Cat serum albumin and application thereof

Abstract

The invention provides cat serum albumin and application thereof, and relates to the technical field of protein engineering. According to the invention, mutation is carried out on the cat serum albumin for the first time to obtain a beneficial mutation site, so that the cat serum albumin mutant which can be highly expressed in a pichia pastoris system is obtained. In addition, the cat serum albumin mutant I31L also has the effect of treating ascites due to cirrhosis, and the treatment effect is obviously higher than that in the prior art. A new biological material is provided for application of the cat serum albumin, and a foundation is laid for genetic engineering modification of the cat serum albumin.

Description

Technical Field

[0001] This application relates to the technical field of protein engineering, and particularly to a feline serum albumin and its application. Background Art

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

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

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

[0005] On the one hand, this application provides a feline serum albumin, which is obtained by mutating the 31st position of the reference sequence, and the reference sequence is shown as SEQ ID No.1.

[0006] Among them, the reference sequence is the feline serum albumin sequence with GenBank number: NM_001009961. The amino acid sequence of the reference sequence is shown as SEQ ID No.1, and the nucleotide sequence encoding the reference sequence includes the sequence shown as 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%, 99.9% identity with SEQ ID NO.2.

[0007] Further, the feline serum albumin is obtained by mutating the isoleucine (I) residue at position 31 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%, 99.9% identity to SEQ ID NO.4.

[0009] On the other hand, the present application also provides biomaterials, which comprise any one of the following A1)-A5): A1) A nucleic acid molecule encoding the feline serum albumin; A2) An expression cassette containing the nucleic acid molecule of A1); A3) A recombinant vector containing the nucleic acid molecule of A1) and / or the expression cassette of A2); A4) A recombinant microorganism containing the nucleic acid molecule of A1), the expression cassette of A2), and / or the recombinant vector of A3); A5) A recombinant cell containing the nucleic acid molecule of A1), the expression cassette of A2), and / or the recombinant vector of A3).

[0010] Functional elements such as promoters, terminators, and marker genes may also be included in the expression cassettes described herein. Those skilled in the art can make routine selections according to the actual situation as long as the expression of the nucleic acid molecule of A1) can be achieved. The structure and composition of the expression cassette will not be overly restricted here.

[0011] The vector described in this article refers to a vector that can transport exogenous DNA or a target gene into a host cell for amplification and expression. The vector can be any vector that is convenient for recombinant DNA operation and expression of nucleic acid sequences (such as plasmids or viruses). The choice of vector usually depends on the compatibility between the vector and 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 existing outside the chromosome and capable of replicating independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be applied, or two or more vectors or plasmids that collectively contain all the DNA to be introduced into the host cell genome, or a transposon. Those skilled in the art can make a choice according to the actual situation, and no excessive limitation is imposed here.

[0012] In a preferred embodiment, the vector can be pPIC9K.

[0013] Furthermore, the recombinant microorganism is one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0014] In a preferred embodiment, the recombinant microorganism can be Pichia pastoris, specifically Pichia pastoris ( Pichia pastoris ) GS115.

[0015] Those skilled in the art can use known gene editing methods or gene editing systems to complete the construction of the above biological materials.

[0016] On the other hand, this application also provides the use of the described biological materials in the preparation of feline serum albumin, which is obtained by mutating the 31st position of the reference sequence, and the reference sequence is shown as SEQ ID No.1.

[0017] Furthermore, the feline serum albumin is obtained by mutating the isoleucine (I) residue at the 31st position of the reference sequence to a leucine (L) residue.

[0018] On the other hand, this application also provides a method for preparing feline serum albumin, and the method includes: fermenting with the recombinant microorganism in the biological materials to prepare feline serum albumin.

[0019] 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.

