro-peptide region to obtain the 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 a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO. 1; and / or, The canine serum albumin mature peptide sequence is selected from a nucleotide sequence encoding an amino acid sequence as 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 a nucleotide sequence as 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 a nucleotide sequence as 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 Kex2 endopeptidase recognition and cleavage site Lys-Arg sequence 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 by comprising a nucleotide sequence represented by SEQ ID NO:

1. The expression vector and the coding gene of the canine serum albumin as claimed in any one of claims 1-4.

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

7. A recombinant engineering bacteria of canine serum albumin, characterized in that, The expression vector of the canine serum albumin as claimed in claim 5; The host bacteria comprise one or more of Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71 and Pichia pastoris SMD1168.

8. The method for preparing a recombinant engineering bacteria of canine serum albumin according to claim 7, characterized in that, The following operations are included: The Ost1 signal peptide pre-peptide region and the alpha-factor signal peptide pro-peptide region of Saccharomyces cerevisiae are chimeric to obtain the co-translational translocation chimeric signal peptide sequence; The amino acid sequence of the co-translational translocation chimeric signal peptide and the canine serum albumin mature peptide are 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; The nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide are chimeric to obtain the coding gene of the canine serum albumin; The coding gene of the canine serum albumin is connected to the expression vector to obtain a recombinant plasmid; The recombinant plasmid is subjected to enzyme cutting treatment and then transformed into the host bacteria to obtain the canine serum albumin recombinant engineering bacteria.

9. The method for preparing recombinant engineered bacteria for canine serum albumin according to claim 8, characterized in that, Before the nucleotide sequence of the co-translational translocation chimeric signal peptide and the nucleotide sequence of the canine serum albumin mature peptide are chimeric, the following operations are performed: A Kex2 endopeptidase recognition and cleavage Lys-Arg sequence 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. The recombinant engineered bacteria of canine serum albumin according to claim 7, or the recombinant engineered bacteria of canine serum albumin prepared by the method according to any one of claims 8-9, for use in the preparation of canine serum albumin and biological products of canine serum albumin.

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