Cat Serum Albumin Mutant, Its Expression Method in CHO Cells and Application

Abstract

The present invention belongs to the technical field of genetic engineering, and discloses a feline serum albumin mutant, its expression method in CHO cells and its application. The present invention for the first time provides a feline serum albumin mutant Mut-FSA. By replacing the K at the 558th position at the end of the FSA amino acid sequence with R, the optimized Mut-FSA sequence is finally formed. Compared with the unmutated FSA, Mut-FSA can show more significant curative effect advantages in the treatment intervention of feline hypoproteinemia or acute blood loss. In addition, Mut-FSA can not only enable the serum albumin concentration of feline hypoproteinemia patients to be rapidly corrected within 24 hours after treatment intervention, but also effectively improve the serum albumin level and overall prognosis of the affected cats. The recombinant FSA prepared by the present invention can not only provide a more effective innovative therapy for feline hypoproteinemia or acute blood loss, but also has broad application prospects and industrialization value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a feline serum albumin mutant, a CHO cell expression method, and an application thereof. The technical solution of the present invention significantly improves the yield and purity of recombinant FSA, while having the advantages of high safety and low immunogenicity. It can be used clinically as an innovative therapy for treating feline hypoproteinemia or acute blood loss, providing the pet medical industry with a more economical, safe, and efficient therapeutic drug option. Background Art

[0002] In the clinical medical field, feline serum albumin (FSA) is widely used to treat conditions such as hypoproteinemia, hemorrhagic shock, burns, ascites due to cirrhosis, and nephrotic edema. FSA is mainly obtained through blood extraction, which faces the problem of extremely scarce supply and potential risks such as blood-borne viruses, making it impossible to meet the growing market demand.

[0003] Therefore, developing efficient, safe, and easily scalable FSA production technology has become a key issue that needs to be urgently addressed in the field of pet healthcare. CHO cells, as an important expression system in the biopharmaceutical field, have advantages such as complete protein glycosylation modification functions, accurate post-transcriptional modification, low risk of viral contamination, and low endotoxin production. In addition, the CHO cell expression system has powerful gene amplification capabilities and high exogenous protein expression capabilities, enabling efficient and stable expression of recombinant proteins. Its fibroblast characteristics almost do not secrete endogenous proteins, which greatly simplifies the separation and purification process of recombinant proteins and reduces production costs and process difficulty. In view of this, the use of CHO cell expression systems to produce recombinant FSA has significant technical advantages and broad market prospects.

[0004] In summary, in order to solve the problems of limited FSA sources, safety risks of traditional extraction methods, and limitations of alternative treatment options, the present invention proposes a feline serum albumin mutant, and its CHO cell expression method and application. Summary of the Invention

[0005] In view of the current problems of limited FSA sources, high production costs, and insufficient safety, the present invention provides a feline serum albumin mutant Mut-FSA. The amino acid sequence of Mut-FSA is shown in SEQ ID NO.5.

[0006] Another object of the present invention is to provide the use of Mut-FSA in the preparation of a medicament for treating feline hypoproteinemia or acute blood loss.

[0007] To achieve the above object, the following invention is provided:

[0008] The present invention for the first time provides a cat serum albumin mutant Mut-FSA. By replacing the K at the 558th position at the end of the FSA amino acid sequence with R, the optimized Mut-FSA sequence is finally formed. The amino acid sequence of Mut-FSA is shown in SEQ ID NO.5. Compared with the unmutated FSA, Mut-FSA can show more significant curative effect advantages (P<0.05) in the treatment intervention of feline hypoproteinemia or acute blood loss. In addition, Mut-FSA can not only enable the serum albumin concentration of feline hypoproteinemia patients to be rapidly corrected within 24 hours after treatment intervention, but also effectively improve the serum albumin level and overall prognosis of the affected cats.

[0009] The protection scope of the present invention also includes:

[0010] A fusion protein obtained by fusing the mutant protein described in SEQ ID NO.5 with a protein tag.

[0011] For the above-mentioned fusion protein, preferably, it is shown in SEQ ID NO.3.

[0012] The coding gene of the mutant or fusion protein described in SEQ ID NO.5.

[0013] For the coding gene of the above-mentioned fusion protein, preferably, it is shown in SEQ ID NO.4.

[0014] An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above-mentioned coding gene.