[0020] In a preferred embodiment, a method for preparing feline serum albumin includes: Inoculate the recombinant microorganism into a culture medium and culture at 25°C - 40°C and 100 - 300 rpm until OD 600 = 9 - 11, then centrifuge at 1000 - 2000 g for 1 - 10 min, discard the supernatant, add culture medium to resuspend the cells, culture at 25°C - 40°C and 100 - 300 rpm, and add 0.1% - 0.5% methanol every 12 - 24 h for 36 - 72 h of induction.

[0021] In a preferred embodiment, a method for preparing feline serum albumin includes: Inoculate the recombinant microorganism into a culture medium and culture at 28°C and 210 rpm until OD 600 = 10, then centrifuge at 1500 g for 5 min, discard the supernatant, add culture medium to resuspend the cells, culture at 28°C and 210 rpm, and add 0.5% methanol every 24 h for 72 h of induction.

[0022] 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.

[0023] On the other hand, the present application also provides the use of the feline serum albumin or the biomaterial or the method in the preparation of drugs for treating liver cirrhosis ascites and / or in the preparation of pet drugs for cats.

[0024] Furthermore, the treatment of liver cirrhosis ascites includes increasing the concentration of albumin (ALB) in the subject to be treated.

[0025] Preferably, the subject to be treated can be a mammal; more preferably, the subject to be treated is a cat.

[0026] The present invention has the following beneficial effects: The present invention has obtained beneficial mutation sites for the first time by mutating feline serum albumin, and thus obtained a mutant of feline serum albumin that can be highly expressed in the Pichia pastoris system. Moreover, the feline serum albumin mutant I31L in the present invention also has the effect of treating liver cirrhosis ascites, and the treatment effect is significantly higher than that of the prior art. It provides a new biomaterial for the application of feline serum albumin and lays a foundation for the genetic engineering transformation of feline serum albumin. Detailed implementation manners

[0027] Technical terms: Identity: In molecular evolution research, it refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules.

[0028] Recombination: Generally speaking, any gene exchange process that causes genotype changes is called recombination.

[0029] Expression cassette: An expression cassette refers to a set of DNA sequences composed of a promoter, a target gene, a reporter gene, etc., which can be expressed in specific tissues and are easy to detect.

[0030] Recombinant vector: A recombinant vector is a vector that transfers a target gene on the basis of the basic backbone of a cloning vector, so that the target gene can be expressed.

[0031] Recombinant microorganism: A fungal cell line in which a foreign gene is highly expressed by genetic engineering methods.

[0032] Recombinant cell: The term "recombinant cell" means any cell type that is easily transformed, transfected, transduced, etc. with a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parental cell that is not identical to the parental cell due to mutations that occur during replication.

[0033] To more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0034] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than for limiting the protection scope of the present invention.

[0035] Unless otherwise specified, in the following embodiments, reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase. For those not indicating specific conditions in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0036] Plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are carried out according to the kit instructions. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0037] In addition, the "water" described in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, purified water, etc.

[0038] In the following examples, unless otherwise specifically stated, % represents wt%, that is, weight percentage.

[0039] In this specification, the amino acids at the corresponding sites are represented by the recognized single-letter abbreviations of IUPAC. Among them, each amino acid and its abbreviation are as follows: 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).

[0040] In this specification, for the mutation of amino acids, it is represented by "original amino acid, site, substituted amino acid". For example, the mutation of isoleucine I at the 31st site counted sequentially from the N (nitrogen) terminus to the C (carbon) terminus of the sequence to leucine L is represented by I31L.

[0041] Example 1: Construction and Identification of Recombinant Plasmid Using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), search for and obtain the Feline serum albumin (FSA) sequence in GenBank (GenBank: NM_001009961, and the amino acid sequence is shown as SEQ ID No.1). Perform structural simulation and screening of mutable sites on this sequence. Initially, the 31st amino acid and the 77th amino acid were selected for mutation to obtain mutants I31L and C77S. The amino acid sequence of I31L is shown as SEQ ID No.3, and the nucleotide sequence is shown as SEQ ID No.4. The amino acid sequence of C77S is shown as SEQ ID No.5, and the nucleotide sequence is shown as SEQ ID No.6.

