Preparation and characterization method of cat serum albumin

Abstract

The invention relates to the technical field of gene engineering, in particular to a cat serum albumin preparation and characterization method. The cat serum albumin gene sequence is optimized, a recombinant expression vector is successfully constructed, pichia pastoris genetically engineered bacteria are prepared, high-yield expression of the cat serum albumin gene in pichia pastoris is achieved, a purification system is established and perfected, and a pure product with the purity larger than 98% is obtained through affinity exchange column purification. Compared with the natural cat serum albumin, the cat serum albumin has a secondary structure, stability and antioxidant activity which are consistent with those of the natural cat serum albumin, has the characteristic of low hemolytic activity, can realize large-scale efficient production, can be applied to development of pet foods and medicines, and has wide application value and market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for preparing and characterizing cat serum albumin. Background Art

[0002] Serum albumin (SA) is the main protein in vertebrate plasma, accounting for about 60% of the total plasma protein. The relative molecular mass of SA is about 66 ku, which is composed of about 585 amino acids and is not glycosylated. In addition, SA contains 17 conserved disulfide bridges and a free thiol group at the 34th Cys at the N-terminus. It undertakes many important physiological functions, such as maintaining plasma colloidal osmotic pressure, transporting various substances including hormones, amino acids, fatty acids, divalent metal ions, and scavenging free radicals. In human clinical practice, large doses of SA are used as blood volume expanders to increase blood volume levels in hemorrhagic shock, severe burns and trauma, and after surgery; to treat hypoalbuminemia caused by ascites, cirrhosis, kidney disease, and massive bleeding. In the pharmaceutical industry, researchers fuse SA with drugs to increase the blood half-life of therapeutic proteins, and also use it as a nanocarrier for targeted drug delivery and as a component of vaccine formulations and cell culture media.

[0003] In recent years, the number of pets has continued to increase on the basis of a huge base. In pet clinics, due to the lack of available animal-specific serum albumin products, people choose human serum albumin (HSA) to treat hypoalbuminemia in dogs and cats, although the dose is small, it may also cause allergic symptoms. Moreover, although the sequence homology between feline serum albumin (FSA) and HSA is as high as 80%, there are still differences, such as the smaller Sudlow II site of FSA and the more flexible Cys-34, which makes FSA have different ligand binding affinity.

[0004] Worldwide, the market demand for SA has reached hundreds of tons and continues to increase. Currently, it is mainly derived from plasma, but the supply of plasma is limited and there is a risk of pathogen infection. In order to circumvent these problems, people widely use transgenic technology to heterologously express SA. However, most of the research focuses on the more popular and mature recombinant human serum albumin (rHSA), and how to produce recombinant feline serum albumin (rFSA) on a large scale and efficiently remains a difficulty. Summary of the invention

[0005] In order to produce recombinant feline serum albumin on a large scale and efficiently, the present invention first optimizes the gene sequence encoding feline serum albumin according to the codons preferred by Pichia pastoris, and obtains the optimized nucleic acid sequence encoding feline serum albumin through a large number of optimization designs and screenings, which is shown in SEQ ID NO.2.

[0006] Furthermore, the present invention provides biological materials containing the nucleic acid sequence.

[0007] In some embodiments, the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, or a viral vector.

[0008] Furthermore, the present invention provides a genetically engineered Pichia pastoris containing the nucleic acid sequence.

[0009] In some embodiments, the method for constructing the genetically engineered Pichia pastoris comprises: connecting the nucleic acid sequence to a pPIC9K vector to obtain a recombinant yeast expression vector; and then linearizing the recombinant yeast expression vector and transferring it into Pichia pastoris.

[0010] Furthermore, the present invention provides the use of the genetically engineered Pichia pastoris or the genetically engineered Pichia pastoris obtained by the construction method in the preparation of feline serum albumin.

[0011] Furthermore, the present invention provides a method for preparing feline serum albumin, comprising: fermenting and producing feline serum albumin using the Pichia pastoris genetically engineered bacteria or the Pichia pastoris genetically engineered bacteria obtained by the construction method.

[0012] Preferably, the fermentation is a high density fermentation.

[0013] Preferably, the fermentation production step comprises: inoculating the genetically engineered Pichia pastoris into a YPD liquid medium (preferably the YPD liquid medium contains antibiotics, such as G418, etc.), culturing in a shaking incubator at 28°C to 32°C (preferably culturing in a shaking incubator until OD 600 nm value is 5) to obtain seed liquid; the seed liquid is inoculated into a basal salt culture medium, the pH value is adjusted to 4.8-5.2, and after adding PMT1, the dissolved oxygen is maintained at more than 20% for fermentation culture; when the dissolved oxygen rises to more than 70%, a glucose solution (preferably the concentration of the glucose solution is 45%-55%) containing PMT1 (preferably the concentration is 10-15‰) is added, and then (preferably after starvation for 0.5 h to 1 h), methanol is added for induction, the pH is adjusted to 5.75, and the dissolved oxygen is controlled to be more than 20% until the fermentation ends.

