Cat serum albumin mutant as well as CHO cell expression method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a cat serum albumin mutant as well as a CHO cell expression method and application thereof. The invention provides a cat serum albumin mutant Mut-FSA for the first time, and an optimized Mut-FSA sequence is finally formed by replacing the 558th K at the tail end of an FSA amino acid sequence with R. Compared with non-mutated FSA, the Mut-FSA can show more remarkable curative effect advantages in treatment intervention of cat hypoproteinemia or acute hemorrhage, in addition, the Mut-FSA not only can enable cats suffering from the hypoproteinemia to achieve rapid correction of serum albumin concentration within 24 hours after treatment intervention, but also can effectively improve the serum albumin level and overall prognosis of the cats. The recombinant FSA prepared by the invention not only can provide a more effective innovative therapy for cat hypoproteinemia or acute hemorrhage, but also has wide application prospect 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, and a CHO cell expression method and application thereof. The technical solution of the present invention significantly improves the yield and purity of recombinant FSA, and at the same time has the advantages of high safety and low immunogenicity. It can be used as an innovative therapy for treating feline hypoproteinemia or acute blood loss in clinical practice, and provides a more economical, safe and efficient therapeutic drug option for the pet medical industry. Background Art

[0002] In the field of clinical medicine, feline serum albumin (FSA) is widely used to treat hypoproteinemia, hemorrhagic shock, burns, ascites due to cirrhosis, and nephrotic edema. The acquisition of FSA mainly relies on blood extraction, which faces the problem of extremely scarce supply. There are also potential risks such as blood-borne viruses, which makes it impossible to meet the growing market demand.

[0003] Therefore, the development of efficient, safe and easy-to-scale FSA production technology has become a key issue that needs to be urgently addressed in the pet medical field. As an important expression system in the biopharmaceutical field, CHO cells have the advantages of perfect protein glycosylation modification function, accurate post-transcriptional modification, low risk of viral contamination, and less endotoxin production. In addition, the CHO cell expression system has strong gene amplification capabilities and high exogenous protein expression capabilities, which can achieve 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 difficulties. 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, potential safety hazards of traditional extraction methods and limitations of alternative treatment options, the present invention proposes a feline serum albumin mutant, and a CHO cell expression method and application thereof. Summary of the invention

[0005] Based on the current problems of limited FSA sources, high production costs, insufficient safety, etc., 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, an optimized Mut-FSA sequence is finally formed. The amino acid sequence of Mut-FSA is shown as SEQ ID NO.5. Compared with the unmutated FSA, Mut-FSA can show a more significant curative 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 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 as 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 as SEQ ID NO.4.

[0014] An expression cassette, recombinant vector, recombinant microorganism or isolated 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 as 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 isolated 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 isolated 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 cat 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 has shown good therapeutic effects in dimensions such as physiological function indicators, blood routine indicators, biochemical indicators, and the time for visible mucous membrane to return to normal color. Moreover, compared with the unmutated FSA, Mut-FSA can show more significant therapeutic effect advantages in the treatment intervention of feline hypoproteinemia or acute blood loss (P<0.05). In addition, Mut-FSA can not only enable feline hypoproteinemic cats to achieve rapid correction of serum albumin concentration 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. Brief 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 body 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 body 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 gums, nose tips, 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 body 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 the cat's gingiva, nose tip and conjunctiva are the clinical observation results on the 7th day after treatment. Detailed implementation mode

[0032] The present invention will be further described below through embodiments, and its purpose is only to better understand the research content of the present invention rather than limit the protection scope of the present invention. The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified, and the reagents or materials are all from commercial channels unless otherwise specified.

[0033] Example 1:

[0034] Design and obtain two recombinant cat serum albumins:

[0035] 1.1 Design of the nucleotide sequences of two recombinant cat serum albumins:

[0036] Search for and obtain the gene sequence and amino acid sequence of cat serum albumin (GenBank: CAA59279.1) from NCBI.

[0037] Design two amino acid sequences of cat serum albumin:

[0038] (1) FSA: By connecting the HRV 3C cleavage site at the C-terminus of the FSA original sequence, followed by the GGGGS flexible linker peptide, Twin-Strep-tag and 10×His tag, a fusion protein obtained by fusing FSA with the protein tag was obtained, which is simply referred to as FAS in the embodiments of the present invention. The amino acid sequence of FSA is shown in SEQ ID NO.1, and the nucleotide sequence encoding it (after codon optimization by the CHO system) is shown in SEQ ID NO.2.

[0039] (2) Mut-FSA: By connecting the HRV 3C cleavage site at the C-terminus of the FSA original sequence, followed by the GGGGS flexible linker peptide, Twin-Strep-tag and 10×His tag, and replacing the 558th K at the C-terminus of the FSA amino acid sequence with R, the 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 simply referred to as Mut-FAS in the embodiments of the present invention. The amino acid sequence of Mut-FSA is shown in SEQ ID NO.3, and the polynucleotide encoding it (after codon optimization by the CHO system) 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 recombinant cat serum albumin can be directly commercially synthesized or obtained by microbial expression. In the present invention, it is obtained by commercial synthesis and CHO eukaryotic expression.

