Cat serum albumin and its application

Abstract

The present invention provides feline serum albumin and its applications, relating to the field of protein engineering technology. This invention, for the first time, mutates feline serum albumin to obtain beneficial mutation sites, thereby obtaining a feline serum albumin mutant that can be highly expressed in a Pichia pastoris system. Furthermore, the feline serum albumin mutant I31L of the present invention is effective in treating ascites caused by cirrhosis, with a therapeutic effect significantly superior to that of existing technologies. This provides a new biomaterial for the application of feline serum albumin and lays a foundation for the genetic engineering modification of feline serum albumin.

Description

Technical Field

[0001] The present application relates to the field of protein engineering technology, and in particular to feline serum albumin and its application. Background Art

[0002] Serum albumin, as the most abundant protein in human plasma, plays an important physiological role in the body. It is also the protein with the largest and most extensive clinical application. The functions and effects of albumin mainly include maintaining plasma osmotic pressure, transporting and storing nutrients, maintaining acid-base balance, immunomodulation, antioxidant and anti-inflammatory effects, etc. At present, there are many studies on the production of HSA, and there are relatively mature processes, especially successful in the Pichia pastoris expression system. However, there are few studies on serum albumin at the animal level. The production method of animal serum albumin is still mainly to purify plasma, which has limited sources and complex processes, and has certain application limitations. With the improvement of people's living standards, more and more families pay more attention to the health of their companion pets. There is an urgent need to improve the production and preparation efficiency of animal serum albumin, especially pet serum albumin, to meet the growing needs of consumers.

[0003] The use of genetic engineering technology to introduce the pet serum albumin gene into Pichia pastoris via plasmid or gene integration for expression is a new production method that can efficiently achieve large-scale expression of pet serum albumin, but its expression level still needs to be improved. Summary of the Invention

[0004] The purpose of the present invention is to provide a cat serum albumin with high expression level and outstanding therapeutic effect on liver cirrhosis ascites.

[0005] On the one hand, the present application provides a feline serum albumin, which is obtained by mutating at position 31 of a 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 mutating the 31st isoleucine (I) residue of the reference sequence to a leucine (L) residue.

[0008] In a preferred embodiment, the feline serum albumin comprises the amino acid sequence shown in SEQ ID NO.3; more preferably, the nucleotide sequence encoding the feline serum albumin 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 to SEQ ID NO.4.

[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] 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.

[0016] 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.

[0017] In a preferred embodiment, the vector may be pPIC9K.

[0018] Furthermore, the recombinant microorganism is one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0019] In a preferred embodiment, the recombinant microorganism may be Pichia pastoris, specifically Pichia pastoris ( Pichia pastoris )GS115.

[0020] Those skilled in the art can use known gene editing methods or gene editing systems to complete the construction of the above-mentioned biomaterials.

[0021] On the other hand, the present application also provides the use of the biomaterial in the preparation of feline serum albumin, wherein the feline serum albumin is obtained by mutation at position 31 of the reference sequence, and the reference sequence is shown in SEQ ID No. 1.

[0022] Furthermore, the feline serum albumin is obtained by mutating the 31st isoleucine (I) residue of the reference sequence to a leucine (L) residue.

[0023] On the other hand, the present application also provides a method for preparing feline serum albumin, which comprises: preparing feline serum albumin by fermentation using the recombinant microorganism in the biological material.

[0024] 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.

[0025] In a preferred embodiment, a method for preparing feline serum albumin comprises:

[0026] 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.

[0027] In a preferred embodiment, a method for preparing feline serum albumin comprises:

[0028] 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.

[0029] Furthermore, the yield of feline serum albumin prepared by the method is greater than or equal to 2 g / L; preferably, greater than or equal to 3 g / L; more preferably, greater than or equal to 3.5 g / L.

[0030] On the other hand, the present application also provides the use of the cat serum albumin or the biomaterial or the method in preparing a drug for treating liver cirrhosis and ascites and / or preparing a pet medicine for cats.

[0031] Furthermore, the treating of ascites due to liver cirrhosis comprises increasing the concentration of albumin (ALB) in the subject.

[0032] Preferably, the subject to be treated is a mammal; more preferably, the subject to be treated is a cat.

