Cat serum albumin, separation method and application thereof

Abstract

The present invention provides feline serum albumin, its isolation method, and applications, relating to the field of genetic engineering technology. This invention, for the first time, mutates feline serum albumin to obtain beneficial mutation sites, thereby producing a feline serum albumin mutant that can be highly expressed in a Pichia pastoris system. Furthermore, the feline serum albumin mutant L94H not only exhibits excellent antioxidant effects but also significantly increases albumin concentration in cats, offering promising applications in treating feline hypoproteinemia, cirrhosis, ascites, and other conditions, or alleviating liver damage.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a cat serum albumin and a separation method and application thereof. Background Art

[0002] As people's living standards improve, more and more families are paying more attention to the health of their companion pets. Research in pet medicine and nutrition is also deepening. Albumin, a key blood protein, has potential applications in pet health management due to its antioxidant and physiological regulatory properties.

[0003] Feline serum albumin (FSA) is the most abundant and versatile protein in cat serum. It is primarily used clinically to treat conditions such as shock caused by blood loss or burns, hypoalbuminemia, and edema caused by liver cirrhosis and kidney disease. Currently, most serum albumin preparations used in clinical treatment of cats use either FSA extracted from blood or human serum albumin (HSA). However, the insufficient supply of feline serum, the complex serum source, and the risk of viral transmission through blood have severely limited the industrial production of FSA. Furthermore, the infusion of heterologous HSA can trigger specific allergic reactions, which can be harmful to the cat's health or even life-threatening. Therefore, the development of efficient, safe, and scalable FSA production technology has become a critical issue in the field of pet healthcare. Summary of the Invention

[0004] The purpose of the present invention is to provide a cat serum albumin with good anti-oxidation effect.

[0005] On the one hand, the present application provides a feline serum albumin, which is obtained by mutating the 94th position of the reference sequence, and the reference sequence is shown in SEQ ID No. 1.

[0006] Wherein, the reference sequence is the feline serum albumin sequence with GenBank No. NM_001009961, the amino acid sequence of the reference sequence is shown in SEQ ID No. 1, and the nucleotide sequence encoding the reference sequence includes the sequence shown in SEQ ID NO. 2 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 2.

[0007] Furthermore, the feline serum albumin is obtained by replacing the leucine (L) residue at position 94 of the reference sequence with a histidine (H) residue.

[0008] In a preferred embodiment, the feline serum albumin comprises the amino acid sequence shown in SEQ ID NO.3.

[0009] On the other hand, the present application also provides a biomaterial, which comprises any one of the following A1) to A5):

[0010] A1) a nucleic acid molecule encoding the feline serum albumin;

[0011] A2) an expression cassette, said expression cassette comprising the nucleic acid molecule described in A1);

[0012] A3) a recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0013] A4) a recombinant microorganism, wherein the recombinant microorganism comprises the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3);

[0014] A5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

[0015] In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule described in A1) comprises the sequence shown in SEQ ID NO.4 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.4.

[0016] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 includes the sequence shown in SEQ ID NO.4 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.4.

[0017] The expression cassette described herein may further include functional elements such as a promoter, a terminator, and a marker gene. Those skilled in the art may make routine selections based on actual conditions, as long as they can achieve expression of the nucleic acid molecule in A1). The structure and composition of the expression cassette are not further restricted herein.

[0018] Vectors described herein refer to vectors that can carry exogenous DNA or target genes into host cells for amplification and expression, and the vectors can be any vectors (such as plasmids or viruses) that are convenient for recombinant DNA manipulation and expression of nucleic acid sequences. The selection of vectors generally depends on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can include any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or transposons, that generally comprise the entire DNA that will be introduced into the host cell genome, can be included. Those skilled in the art can select according to actual conditions and are not subject to excessive restrictions.

[0019] In a preferred embodiment, the vector may be pPIC9K.

[0020] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0021] In a preferred embodiment, the recombinant microorganism may be Pichia pastoris, specifically Pichia pastoris ( Pichia pastoris )GS115.

[0022] Those skilled in the art can use known gene editing methods or gene editing systems to complete the construction of the above-mentioned biomaterials.

[0023] On the other hand, the present application also provides the use of the biomaterial in preparing feline serum albumin.

[0024] On the other hand, the present application also provides the use of the feline serum albumin or the biomaterial in any of the following aspects:

[0025] C1) Application in the preparation of antioxidant products;

[0026] C2) Application in the preparation of products for increasing albumin content in animals;

[0027] C3) Use in the preparation of products for treating and / or preventing hypoalbuminemia and / or ascites in animals.

[0028] Furthermore, the treatment and / or prevention of hypoalbuminemia and / or cirrhosis-induced ascites disease comprises increasing the albumin (ALB) concentration in the subject.