[0015] For the above-mentioned recombinant cell, preferably, it is a CHO cell.

[0016] A method for preparing the fusion protein described in SEQ ID NO.3, including culturing CHO containing the gene shown in SEQ ID NO.4.

[0017] The application of the above-mentioned mutant, fusion protein, the coding gene of the mutant described in SEQ ID NO.5, or the coding gene of the fusion protein, an expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above-mentioned coding gene in the preparation of a cat serum albumin mutant.

[0018] The application of the above-mentioned mutant, fusion protein, the coding gene of the mutant described in SEQ ID NO.5, or the coding gene of the fusion protein, an expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the above-mentioned coding gene in the preparation of a drug for treating feline hypoproteinemia or acute blood loss.

[0019] Compared with the prior art, the beneficial effects of the present invention:

[0020] The present invention for the first time provides a feline serum albumin mutant Mut-FSA, which has low immunogenicity and high safety, and provides an effective solution for the treatment of feline hypoproteinemia or acute blood loss. For the conditions of feline hypoproteinemia or acute blood loss, Mut-FSA shows good therapeutic effects in terms of physiological function indexes, blood routine indexes, biochemical indexes, and the time for visible mucous membrane to return to normal color. Moreover, compared with the unmutated FSA, Mut-FSA shows a more significant therapeutic effect advantage (P < 0.05) in the treatment intervention of feline hypoproteinemia or acute blood loss. In addition, Mut-FSA can not only enable the serum albumin concentration of feline hypoproteinemia cats to be rapidly corrected within 24 hours after treatment intervention, but also effectively improve the serum albumin level and overall prognosis of the affected cats.

[0021] The mutant or Mut-FSA provided by the present invention can be produced using the CHO cell expression system, and has significant advantages in terms of the simplicity of protein purification, safety, production cost, etc., providing reliable technical support for large-scale production. It solves the problems such as limited source of FSA, high production cost, and insufficient safety in traditional methods. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the detection results of SDS-PAGE and Western Blot after purification of FSA and Mut-FSA.

[0023] Figure 2 It is a schematic diagram of the measurement results of body temperature and weight in the feline safety experiment.

[0024] Figure 3 It is a schematic diagram of the representative tissue autopsy observation and HE staining examination results in the feline safety experiment.

[0025] Figure 4 It is a schematic diagram of the measurement results of body temperature and weight in the feline modeling experiment.

[0026] Figure 5 It is a schematic diagram of the representative clinical observation results in the feline hypoproteinemia animal model experiment.

[0027] Note: The pictures of feline gingiva, nose tip, and conjunctiva are schematic diagrams of the clinical observation results at 24 h after modeling.

[0028] Figure 6 It is a schematic diagram of the measurement results of body temperature and weight in the feline treatment experiment;

[0029] Note: -1 represents before modeling; 0 represents after modeling.

[0030] Figure 7 It is a schematic diagram of the representative clinical observation results in the feline treatment experiment;

[0031] Note: The pictures of cats’ gums, noses and conjunctiva are the clinical observation results on the 7th day after treatment. DETAILED DESCRIPTION

[0032] The present invention is further described below by way of examples, which are intended only to provide a better understanding of the present invention and are not intended to limit the scope of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all sourced from commercial sources.

[0033] Embodiment 1:

[0034] Design and production of two recombinant feline serum albumins:

[0035] 1.1 Design of two recombinant feline serum albumin nucleotide sequences:

[0036] The gene sequence and amino acid sequence of feline serum albumin (GenBank: CAA59279.1) were searched and obtained from NCBI.

[0037] Design two feline serum albumin amino acid sequences:

[0038] (1) FSA: A fusion protein consisting of FSA and a protein tag was obtained by ligating an HRV 3C restriction site to the C-terminus of the original FSA sequence, followed by a GGGGS flexible linker peptide, a Twin-Strep-tag, and a 10×His tag. This fusion protein is referred to as FAS in the examples of the present invention. The amino acid sequence of FSA is shown in SEQ ID NO. 1, and the nucleotide sequence encoding it (after CHO system codon optimization) is shown in SEQ ID NO. 2.