[0033] The present invention has the following beneficial effects:

[0034] This invention, for the first time, 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 I31L of this invention is effective in treating ascites caused by cirrhosis, with a therapeutic effect significantly superior to that of existing technologies. This invention provides a new biomaterial for the application of feline serum albumin and lays a foundation for the genetic engineering of feline serum albumin. DETAILED DESCRIPTION

[0035] Technical terms:

[0036] 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.

[0037] Recombination: In a broad sense, any gene exchange process that causes genotype changes is called recombination.

[0038] 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.

[0039] 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.

[0040] Recombinant microorganisms: Fungal cell lines that use genetic engineering methods to efficiently express exogenous genes.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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).

[0048] 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.

[0049] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0050] 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).

[0051] In this specification, amino acid mutations are referred to as "original amino acid, position, and substituted amino acid." For example, a mutation of isoleucine I to leucine L at position 31, counting sequentially from the N (nitrogen) terminus toward the C (carbon) terminus, is represented by I31L.

[0052] Example 1: Construction and identification of recombinant plasmids

[0053] 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. Amino acids 31 and 77 were initially selected for mutation, resulting in mutants I31L and C77S. The amino acid sequence of I31L is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4. The amino acid sequence of C77S is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6.

[0054] 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 products were purified by gel recovery to obtain recombinant plasmids pPIC9K-I31L and pPIC9K-C77S. Sac I and Not I The pPIC9K-FSA, pPIC9K-I31L, and pPIC9K-C77S 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.

[0055] Table 1 Mutation primers and mutant information

[0056]

[0057] Example 2: Construction and screening of engineered strains

[0058] In this example, the pPIC9K-FSA, pPIC9K-I31L, and pPIC9K-C77S 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.

[0059] Specifically, pPIC9K-FSA, pPIC9K-I31L, pPIC9K-C77S 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. The cells were centrifuged at 5000 rpm for 5 min, and part of the supernatant was discarded. Part of the bacterial solution was spread on MD plates and cultured at 28°C for 2-4 days. The plates were then spread on YPD plates containing 3 mg / mL G418 and cultured at 28°C for 2-4 days to obtain the engineered bacteria GS115-FSA, GS115-I31L, and GS115-C77S recombinant plasmids expressing feline serum albumin. The engineered bacteria information is shown in Table 2.

[0060] 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.

[0061] 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.

[0062] The results are shown in Table 2.

[0063] Table 2 Engineering bacteria information

[0064]

[0065] As shown in Table 2, mutation of isoleucine I to leucine L at the 31st position of FSA, counting sequentially from the N (nitrogen) end to the C (carbon) end, can effectively increase the yield of feline serum albumin and improve the production efficiency of feline serum albumin.

[0066] Example 3: Application of Feline Serum Albumin—Evaluation of the Effect in Treating Cirrhosis Ascites Model

[0067] The feline serum albumin I31L 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 ascites.

[0068] 1. Use carbon tetrachloride (CCl4) and olive oil solution combined with sodium phenobarbital and alcohol to establish a rat cirrhosis ascites model:

[0069] 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.

[0070] 2. Methods and efficacy evaluation of feline serum albumin in treating liver cirrhosis ascites model:

[0071] The successfully established rat models were randomly divided into a negative control group, a positive control group, and two experimental groups. The negative control group received normal saline, the positive control group received 20% rHSA, and the two experimental groups received 20% FSA and 20% I31L mutant, respectively. Each group received a 1.5 mL dose, 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 3.

[0072] Table 3 Effects of treating liver cirrhosis ascites model

[0073]

[0074] As shown in Table 3, the I31L (FSA) mutant has a more excellent effect of increasing ALB concentration than the wild type or human serum albumin, and can effectively treat ascites caused by liver cirrhosis.

[0075] 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 amino acid sequence of feline serum albumin is shown in SEQ ID NO.

3.

2. 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; 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).

3. The biomaterial according to claim 2, characterized in that The recombinant microorganism is one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

4. Use of the biomaterial according to claim 2 or 3 in the preparation of feline serum albumin, characterized in that: The amino acid sequence of feline serum albumin is shown in SEQ ID NO.

3.

5. A method for preparing feline serum albumin, characterized in that: The method comprises: using the recombinant microorganism in the biological material according to claim 2 or 3 to ferment and prepare feline serum albumin.

6. Application of cat serum albumin as claimed in claim 1 or biomaterial as claimed in claim 2 or 3 or method as claimed in claim 5 in preparing a medicine for treating liver cirrhosis and ascites.

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