[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 codon-optimized target gene sequences SEQ ID NO.2 or SEQ ID NO.4 for the CHO system were respectively inserted into the pXC17.4 vector 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 respectively introduced into CHO cells 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 through 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 high-purity 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 expectations, 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 house was strictly disinfected by formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity in the animal house were controlled at 24±2°C and 40-60% respectively, and the experimental animals were fed and watered freely.

[0052] Ten healthy cats at 10 months old with a body weight of 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 2-fold 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. The body temperature, body weight, blood pressure, and pulse of all cats were measured on days 0 (before the examination), 1, 3, 5, and 7 (after the examination) respectively. 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 observations were carried out, the gross changes of the visceral tissues were recorded, and photos were taken. 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 respectively and fixed in 4% paraformaldehyde solution for more than 24 h, and then HE staining was performed on each visceral 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 after 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 in 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 between-group differences of 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 between-group differences of 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: The data analysis is the same as that 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: The data analysis is the same as that 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: The data analysis is the same as that 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 between-group differences of 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: The data analysis is the same as that in Table 3-1

[0075] Table 3-3 Analysis of the Detection Results of Biochemical Indexes in the Cat Safety Experiment

[0076]

[0077] Note: The data analysis is the same as that in Table 3-1

[0078] Example 3:

[0079] Establishment of a Cat Hypoproteinemia Animal Model

[0080] Before the experiment, the animal house was strictly disinfected with formaldehyde fumigation method and potassium permanganate. During the experiment, the temperature and humidity of the animal house were controlled at 24±2°C and 40-60% respectively. The experimental animals were fed and watered freely.

[0081] Ten healthy cats at 10 months old and weighing about 2.5-3.5 kg were selected for the experiment and randomly divided into a modeling group (n = 5) and a control group (n = 5). After the experimental cats were acclimatized in the animal house for 1 week, the cats in the modeling group were anesthetized with Zoletil® 50 anesthetic (5-7.5 mg / kg). Subsequently, 30% of the total body blood volume of the cats in the modeling group was collected at one time through the jugular vein (the total body blood content accounts for 6-7% of the body weight). The cats in the control group were not treated. The body temperature, body weight, blood pressure and pulse of all experimental cats were measured at 0 (before modeling), 12, 24, 36 and 48 h (after modeling), respectively. At the same time, the gums, nose tips and conjunctivae were observed and photographed. In addition, anticoagulated blood and serum samples of all experimental cats were collected at the specified time points for routine blood and biochemical index detection. The following results were obtained from the experiment:

[0082] Compared with the control group, the body temperature and body weight of the cats in the modeling group showed a downward trend, but there was no significant difference (P>0.05) (see Figure 4 ); starting from 12 h after modeling, the systolic blood pressure and diastolic blood pressure of the cats in the modeling group were significantly lower than those in the control group (P<0.01), and the pulse rate increased significantly (P<0.01) (see Table 4); the clinical observation results showed that compared with before modeling, at 24 h after modeling, obvious pallor could be observed in the gums, nose tips and conjunctivae of the cats in the modeling group. In addition, the cats in the modeling group also showed symptoms such as listlessness, decreased appetite and reduced activity (see Figure 5); The test results of blood routine indicators showed that at 12 h after modeling, the number of red blood cells (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 h after modeling, the test values of various blood routine indicators of RBC, HGB, and HCT deviated from the normal reference range (see Tables 5-1 and 5-2); The test results of biochemical indicators showed that at 12 h after modeling, the total protein (TB), albumin (ALB), and globulin (GLB) in the serum of cats in the modeling group were significantly decreased compared with the control group (P < 0.05 or P < 0.01). And starting from 24 h after modeling, the test values of various biochemical indicators of TB, ALB, and GLB deviated from the normal reference range (see Table 6).

[0083] In summary, compared with the control group, significant changes occurred in the clinical observation symptoms, physiological function indicators, blood routine, and biochemical indicators of cats in the modeling group. Based on the above experimental results, it was determined in this experiment that the establishment of the cat hypoproteinemia animal model was successful. In addition, this experiment further found that at 24 h after modeling, the differences in various indicators between the modeling group and the control group were the most significant. Therefore, this experiment used 24 h after modeling (24 h after blood sample collection) as the key evaluation node for evaluating the therapeutic effects of FSA and Mut-FSA.

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

[0085]

[0086] 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 groups of data. A P value < 0.05 indicates a significant difference; a P value < 0.01 indicates a highly significant difference; a P value > 0.05 indicates no significant difference.