[0039] (2) Mut-FSA: By connecting the HRV 3C restriction site to the C-terminus of the original FSA sequence, followed by a GGGGS flexible linker peptide, a Twin-Strep-tag, and a 10×His tag, and replacing the 558th K at the end of the FSA amino acid sequence with an R, an optimized Mut-FSA sequence was finally formed, and a fusion protein obtained by fusing the FSA mutant with the protein tag was obtained, which is referred to as Mut-FAS in the examples of the present invention. The amino acid sequence of Mut-FSA is shown in SEQ ID NO.3, and the polynucleotide encoding it (after CHO system codon optimization) is shown in SEQ ID NO.4. The amino acid sequence of Mut-FSA without the purification tag is shown in SEQ ID NO.5, and the polynucleotide encoding it is shown in SEQ ID NO.6.

[0040] The above-mentioned recombinant feline serum albumin can be directly synthesized commercially or obtained through microbial expression. The present invention adopts commercial synthesis and CHO eukaryotic expression to obtain it.

[0041] 1.2 Synthesis of Gene Sequences and Construction of Plasmids

[0042] The DNAman software was used to analyze the restriction enzyme cleavage sites of the target gene. According to the analysis results and the multiple cloning sites on the pXC17.4 transfer vector, HindIII (5' end) and EcoRI (3' end) cleavage sites were introduced at both ends of the target gene sequence to ensure that the selected cleavage sites did not appear in the target gene sequence. The identified target gene sequences SEQ ID NO.2 or SEQ ID NO.4 after codon optimization for the CHO system were inserted into the pXC17.4 vector respectively to construct recombinant plasmids, and the expressed proteins were named FSA and Mut-FSA respectively. These plasmids were used for subsequent transfection of the CHO cell expression system and high-efficiency expression of recombinant proteins.

[0043] 1.3 Expression of FSA and Mut-FSA in CHO

[0044] Under sterile conditions, electroporation was performed using an X cell gene pulser (Bio-Rad). The electroporation voltage was set to 300 V, the capacitance was 900 μF (single pulse), and the resistance was set to infinity. A disposable 4 mm electroporation cuvette (Bio-Rad) was taken and 20 μg of plasmid DNA and 0.4 mL of CHO cell suspension (5×10 6 cells / mL) were added. The plasmids were introduced into CHO cells respectively by electroporation technology. After transfection, the cells in the electroporation cuvette were transferred to a shake flask, 10 mL of basal culture medium was added, and the cells were cultured in a shaker at 37 °C and 5% CO2.

[0045] 1.4 Purification and Identification of FSA and Mut-FSA

[0046] The electroporated CHO cells were inoculated into a 2 L shake flask containing 350 - 400 mL of CHO culture medium at a density of 0.5 - 1×10 6 cells / mL. Under the conditions of 36 - 37 °C and 5% CO2, the cells were cultured in a shaker for 3 - 4 days, and the cell viability and density were monitored daily. When the cell density reached 4×10 6 cells / mL, the feeding operation was started. The feeding process was as follows: First, 2% of solution A was added, and the cells were cultured in a shaker at 37 °C and 5% CO2 for 10 min, and then 0.2% of solution B was added. When the cell density reached 1×10 7 cells / mL, 2% of solution A was added and the cells were continuously cultured for 7 - 8 days. When the proportion of viable cells decreased to 80 - 90%, the culture was terminated.

[0047] After the cultivation, cell debris was removed by centrifugation, the supernatant was collected, and filtered using a 0.45 μm filter membrane. The filtered protein supernatant was loaded onto a nickel column pre-equilibrated with PBS buffer. After washing until the A280 absorbance reached the baseline level, it was eluted with 300 mM imidazole, and finally, highly pure FSA and Mut-FSA were obtained. After the purified protein solution was sterilized and filtered through a 0.22 μm filter membrane, SDS-PAGE and Western Blot (WB) assays were performed. Subsequently, the total protein concentrations of FSA and Mut-FSA were determined by the microplate protocol (Thermo Scientific Pierce BCA Protein Assay Kit). The test results are as follows:

[0048] The results of SDS-PAGE and Western Blot showed that the molecular weights of FSA and Mut-FSA were consistent with the expected values, both around 70 kDa (see Figure 1 ). The test results of the total protein concentrations of FSA and Mut-FSA showed that 5.75 g of FSA and 5.83 g of Mut-FSA proteins could be obtained from each liter of CHO cell supernatant, respectively.