[0029] Preferably, the subject to be treated is a mammal; more preferably, the subject to be treated is a cat.

[0030] On the other hand, the present application also provides a method for preparing feline serum albumin, which comprises: the method at least comprises constructing a recombinant microorganism that expresses the feline serum albumin and culturing the recombinant microorganism.

[0031] Furthermore, the method further comprises the step of separating and purifying the cultured recombinant microorganism fermentation broth. Preferably, the separation and purification step comprises the step of centrifuging the cultured recombinant microorganism fermentation broth and purifying it using a DEAE column.

[0032] Those skilled in the art can select a general method for fermentation to prepare feline serum albumin according to the characteristics of the recombinant microorganism.

[0033] In a preferred embodiment, a method for preparing feline serum albumin comprises:

[0034] Inoculate the recombinant microorganism into the culture medium and culture at 25℃-40℃, 100-300 rpm to obtain the OD 600 =9-11, centrifuge at 1000-2000 g for 1-10 min, discard the supernatant, add culture medium to resuspend the bacteria, culture at 25℃-40℃, 100-300 rpm, add 0.1%-0.5% methanol every 12-24 h, and induce for 36-72 h.

[0035] In a preferred embodiment, a method for preparing feline serum albumin comprises:

[0036] The recombinant microorganism was inoculated into the culture medium and cultured at 28°C and 210 rpm to obtain the OD 600 =10, centrifuge at 1500 g for 5 min, discard the supernatant, add culture medium to resuspend the bacteria, and culture at 28°C, 210 rpm. Add 0.5% methanol every 24 h for 72 h of induction.

[0037] Furthermore, the yield of feline serum albumin prepared by the method is greater than or equal to 2.5 g / L; preferably, greater than or equal to 3 g / L.

[0038] The present invention has the following beneficial effects:

[0039] The present invention mutates feline serum albumin to identify beneficial mutation sites, thereby producing a feline serum albumin mutant that can be highly expressed in the Pichia pastoris system. Furthermore, the feline serum albumin mutant L94H not only exhibits excellent antioxidant effects but also significantly increases albumin concentration in cats, offering promising applications in treating feline hypoproteinemia, cirrhosis with ascites, and other conditions, alleviating liver damage. DETAILED DESCRIPTION

[0040] Technical terms:

[0041] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution research.

[0042] Recombination: In a broad sense, any gene exchange process that causes genotype changes is called recombination.

[0043] Expression cassette: An expression cassette is a set of DNA sequences consisting of a promoter, target gene, and reporter gene that can be expressed in specific tissues and easily detected.

[0044] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0045] Recombinant microorganisms: Fungal cell lines that use genetic engineering methods to efficiently express exogenous genes.

[0046] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0047] In order to more clearly illustrate the overall concept of the application, the following is described in detail in the form of embodiments. In the following description, a large amount of specific details are provided to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be implemented without the need for one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0048] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0049] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0050] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0051] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercial products, and the specific operations were performed according to the kit instructions.

[0052] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0053] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0054] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0055] In this specification, the amino acids at the corresponding positions are represented by the generally accepted IUPAC single-letter abbreviations, wherein the amino acids and their abbreviations are: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).

[0056] In this specification, amino acid mutations are referred to as "original amino acid, position, and substituted amino acid." For example, a mutation of isoleucine L to leucine H at position 94, counting sequentially from the N (nitrogen) terminus toward the C (carbon) terminus, is represented by L94H.

[0057] Example 1: Construction and identification of recombinant plasmids

[0058] The feline serum albumin (FSA) sequence (GenBank: NM_001009961, amino acid sequence shown in SEQ ID No. 1) was retrieved from GenBank using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Structural modeling and screening for potential mutation sites were performed on this sequence. The amino acid at position 94 was initially selected, resulting in the mutant L94H. The amino acid sequence of L94H is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4.

[0059] Specifically, the FSA (GenBank: NM_001009961) gene sequence was delivered to Nanjing GenScript Biotechnology Co., Ltd. for optimization (the nucleotide sequence is shown in SEQ ID No. 2), chemically synthesized and cleaved by enzyme sites. SnaB Ⅰ and Not Ⅰ Inserted into the vector pPIC9K, the plasmid was constructed and named pPIC9K-FSA. Single site mutation primers (as shown in Table 1) were designed using pPIC9K-FSA as a template, and mutation was performed by inverse PCR. The amplified linear plasmid product was purified by gel recovery to obtain the recombinant plasmid pPIC9K-L94H. Sac I and Not I The pPIC9K-FSA and pPIC9K-L94H recombinant plasmids were double-digested. All samples were gently mixed and incubated at 37°C for 1-2 hours. The digested products were then identified by 1% agarose gel electrophoresis and sent to Qingke Bio for sequencing to confirm the correct product sequence.