[0014] Preferably, the flow acceleration of the glucose solution is 12 to 36 mL / L / min, the flow is continued for 6 to 8 hours, and the dissolved oxygen is maintained above 20%.

[0015] Preferably, the flow rate induced by the addition of methanol is gradually increased from 1 mL / L / min in the first hour to 10 mL / L / min in the tenth hour, the rotation speed is increased to above 1000 rpm, the pH is increased to 5.75, and the dissolved oxygen is controlled to be above 20% until the end of the fermentation.

[0016] Preferably, the basal salt culture medium comprises: 40-50 g / L glucose, 45-55 g / L NH4H2PO4, 15-25 g / L K2SO4, 10-20 g / L MgSO4·7H2O, 2-8 g / L KH2PO4, 0.1-0.6 g / L CaSO4 and 0.5-3 g / L KOH.

[0017] Preferably, the fermentation production step further comprises purifying the fermentation broth after the fermentation is completed, and the purification step is as follows: the HiTr Blue chromatography column is equilibrated with a phosphate buffer solution of pH 4.8-5.2 (preferably a concentration of 15-25 mM) for 3-5 column volumes and then loaded with the sample; after the loading is completed, the impurity band is first eluted with a phosphate buffer solution, and then the target band is eluted with a NaCl solution of pH 6.5-7.0 (preferably a concentration of 1-3 M), and the eluate is collected (preferably the elution is monitored according to UV280 nm); the eluate is dialyzed and desalted in a phosphate buffer solution, and after adjusting the pH to 4.8-5.2, purification is performed for more than 2 times.

[0018] After screening, the present invention found that the hydrophobic column HiTrap Phenyl HP, the anion column HiTrap DEAE FF, and the cationic column HiPrep SP FF were unable to specifically bind to the target protein, and the purity of the product was less than 90% when used alone or in combination. The HiTrBlue column material has the characteristic of specifically recognizing feline serum albumin.

[0019] Furthermore, the present invention provides cat serum albumin prepared by the preparation method.

[0020] Preferably, the hemolysis rate of the feline serum albumin at a concentration below 160 μM is <1%; and / or, the free thiol content per mole of feline serum albumin is 1.00-1.20 mol (preferably 1.01 mol); and / or, the protein retention rate of the recombinant feline serum albumin at 25° C.-70° C. is >80%; the protein retention rate at pH 1-11 is >75%; the protein retention rate of the recombinant feline serum albumin after incubation in plasma for 480 minutes is >95%; and / or, the recombinant feline serum albumin has the ability to scavenge free radicals.

[0021] Preferably, the recombinant feline serum albumin has temperature, pH and serum stability.

[0022] Preferably, the recombinant feline serum albumin is + • , •O 2— , DPPH• and OH• free radicals have scavenging ability.

[0023] Preferably, the recombinant feline serum albumin is+ • Free radical scavenging rate>95%, DPPH free radical scavenging rate>35%, OH free radical scavenging rate>60%, O2 — Free radical scavenging rate>40%.

[0024] Further, the present invention provides the use of the cat serum albumin in at least one of the following aspects: (1) Preparing cat food (such as kitten food, adult cat food, senior cat food or sterilized cat food, etc.); (2) Preparation of cat immunomodulators, immunoenhancers, nutritional supplements for illness, postpartum nutritional supplements or postoperative nutritional supplements; (3) Preparation of nanocarrier targeted drugs; (4) Preparation of vaccine adjuvants; (5) Prepare cell culture medium.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The invention optimizes the gene sequence of cat serum albumin, successfully constructs a recombinant expression vector, prepares Pichia pastoris genetic engineering bacteria, realizes high-yield expression of the cat serum albumin gene in Pichia pastoris, establishes and improves a purification system, obtains a pure product with a purity greater than 98% through affinity exchange column purification, and compares the pure product with natural cat serum albumin. The pure product has the secondary structure, stability and antioxidant activity consistent with those of natural cat serum albumin, and has the characteristics of low hemolysis, can realize large-scale and efficient production, can be applied to pet food and drug development, and has broad application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the recombinant expression vector pPIC9K-rFSA.

[0027] Figure 2 is the result of extraction and linearization electrophoresis of the recombinant expression vector pPIC9K-rFSA; where M: Trans 15K DNA Ladder; 1-2: circular and linearized pPIC9K; 3-4: circular and linearized pPIC9K-rFSA.