[0049] Example 2:

[0050] Safety experiment of Mut-FSA in cats

[0051] Before the experiment, the animal room was strictly disinfected with formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity of the animal room were controlled at 24 ± 2 °C and about 40 - 60% respectively. The experimental animals were fed and watered ad libitum.

[0052] Ten healthy cats aged 10 months and weighing about 2.5 - 3.5 kg were selected for the experiment and randomly divided into the Mut-FSA group (n = 5) and the control group (n = 5). After all the experimental cats were acclimatized in the animal house for 1 week, they were anesthetized with a compound anesthetic of tiletamine hydrochloride and zolazepam hydrochloride (Zoletil® 50) (5 - 7.5 mg / kg). Subsequently, a disposable intravenous infusion set was used to perform intravenous infusion at a flow rate of 35 mL / h. Among them, the cats in the Mut-FSA group were intravenously infused with Mut-FSA at a double dose (0.8 g / kg), and the cats in the control group were injected with an equal volume of 0.9% normal saline in the same manner. On days 0 (before the examination), 1, 3, 5, and 7 (after the examination) of the examination, the body temperature, body weight, blood pressure, and pulse of all cats were measured. At the same time, anticoagulated blood and serum samples of all experimental cats were collected for routine blood and biochemical index detection. After the experiment, the cats were euthanized, and then autopsy was performed to observe and record the gross changes of the organ tissues and take pictures. During the above-mentioned autopsy, the hearts, livers, spleens, lungs, and kidneys of the cats in the control group and the Mut-FSA group were collected and fixed in 4% paraformaldehyde solution for more than 24 h, and then HE staining was performed on each organ tissue. The following results were obtained from the experiment:

[0053] Compared with the control group, there were no significant differences in the body temperature, body weight, blood pressure, and pulse of the cats in the Mut-FSA group (P > 0.05) (see Figure 2 and Table 1); compared with the control group, the test results of the routine blood and biochemical indexes of the cats in the Mut-FSA group were within the normal range, and there were no significant differences (except for the biochemical index test values of total protein and albumin on days 1 and 3 of the examination) (P > 0.05) (see Tables 2-1, 2-2, 2-3, 2-4 and Tables 3-1, 3-2, 3-3); the autopsy observation results showed that the macroscopic structures of the hearts, livers, spleens, lungs, and kidneys of all cats in the Mut-FSA group were normal (see Figure 3 ); the HE staining results showed that no obvious pathological changes were observed in the hearts, livers, spleens, lungs, and kidneys of the cats in the Mut-FSA group (see Figure 3 ). According to the same experimental protocol as the Mut-FSA group, after the cats in the FSA group were intravenously infused with FSA, the cats in the FSA group did not show obvious adverse reactions, and there were no significant differences in the above-mentioned detection indexes compared with the Mut-FSA group (P > 0.05). The above results indicate that after intravenous injection of Mut-FSA or FSA, the cats did not show obvious adverse reactions and had good safety in the animal body.

[0054] Table 1 Analysis of blood pressure and pulse detection results of the cat safety experiment

[0055]

[0056] Note: All data were analyzed using SPSS 22.0 software, and all results were expressed as mean (M) ± standard deviation (SD). The t-test was used to analyze the inter-group differences between the two sets of data. A P-value < 0.05 indicates significant differences; a P-value < 0.01 indicates extremely significant differences; a P-value > 0.05 indicates no significant differences.

[0057] Table 2-1 Analysis of the detection results of blood routine indicators in the cat safety experiment

[0058]

[0059] Note: All data were analyzed using SPSS 22.0 software, and all results were expressed as mean (M) ± standard deviation (SD). The t-test was used to analyze the inter-group differences between the two sets of data. A P-value < 0.05 indicates significant differences; a P-value < 0.01 indicates extremely significant differences; a P-value > 0.05 indicates no significant differences.

[0060] Table 2-2 Analysis of the detection results of blood routine indicators in the cat safety experiment

[0061]

[0062] Note: Data analysis is the same as in Table 2-1

[0063] Table 2-3 Analysis of the detection results of blood routine indicators in the cat safety experiment

[0064]

[0065] Note: Data analysis is the same as in Table 2-1

[0066] Table 2-4 Analysis of the detection results of blood routine indicators in the cat safety experiment

[0067]

[0068] Note: Data analysis is the same as in Table 2-1

[0069] Table 3-1 Analysis of the detection results of biochemical indicators in the cat safety experiment

[0070]

[0071] Note: All data were analyzed using SPSS 22.0 software, and all results were expressed as mean (M) ± standard deviation (SD). The t-test was used to analyze the inter-group differences between the two sets of data. A P-value < 0.05 indicates significant differences; a P-value < 0.01 indicates extremely significant differences; a P-value > 0.05 indicates no significant differences.