[0060] Table 1 Mutation primers and mutant information

[0061]

[0062] Example 2: Construction and screening of engineered strains

[0063] In this example, the pPIC9K-FSA and pPIC9K-L94H recombinant plasmids obtained in Example 1 were electroporated into Pichia pastoris ( Pichia pastoris ) Construction of an engineered bacterium expressing feline serum albumin in the GS115 strain.

[0064] Specifically, pPIC9K-FSA, pPIC9K-L94H recombinant plasmids and restriction enzymes Sac I Place on ice and add 50 μl of DNA per 100 μl of PBS according to the DNA concentration and restriction endonuclease. Sac Ⅰ Follow the instructions, mix well, and incubate at 37°C for 1 hour. After enzyme digestion, identify the plasmid by 1% agarose gel electrophoresis. Recover the linearized plasmid using a DNA purification kit according to the manufacturer's instructions, and measure the DNA concentration. Mix 7 μg of the linearized plasmid with 80 μL of GS115 competent medium, transfer to a pre-chilled electroporation cuvette, mix thoroughly on ice for 10 minutes, and electrotransform at 1500 V, 25 μF, 200 Ω, and 5 ms. Add 1 mL of pre-chilled 1 M sorbitol to the suspended cells, transfer to a test tube, add 1 mL of YPD liquid medium, and incubate at 30°C and 150 rpm for 2 hours. Centrifuge at 5000 rpm for 5 min, discard part of the supernatant, aspirate part of the bacterial solution and spread it on MD plates, culture at 28°C for 2-4 days, spread it on YPD plates containing 3 mg / mL G418, and culture at 28°C for 2-4 days to obtain the engineered bacteria GS115-FSA and GS115-L94H recombinant plasmids expressing feline serum albumin. The engineered bacteria information is shown in Table 2.

[0065] The positive monoclonal colonies of the above engineering bacteria were picked up and cultured in BMGY liquid medium at 28°C and 210 rpm to obtain the OD 600 =10, centrifuge at 1500 g for 5 min, discard the supernatant, add 100 mL BMMY liquid medium to resuspend the bacteria, culture at 28°C, 210 rpm, add 0.5% methanol every 24 h, induction after 72 h, centrifuge the fermentation broth, and centrifuge at 4°C, 7000 rpm for 10 min.

[0066] The fermentation broth was purified using a DEAE column. Specifically, the DEAE column was installed in an SDL-100 purification system and equilibrated with 0.2 mM MPB. The sample was passed through the column at a flow rate of 0.5 mL / min. Protein was linearly eluted using 0.5 M NaCl as an eluent to fully elute the purification column. The column was then sealed with 20% ethanol to obtain purified feline serum albumin. The feline serum albumin concentration (yield) was determined using the BCA assay. The results are shown in Table 2.

[0067] The results are shown in Table 2.

[0068] Table 2 Engineering bacteria information

[0069]

[0070] As shown in Table 2, FSA can effectively increase the yield of feline serum albumin and improve the production efficiency of feline serum albumin by replacing the leucine (L) residue at position 94 with a histidine (H) residue in the direction from the N (nitrogen) end to the C (carbon) end of the sequence.

[0071] Example 3: Antioxidant Application of Feline Serum Albumin—Scavenging Free Radicals

[0072] The feline serum albumin L94H mutant obtained in Example 2 was used as a sample, and the feline serum albumin sequence (GenBank: NM_001009961) (FSA) was used as a control to conduct antioxidant tests.

[0073] (1) In vitro DPPH scavenging ability

[0074] FSA and FSA mutant L94H were diluted with PBS to 0.5, 1, and 2 mg / mL, respectively. For the experimental group, 100 μL of 0.1 mM DPPH solution and 100 μL of varying concentrations of FSA mutant L94H were added to a 96-well plate. The mixture was mixed and incubated in the dark at room temperature for 30 minutes. The mixture was then centrifuged (10,000 rpm, 5 minutes) to remove the precipitate (if the solution was turbid). The absorbance at 517 nm was measured. The blank control group consisted of 100 μL DPPH• + 100 μL ethanol (maximum absorbance was measured); the control group consisted of 100 μL DPPH• + 100 μL FSA. The DPPH• clearance rate was calculated as follows:

[0075]

[0076] The results are shown in Table 3.