[0028] Figure 3 is the SDS-PAGE electrophoresis result of the supernatant of the rFSA recombinant strain induced fermentation at the well plate level, where M: Blue Plus Protein Marker; 1: electrophoresis band of the supernatant of the fermentation broth of an empty transformant containing pPIC9K; 2-11: electrophoresis bands of the supernatant of the fermentation broth of ten randomly selected highly resistant transformants.

[0029] Figure 4It is the change of protein expression level of the recombinant strain at induced fermentation time of 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 113 h (sample was loaded with ten-fold dilution).

[0030] Figure 5 This is the time change curve of the wet weight and total protein of the recombinant strain at different induction times of 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h and 113 h.

[0031] Figure 6 is the result of SDS-PAGE electrophoresis after rFSA purification; where M: Blue Plus Protein Marker; 1-2: fermentation supernatant and breakthrough peak, respectively; 3-4: 100% B solution elution peak and 0.1 M NaOH elution peak in the first purification; 5-6: 100% B solution elution peak and 0.1 M NaOH elution peak in the second purification.

[0032] Figure 7 This is the RP-HPLC analysis result of purified rFSA.

[0033] Figure 8 This is the result of the hemolytic test of purified rFSA.

[0034] Fig. 9 is the circular dichroism spectroscopy of purified rFSA and FSA.

[0035] Fig.10 This is the temperature stability result of purified rFSA.

[0036] Fig.11 is the pH stability result of purified rFSA.

[0037] Fig.12 is the plasma stability result of purified rFSA.

[0038] Fig.13 Purified rFSA to ABTS + •Inhibition rate results.

[0039] Fig.14 This is the inhibition rate of purified rFSA on DPPH•.

[0040] Fig.15 This is the inhibition rate of purified rFSA on OH•.

[0041] Fig.16 Is purified rFSA effective for •O 2- The inhibition rate results. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental materials, reagents and instruments used in the examples of the present invention are all commercially available. Unless otherwise specified, the technical means used in the examples are all conventional means well known to those skilled in the art.

[0044] The culture medium and buffer formulations involved in the following examples are as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; solid LB medium was added with 2% agar powder.

[0045] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose; solid YPD medium is added with 2% agar powder.

[0046] YPDS medium: peptone 20 g / L, yeast extract 10 g / L, sorbitol 182.2 g / L, glucose 20 g / L, agar powder 20 g / L.

[0047] BMGY medium / L: yeast extract 10 g, peptone 20 g, glycerol 10 mL, 13.4% amino acid-free yeast nitrogen base (YNB) 100 mL, 0.02% biotin 2 mL, 1 M phosphate buffer, pH 6.0, 100 mL.

[0048] For the use of LB medium, YPD, YPDS and other culture media, refer to the Invitrogen Pichia pastoris operation manual.

[0049] 20 mM phosphate buffer (solution A): 0.36 g Na2HPO4•12H2O, 2.96 g NaH2PO4•2H2O, add 900 mL of deionized water, place on a magnetic stirrer until completely dissolved, adjust the pH to 5.0, and make up to 1000 mL.

[0050] 20 mM phosphate buffer (solution B): 116.88 g NaCl, 3.51 g Na2HPO4•12H2O, 1.59 g NaH2PO4•2H2O, add 850 mL of deionized water, place on a magnetic stirrer until completely dissolved, adjust the pH to 6.8, and make up to 1000 mL.

[0051] The protein detection method involved in the following examples is SDS-PAGE, referring to (Schägger H., et al, 2006).

[0052] The protein concentration determination method involved in the following examples is the Coomassie Brilliant Blue method.

[0053] The protein purity detection method involved in the following examples is the RP-HPLC method.

[0054] The protein purification method involved in the following examples is affinity chromatography.

[0055] The fermentation method involved in the following examples is a high-density fermentation method.

[0056] The bacterial species and plasmids involved in the following examples are shown in Table 1.

[0057] Table 1 Test strains and plasmids

[0058] Example 1 Construction of recombinant expression vector The amino acid sequence of feline serum albumin FSA is shown in SEQ ID No. 1. According to the codon preference of the expression system Pichia pastoris, the gene coding sequence of FSA was designed, and the DNA sequence (SEQ ID NO. 2) was obtained by codon optimization.

[0059] In order to effectively terminate the translation expression of FSA, the stop codon TAA was added to the 3' end of the FSA gene sequence. Eco RI, Not I endonuclease site. The resulting gene expression cassette gene sequence is shown in SEQ ID NO. 3. The above sequence was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0060] The synthetic FSA gene sequence was inserted into the Eco RI and Not I double-digested pPIC9K vector, construct a recombinant expression vector pPIC9K-rFSA (such as Figure 1The transformed cells were plated on LB solid plates containing 50 μg / mL ampicillin and cultured overnight at 37°C, 250 rpm. Single clones grown on the LB plates were picked and cultured overnight in LB liquid medium containing 50 μg / mL ampicillin at 37°C, 250 rpm. The recombinant plasmids were extracted and identified by 0.7% agarose gel electrophoresis, as shown in Figure 2. Figure 2 , for linear electroporation P. pas toris prepared.