[0072] Table 3-2 Analysis of the detection results of biochemical indicators in the cat safety experiment

[0073]

[0074] Note: Data analysis is the same as Table 3-1

[0075] Table 3-3 Analysis of biochemical index test results in cat safety experiments

[0076]

[0077] Note: Data analysis is the same as Table 3-1

[0078] Example 3:

[0079] Establishment of a feline hypoproteinemia animal model

[0080] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity in the animal room were controlled at 24±2°C and 40-60%, respectively. The experimental animals were maintained with free access to food and water.

[0081] Ten healthy cats aged 10 months and weighing approximately 2.5-3.5 kg were randomly divided into a modeling group (n=5) and a control group (n=5). After the experimental cats acclimatized in the animal room for one week, the modeling group cats were anesthetized using Cetuximab® 50 anesthetic (5-7.5 mg / kg). Subsequently, 30% of the total blood volume (total blood content accounts for 6-7% of body weight) was collected from the modeling group cats through the jugular vein at one time. The control group cats did not receive any treatment. Body temperature, weight, blood pressure, and pulse were measured for all experimental cats at 0 (before modeling), 12, 24, 36, and 48 hours (after modeling). The gums, nose, and conjunctiva were observed and photographed. In addition, anticoagulant blood and serum samples were collected from all experimental cats at designated time points for blood routine and biochemical index testing. The following results were obtained:

[0082] Compared with the control group, the body temperature and weight of cats in the modeling group showed a downward trend, but no significant difference was found (P>0.05) (see Figure 4 ); Starting from the 12th hour after modeling, the systolic and diastolic blood pressures of the cats in the modeling group were significantly lower than those in the control group (P < 0.01), and the pulse rate was significantly increased (P < 0.01) (see Table 4); Clinical observation results showed that compared with before modeling, at the 24th hour after modeling, the gums, noses and conjunctiva of the cats in the modeling group were significantly pale. In addition, the cats in the modeling group also showed symptoms such as depression, decreased appetite, and decreased activity (see Figure 5The results of routine blood test showed that at 12 hours after modeling, the red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT) and platelet count (PLT) in the blood of cats in the modeling group were significantly lower than those in the control group (P < 0.05 or P < 0.01), and starting from 24 hours after modeling, the test values of the red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT) of the blood deviated from the normal reference range (see Table 5-1 and Table 5-2); the results of biochemical test showed that at 12 hours after modeling, the total protein (TB), albumin (ALB) and globulin (GLB) in the serum of cats in the modeling group were significantly lower than those in the control group (P < 0.05 or P < 0.01), and starting from 24 hours after modeling, the test values of the biochemical indicators of total protein (TB), albumin (ALB) and globulin (GLB) of the blood deviated from the normal reference range (see Table 6).

[0083] In summary, compared with the control group, the modeling group showed significant changes in clinical symptoms, physiological function indicators, blood routine, and biochemical parameters. Based on these experimental results, this study concluded that the feline hypoproteinemia animal model was successfully established. Furthermore, this study further revealed that the differences in various indicators between the modeling group and the control group were most significant at 24 hours after modeling. Therefore, this study used the 24th hour of modeling (the 24th hour after blood sample collection) as the key evaluation point for assessing the therapeutic effects of FSA and Mut-FSA.

[0084] Table 4 Analysis of blood pressure and pulse test results in cat modeling experiments

[0085]

[0086] Note: All data were analyzed using SPSS 22.0 software. All results are expressed as mean (M) ± standard deviation (SD). Student's t-test was used to analyze differences between groups. A P value < 0.05 indicated a significant difference; a P value < 0.01 indicated an extremely significant difference; and a P value > 0.05 indicated no significant difference.