[0077] Table 3 Results of cat serum albumin's ability to scavenge DPPH

[0078]

[0079] (2) ROS damage detection in cat hepatocyte model

[0080] Cat hepatocytes were seeded in 96-well plates (1 × 10 4 / well), cultured for 24 hours. Experimental group: replaced with fresh culture medium containing L94H mutant (0.5, 1, 2 mg / mL), pretreated for 2 hours. Add H2O2 (200 μM) and incubate for 4 hours. Discard the culture medium, wash once with PBS, add DCFH-DA (10 μM, incubate at 37°C for 30 min), add 10 μL CCK-8 reagent to each well, incubate at 37°C for 1-2 hours. Detect the OD value at 450 nm with a microplate reader. Blank control group: (no H2O2 and no FSA); damage group (H2O2, no FSA) positive control group (H2O2 + FSA) normal control group (no H2O2 and FSA). The cell survival rate is calculated as follows:

[0081]

[0082] The results are shown in Table 4.

[0083] Table 4 Results of cat serum albumin on reducing ROS damage in stem cells

[0084]

[0085] As shown in Tables 3 and 4, the feline serum albumin mutant L94H of the present invention has a good antioxidant effect and can effectively reduce the damage of ROS to the liver.

[0086] Example 4: Application of Feline Serum Albumin—Evaluation of the Effect of Treating Cirrhosis Ascites Model

[0087] The feline serum albumin L94H mutant obtained in Example 2 was used as a sample, and the feline serum albumin sequence (GenBank: NM_001009961) (FSA) was used as a control to conduct an experiment on treating liver cirrhosis and ascites.

[0088] 1. Use carbon tetrachloride (CCl4) and olive oil solution combined with sodium phenobarbital and alcohol to establish a rat cirrhosis ascites model:

[0089] Male SD rats were randomly divided into two groups: the model group received intraperitoneal injections of 40% CCl₄ and olive oil, and received 0.3 g / L of sodium phenobarbital in drinking water for the first two weeks, followed by 10% alcohol starting in the third week; the control group received intraperitoneal injections of olive oil solution and normal drinking water. Each experimental group received an intraperitoneal injection dose of 2 mL / kg twice weekly. Successful modeling was considered when rats in the experimental group showed positive ascites and pseudolobules on liver pathological sections.

[0090] 2. Methods and efficacy evaluation of feline serum albumin in treating liver cirrhosis ascites model:

[0091] The successfully established rat models were randomly divided into a negative control group, two positive control groups, and two experimental groups. The negative control group was given normal saline, the positive control group was given 20% rHSA and 20% FSA, respectively, and the two experimental groups were given 20% L94H mutant and 20% I288W mutant, respectively. The dose for each group was 1.5 mL, administered via tail vein bolus injection within 2 hours, once daily for one week. Rats were weighed before daily dosing. After the last dose, the rats were deprived of food and water, and blood was drawn from the fundus vein 18 hours later. ALB concentrations were measured using a kit from the Nanjing Jiancheng Bioengineering Institute, and the results are shown in Table 5.

[0092] Table 5 Effects of treating liver cirrhosis ascites model

[0093]

[0094] As shown in Table 3, the L94H mutant has a more excellent effect of increasing ALB concentration than the wild type or human serum albumin, and can effectively treat ascites and hypoalbuminemia caused by liver cirrhosis.

[0095] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A feline serum albumin, characterized in that The feline serum albumin is obtained by replacing the leucine (L) residue at position 94 of the reference sequence with a histidine (H) residue, and the reference sequence is shown in SEQ ID No.

1.

2. The cat serum albumin according to claim 1, wherein The amino acid sequence of the feline serum albumin is shown in SEQ ID NO.

3.

3. Biomaterial, characterized in that The biological material includes any one of the following A1) to A5): A1) a nucleic acid molecule encoding the feline serum albumin according to claim 1 or 2; A2) an expression cassette, said expression cassette comprising the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) a recombinant microorganism, wherein the recombinant microorganism comprises the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

4. The biomaterial according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule described in A1) is shown in SEQ ID NO.

4.

5. The biomaterial according to claim 3, characterized in that A4) The recombinant microorganism is selected from one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

6. Use of the arbitrary described biomaterial of claim 3-5 in the preparation of feline serum albumin.

7. Application of cat serum albumin as claimed in claim 1 or 2 or the arbitrary described biomaterial of claim 3-5 in the preparation of medicines for the treatment and / or prevention of hypoalbuminemia and / or cirrhosis of the liver ascites disease.

8. A method for preparing feline serum albumin, characterized in that: The method comprises: constructing a recombinant microorganism expressing the feline serum albumin according to claim 1 or 2, and culturing the recombinant microorganism.

9. The method according to claim 8 is characterized in that The method further comprises the step of separating and purifying the cultured recombinant microbial fermentation liquid.

10. The method according to claim 9, characterized in that The separation and purification step comprises the steps of centrifuging the cultured recombinant microbial fermentation liquid and purifying it with a DEAE column.

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