[0042] Specifically, the FSA (GenBank: NM_001009961) gene sequence was delivered to Nanjing Genscript Biotech Co., Ltd. for optimization (the nucleotide sequence is shown as SEQ ID No.2), chemically synthesized and inserted into the vector pPIC9K through restriction enzyme sites SnaB Ⅰ and Not Ⅰ to construct a plasmid named pPIC9K-FSA. Single-site mutation primers were designed using pPIC9K-FSA as a template (as shown in Table 1), and mutations were carried out by inverse PCR. The amplified linear plasmid products were recovered and purified by gel electrophoresis, and recombinant plasmids pPIC9K-I31L and pPIC9K-C77S were obtained. The recombinant plasmids pPIC9K-FSA, pPIC9K-I31L, and pPIC9K-C77S were double-digested with restriction enzymes Sac I and Not I After gently mixing all samples and incubating at 37°C for 1 - 2 h, the digested products were identified by 1% agarose gel electrophoresis and sent to Tsingke Biotechnology for sequencing to confirm that the product sequences were correct.

[0043] Table 1 Mutation primers and mutant information

[0044] Example 2: Construction and screening of engineering strains In this example, the recombinant plasmids pPIC9K-FSA, pPIC9K-I31L, and pPIC9K-C77S obtained in Example 1 were electrotransformed into the Pichia pastoris ( Pichia pastoris ) GS115 strain to construct engineering bacteria expressing feline serum albumin.

[0045] Specifically, the recombinant plasmids pPIC9K-FSA, pPIC9K-I31L, pPIC9K-C77S, and restriction enzymesSac I Place on ice. According to the DNA concentration and the instruction manual of the restriction endonuclease, Sac Ⅰ after mixing evenly and incubating at 37°C for 1 h, use 1% agarose gel electrophoresis for identification. The linearized plasmid uses a DNA purification kit to recover DNA fragments according to the instruction manual, and the DNA concentration is detected. Take 7 μg of the linearized plasmid and mix it evenly with 80 μL of GS115 competent cells, transfer it to a pre-cooled electroporation cuvette, mix well and incubate on ice for 10 min, perform electroporation under the conditions of 1500 V, 25 μF, 200 Ω, and 5 ms, add 1 mL of pre-cooled 1 M sorbitol to suspend the cells and transfer them to a test tube, add 1 mL of YPD liquid medium, and incubate at 30°C and 150 rpm for 2 h. Centrifuge at 5000 rpm for 5 min, discard part of the supernatant, pipette part of the bacterial liquid and spread it on an MD plate, culture at 28°C for 2 - 4 d, spread it on a YPD plate containing 3 mg / mL G418, and after culturing at 28°C for 2 - 4 d, the recombinant plasmids of the engineered bacteria GS115-FSA, GS115-I31L, and GS115-C77S expressing feline serum albumin are obtained. The information of the engineered bacteria is shown in Table 2.

[0046] Respectively pick the positive monoclonal colonies of the above-mentioned engineered bacteria into BMGY liquid medium, culture at 28°C and 210 rpm until OD 600 = 10, centrifuge at 1500 g for 5 min, discard the supernatant, add 100 mL of BMMY liquid medium to resuspend the cells, culture at 28°C and 210 rpm, add 0.5% methanol every 24 h, after inducing for 72 h, centrifuge the fermentation broth, centrifuge at 4°C and 7000 rpm for 10 min.

[0047] Purify the above fermentation broth using a DEAE column. Specifically, install the DEAE column on the SDL-100 purification system, equilibrate the column with 0.2 mM PB, pass the sample through the column at a flow rate of 0.5 mL / min, perform protein linear elution using a 0.5 M NaCl eluent, fully elute the purification column, and seal the column with 20% ethanol to obtain purified feline serum albumin. And use the BCA method to measure the concentration (yield) of feline serum albumin. The results are shown in Table 2.

[0048] The results are shown in Table 2.