[0087] Table 5-1 Analysis of blood routine index test results in cat modeling experiments

[0088]

[0089] Note: All data were analyzed using SPSS 22.0 software. All results are expressed as mean (M) ± standard deviation (SD). Student's t-test was used to analyze differences between groups. A P value < 0.05 indicated a significant difference; a P value < 0.01 indicated an extremely significant difference; and a P value > 0.05 indicated no significant difference.

[0090] Table 5-2 Analysis of blood routine index test results in cat modeling experiments

[0091]

[0092] Note: Data analysis is the same as Table 5-1

[0093] Table 6 Analysis of biochemical index test results in cat modeling experiment

[0094]

[0095] Note: All data were analyzed using SPSS 22.0 software. All results are expressed as mean (M) ± standard deviation (SD). Student's t-test was used to analyze differences between groups. A P value < 0.05 indicated a significant difference; a P value < 0.01 indicated an extremely significant difference; and a P value > 0.05 indicated no significant difference.

[0096] Embodiment 4:

[0097] Evaluation of the therapeutic effects of FSA and Mut-FSA prepared in Example 1 in a feline hypoproteinemia animal model

[0098] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity in the animal room were controlled at 24±2°C and 40-60%, respectively. The experimental animals were maintained with free access to food and water.

[0099] Fifteen healthy cats, 10 months old and weighing approximately 2.5-3.5 kg, were randomly divided into a Mut-FSA group (n=5), a FSA group (n=5), and a control group (n=5). After acclimating to the animal room for one week, the cats were anesthetized with Zota® 50 anesthetic (5-7.5 mg / kg). Subsequently, 30% of the cats' systemic blood volume was collected via the jugular vein. Twenty-four hours after blood collection, each cat received an intravenous infusion of 0.4 g / kg of Mut-FSA or FSA, respectively, in the Mut-FSA and FSA groups. The control group received an intravenous infusion of the same volume of 0.9% saline. Temperature, weight, blood pressure, and pulse were measured before and after modeling, and on days 1, 3, 5, and 7 after Mut-FSA and FSA treatment. The gingiva, nasal tip, and conjunctiva were observed and photographed. In addition, anticoagulated blood and serum samples were collected at designated time points for routine blood tests and biochemical indices. The following results were obtained:

[0100] The results of body temperature and weight measurements showed that on the first day after treatment, the body temperature and weight of cats in the Mut-FSA group, FSA group and control group showed an upward trend, but no significant difference was found (P>0.05). On the third day after treatment, the body temperature and weight tended to be stable (seeFigure 6 ); Blood pressure and pulse measurement results showed that compared with before modeling, the blood pressure of cats in the Mut-FSA group, FSA group, and control group decreased after modeling, and the pulse accelerated. On the 3rd day after treatment, the blood pressure and pulse of cats in the Mut-FSA group and FSA group returned to the normal frequency, while the recovery speed of blood pressure and pulse in the control group was slower than that in the Mut-FSA group and FSA group (P < 0.01), and did not return to the normal frequency during the entire experimental period. It is worth noting that during the entire treatment cycle, the recovery speed of blood pressure and pulse in cats in the Mut-FSA group was faster than that in the FSA group, but there was no significant difference (P > 0.05) (see Table 7); Clinical observation results showed that after treatment, the color of the gums, nose tip, and conjunctiva of cats in the Mut-FSA group and FSA group was significantly improved, returning from pale white to physiological pink, while the color of the mucosa in the control group did not fully return to physiological pink (see Figure 7 ); The results of routine blood index detection showed that compared with the control group, cats in the Mut-FSA group and FSA group showed significant advantages in improving hematological indexes. Specifically, the number of red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) in the Mut-FSA group and FSA group began to return to the normal reference range on the 1st day after treatment, while the control group did not start to show a recovery trend until the 5th day. Longitudinal observation showed that there were statistically significant differences in the dynamic changes of hematological parameters in the Mut-FSA group and FSA group during the entire treatment cycle compared with the control group (P < 0.05 or P < 0.01). In addition, during the entire experimental cycle, the recovery speed of hematological indexes in cats in the Mut-FSA group was faster than that in the FSA group, but there was no significant difference (P > 0.05) (see Tables 8-1 and 8-2); The results of biochemical index detection showed that during the entire treatment cycle, compared with the control group, there were statistically significant differences in the test values of various biochemical indexes of total protein (TB), albumin (ALB), and globulin (GLB) in the blood of cats in the Mut-FSA group and FSA group (P < 0.01). In addition, the test value of albumin (ALB) in the blood of cats in the Mut-FSA group and FSA group began to return to the normal range on the 1st day after treatment, while the control group remained lower than the physiological threshold during the entire observation period. It should be emphasized that on the 1st, 3rd, and 5th days after treatment, there were significant differences in the test value of albumin (ALB) in the blood of cats in the Mut-FSA group compared with the FSA group (P < 0.05) (see Table 9).