[0087] Table 5-1 Analysis of blood routine indicator test results in the cat modeling experiment

[0088]

[0089] 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 groups of data. A P value < 0.05 indicates a significant difference; a P value < 0.01 indicates a highly significant difference; a P value > 0.05 indicates no significant difference.

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

[0091]

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

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

[0094]

[0095] 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 difference between the two groups of data. P value < 0.05 indicates a significant difference; P value < 0.01 indicates an extremely significant difference; P value > 0.05 indicates 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 by formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity of the animal room were controlled at 24±2℃ and 40~60%, respectively, and the experimental animals were fed and watered freely.

[0099] Fifteen healthy cats aged 10 months and weighing about 2.5-3.5 kg were selected for the experiment and randomly divided into Mut-FSA group (n=5), FSA group (n=5) and control group (n=5). After the experimental cats were adapted to the animal room for 1 week, they were anesthetized with Shutai® 50 anesthetic (5-7.5 mg / kg). Subsequently, 30% of the cats' total blood volume was collected at one time through the jugular vein. 24 hours after the blood sample was collected, each cat in the Mut-FSA group and FSA group was intravenously infused with Mut-FSA and FSA at a dose of 0.4 g / kg, respectively, and each cat in the control group was intravenously infused with the same volume of 0.9% saline. Before and after modeling and on the 1st, 3rd, 5th and 7th days after Mut-FSA and FSA treatment, the body temperature, weight, blood pressure and pulse of all experimental cats were measured, and the gums, nose and conjunctiva were observed and photographed. In addition, anticoagulated blood and serum samples were collected at designated time points for blood routine and biochemical index testing. The following results were obtained:

[0100] The results of body temperature and weight measurement 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 stabilize (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 of cats in the control group was slower than that of 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 of cats in the Mut-FSA group was faster than that of 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 was not completely restored to physiological pink (see Figure 7 ); The results of routine blood test 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 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 period, the recovery speed of hematological indexes of cats in the Mut-FSA group was faster than that of the FSA group, but there was no significant difference (P > 0.05) (see Table 8-1 and Table 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 group and the FSA group demonstrated good therapeutic effects in terms of physiological function indicators, blood routine indicators, biochemical indicators, and the time of visible mucosal color restoration. It should be emphasized that, compared with FSA, Mut-FSA showed a more significant therapeutic advantage in the treatment of feline hypoproteinemia (P<0.05). In addition, Mut-FSA can not only rapidly correct the serum albumin concentration in feline hypoproteinemia patients within 24 hours after treatment intervention, but also effectively improve the serum albumin level and overall prognosis of the patients.

[0102] Table 7 Analysis of blood pressure and pulse detection results in feline treatment experiments

[0103]

[0104] 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 groups of data. A P value < 0.05 indicates a significant difference; a P value < 0.01 indicates a highly significant difference; a P value > 0.05 indicates no significant difference. Among them, the P1 value is the analysis of the significance of the difference between the Mut-FSA group and the control group, the P2 value is the analysis of the significance of the difference between the FSA group and the control group, and the P3 value is the analysis of the significance of the difference between the Mut-FSA group and the FSA group.

[0105] Table 8-1 Analysis of blood routine indicator detection results in feline treatment experiments

[0106]

[0107] 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 groups of data. A P value < 0.05 indicates a significant difference; a P value < 0.01 indicates a highly significant difference; a P value > 0.05 indicates no significant difference. Among them, the P1 value is the analysis of the significance of the difference between the Mut-FSA group and the control group, the P2 value is the analysis of the significance of the difference between the FSA group and the control group, and the P3 value is the analysis of the significance of the difference between the Mut-FSA group and the FSA group.

[0108] Table 8-2 Analysis of blood routine indicator detection results in feline treatment experiments

[0109]

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

[0111] Table 9 Analysis of biochemical indicator detection results in feline 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 inter-group differences between the two groups of data. A P-value < 0.05 indicates a significant difference; a P-value < 0.01 indicates a highly significant difference; a P-value > 0.05 indicates no significant difference. Among them, the P1 value is the analysis of the significance of the difference between the Mut-FSA group and the control group, the P2 value is the analysis of the significance of the difference between the FSA group and the control group, and the P3 value is the analysis of the significance of the difference between the Mut-FSA group and the FSA group.

Claims

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

5.

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

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

3.

4. A gene encoding the mutant according to claim 1 or the fusion protein according to claim 2.

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

4.

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

7. The recombinant cell according to claim 6, characterized in that For CHO cells.

8. A method for preparing the fusion protein of SEQ ID NO.3, comprising culturing CHO containing the gene of 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 the coding gene of the fusion protein according to claim 2, an expression cassette, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell having the coding gene according to claim 4 in preparing a feline 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 the coding gene of the fusion protein according to claim 2, an expression cassette, a recombinant vector, a recombinant microorganism or an in vitro recombinant cell having the coding gene according to claim 4 in the preparation of a medicament for treating feline hypoproteinemia or acute blood loss.

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