[0049] Table 2 Information of engineered bacteria

[0050] As can be seen from the results in Table 2, after the isoleucine I at the 31st site counting from the N (nitrogen) - terminal to the C (carbon) - terminal direction of the FSA sequence mutates to leucine L, it can effectively increase the yield of feline serum albumin and improve the production efficiency of feline serum albumin.

[0051] Example 3: Application of Feline Serum Albumin - Evaluation of the Effect in Treating Liver Cirrhosis Ascites Model The I31L mutant of feline serum albumin 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.

[0052] 1. A rat liver cirrhosis ascites model was constructed by using a mixture of carbon tetrachloride (CCl4) and olive oil solution combined with sodium phenobarbital and alcohol: SD male rats were randomly divided into two groups: The model group was intraperitoneally injected with 40% CCl4 and olive oil, given 0.3 g / L sodium phenobarbital drinking water in the first two weeks, and 10% alcohol starting from the third week; the control group was intraperitoneally injected with olive oil solution and given normal drinking water. The intraperitoneal injection dose for each experimental group was 2 mL / kg, twice a week. When the rats in the experimental group showed positive for ascites and false lobules appeared in the liver pathological section, the model was successfully established.

[0053] 2. Method and effect evaluation of feline serum albumin in treating liver cirrhosis ascites model: The successfully modeled rat models were randomly divided into a negative control group, a positive control group, and two experimental groups. The negative control group was administered normal saline, the positive control group was administered 20% rHSA, and the two experimental groups were respectively administered 20% FSA and 20% I31L mutant. The administration dose for each group was 1.5 mL, and the administration method was intravenous injection through the tail vein, which was completed within 2 h, once a day for one week continuously. The body weight of the rats was measured before each administration. After the last administration, the rats were fasted and water-deprived, and blood was taken from the fundus vein plexus after 18 h. The ALB concentration was detected using the kit from Nanjing Jiancheng Bioengineering Institute, and the results are shown in Table 3.

[0054] Table 3 Effect of Treating Liver Cirrhosis Ascites Model

[0055] As can be seen from the results in Table 3, compared with the wild type or human serum albumin, the I31L (FSA) mutant has a more excellent effect of increasing the ALB concentration and can effectively treat liver cirrhosis ascites.

[0056] The above are only the embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A cat serum albumin, characterized in that, The feline serum albumin is obtained by mutating the 31st position of the reference sequence, and the reference sequence is as shown in SEQ ID No.

1.

2. The cat serum albumin according to claim 1, wherein The feline serum albumin is obtained by mutating the isoleucine residue at the 31st position of the reference sequence to a leucine residue.

3. A biomaterial, characterized in that, The biological material includes any one of the following A1)-A5): A1) A nucleic acid molecule encoding the feline serum albumin according to claim 1 or 2; A2) An expression cassette containing the nucleic acid molecule of A1); A3) A recombinant vector containing the nucleic acid molecule of A1) and / or the expression cassette of A2); A4) A recombinant microorganism containing the nucleic acid molecule of A1), the expression cassette of A2), and / or the recombinant vector of A3); A5) A recombinant cell containing the nucleic acid molecule of A1), the expression cassette of A2), and / or the recombinant vector of A3).

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

5. Use of the biomaterial according to claim 3 or 4 in the preparation of feline serum albumin, characterized in that, The feline serum albumin is obtained by mutating the 31st position of the reference sequence, and the reference sequence is as shown in SEQ ID No.

1.

6. The application according to claim 5, characterized in that, The feline serum albumin is obtained by mutating the isoleucine residue at the 31st position of the reference sequence to a leucine residue.

7. A method for preparing feline serum albumin, characterized in that, The method includes: fermenting the recombinant microorganism in the biological material according to claim 3 or 4 to prepare feline serum albumin.

8. Use of the feline serum albumin according to claim 1 or 2, the biological material according to claim 3 or 4, or the method according to claim 7 in the preparation of a drug for treating liver cirrhosis ascites and / or the preparation of pet drugs for cats.

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