[0101] Comprehensive experimental data showed that compared with the control group, both the Mut-FSA and FSA groups demonstrated favorable therapeutic effects in terms of physiological function indicators, routine blood tests, biochemical parameters, and time to visual mucosal repigmentation. It is important to note that Mut-FSA demonstrated a significantly greater efficacy than FSA in the treatment of feline hypoproteinemia (P < 0.05). Furthermore, Mut-FSA not only rapidly corrected serum albumin concentrations in hypoproteinemic cats within 24 hours of treatment but also effectively improved serum albumin levels and overall prognosis.

[0102] Table 7 Analysis of blood pressure and pulse test results in cat treatment experiments

[0103]

[0104] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). Intergroup differences were analyzed using the t-test. P values < 0.05 indicated a significant difference; P values < 0.01 indicated an extremely significant difference; and P values > 0.05 indicated no significant difference. P1 indicates a significant difference between the Mut-FSA group and the control group, P2 indicates a significant difference between the FSA group and the control group, and P3 indicates a significant difference between the Mut-FSA group and the FSA group.

[0105] Table 8-1 Analysis of blood routine index test results in cat treatment experiments

[0106]

[0107] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). Intergroup differences were analyzed using the t-test. P values < 0.05 indicated a significant difference; P values < 0.01 indicated an extremely significant difference; and P values > 0.05 indicated no significant difference. P1 indicates a significant difference between the Mut-FSA group and the control group, P2 indicates a significant difference between the FSA group and the control group, and P3 indicates a significant difference between the Mut-FSA group and the FSA group.

[0108] Table 8-2 Analysis of blood routine index test results in cat treatment experiments

[0109]

[0110] Note: Data analysis is the same as Table 8-1

[0111] Table 9 Analysis of biochemical index test results in cat treatment experiments

[0112]

[0113] Note: All data were analyzed using SPSS 22.0 software, and all results were expressed as mean (M) ± standard deviation (SD). The t-test was used to analyze the between-group differences of the two groups of data. A P value < 0.05 indicates significant differences; a P value < 0.01 indicates extremely significant differences; a P value > 0.05 indicates no significant differences. Among them, the P1 value is the analysis of the significance of differences between the Mut-FSA group and the control group, the P2 value is the analysis of the significance of differences between the FSA group and the control group, and the P3 value is the analysis of the significance of differences between the Mut-FSA group and the FSA group.

Claims

1. An artificially synthesized cat serum albumin mutant Mut-FSA, the amino acid sequence of the mutant Mut-FSA is shown as SEQ ID NO.

5.

2. A fusion protein obtained by fusing the mutant protein shown in SEQ ID NO.5 with a protein tag.

3. The fusion protein according to claim 2, the amino acid sequence of the fusion protein is shown as SEQ ID NO.

3.

4. The coding gene of the mutant protein shown in SEQ ID NO.5 or the fusion protein according to claim 3.

5. The coding gene according to claim 4, characterized in that The coding gene of the fusion protein is shown as SEQID NO.

4.

6. An expression cassette, recombinant vector or isolated recombinant cell having the coding gene according to claim 4.

7. The recombinant cell according to claim 6, wherein It is a CHO cell.

8. A method for preparing the fusion protein shown in SEQ ID NO.3, including culturing CHO containing the gene shown in SEQ ID NO.

4.

9. Use of the mutant according to claim 1, the fusion protein according to claim 2, the coding gene of the mutant according to claim 1, or an expression cassette, recombinant vector or isolated recombinant cell of the coding gene of the fusion protein according to claim 2 in the preparation of a cat serum albumin mutant.

10. Use of the mutant according to claim 1, the fusion protein according to claim 2, the coding gene of the mutant according to claim 1, or an expression cassette, recombinant vector or isolated recombinant cell of the coding gene of the fusion protein according to claim 2 in the preparation of a drug for treating feline hypoproteinemia.

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