[0061] Example 2 Construction of recombinant expression strain 2.1 Linearization of the recombinant expression vector pPIC9K-rFSA Using endonucleases Pme I The constitutive recombinant expression vector pPIC9K-rFSA was digested with enzymes. The enzyme digestion system and reaction conditions are shown in Table 2.

[0062] Table 2

[0063] After the above enzyme digestion system was added, it was placed in a 37°C water bath for 4 h and detected by 0.7% agarose gel electrophoresis under the conditions of 135 V for 35 min. Figure 2 ) shows that the pPIC9K-rFSA recombinant vector is completely linearized.

[0064] 2.2 Linearized plasmid concentration Place the linearized plasmid into a vacuum freeze dryer and wait until the sample becomes powdery, then take it out for later use.

[0065] 2.3 Preparation of competent Pichia pastoris GS115 1) Pick a single colony of GS115 from the YPD plate and inoculate it into 10 mL YPD liquid medium. Culture overnight at 30°C and 250 rpm. 2) Take the overnight culture of Pichia pastoris GS115 and inoculate it into 200 mL YPD liquid medium at 1% inoculation volume. Cultivate at 30°C, 250 rpm until OD 600 nm The absorbance value is about 1.2; 3) Aliquot into 50 mL sterile centrifuge tubes, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, and resuspend in 50 mL sterile ddH2O water; 4) Centrifuge at 4°C, 5000 rpm for 10 min, remove the supernatant, and resuspend in 50 mL of sterile water; 5) Centrifuge at 4°C, 5000 rpm for 10 min, remove the supernatant, and add 2 mL of 1 M pre-cooled sorbitol to resuspend; 6) Finally, centrifuge at 4°C, 5000 rpm for 10 min, remove the supernatant, add 200 μL 1 M pre-cooled sorbitol and resuspend to obtain Pichia pastoris GS115 competent cells.

[0066] 2.4 Electroporation Take 100 μL of yeast competent cells and add the linearized recombinant plasmid lyophilized powder, mix gently, transfer to an ice-cold sterile electroporation cup, place on ice for 10 min, and electroporate at 1500 V, 25 μF, 400 Ω. Immediately after electroporation, add 1 mL of ice-cold 1 M sorbitol solution, mix well and transfer to a 2 mL centrifuge tube, resuscitate at 30°C for 2 h, take 100 μL, 150 μL, and 200 μL of the resuscitated bacterial solution and spread on MD plates, invert and culture at 30°C until a single colony grows. Pick the transformants and inoculate them on YPD plates containing different concentrations of G418 (0.5, 1, and 2 mg / mL) to further screen high-resistant transformants.

[0067] Example 3 Expression of recombinant yeast strain rFSA 3.1 Inducible expression of high-resistance transformants at the plate level Randomly select 10 positive transformants obtained by screening in Example 2 ( P. pastoris GS115-rFSA). The cells were inoculated into a 48-well microplate with 500 μL of BMGY medium added to each well, and cultured at 30°C, 250 rpm for 24 h. Then, 5 μL of methanol was added to each well for induction (the final concentration of methanol was 1%), which was recorded as induction 0 h. The cells were cultured at 30°C, 250 rpm, and 5 μL of methanol was added every 24 h until the induction was completed at 96 h.

[0068] The obtained recombinant yeast transformants were further analyzed by SDS-PAGE for the recombinant expression level ( Figure 3 ), and the transformant with the highest expression level was selected for high-density fermentation.

[0069] 3.2 High-density fermentation of recombinant yeast strain rFSA 1) Preparation of seed solution: Pick a single colony of the high-expressing yeast transformant after screening and place it in 10 mL YPD medium containing 1 mg / mL G418, culture it at 30°C, 250 rpm, and shake it for 24 h. Inoculate 1% of it in 200 mL YPD liquid medium and culture it at 30°C, 250 rpm, and shake it for 16-18 h until the OD reaches 0. 600 nm The value is 5, which means seed solution; 2) Initial stage of fermentation culture: 200 mL of seed liquid was inoculated into 2 L of basal salt medium (90 g glucose, 100 g NH4H2PO4, 40 g K2SO4, 30 g MgSO4·7H2O, 12 g KH2PO4, 0.4 gCaSO4 and 1.5 g KOH, and water was added to make up to 2 L) at a ratio of 10%. The temperature was controlled at 29°C, the pH value was adjusted to 5.0, the ventilation volume was maintained at 8-10 vvm, the rotation speed was maintained at 600 rpm for 6 h, and then adjusted to 800 rpm, and the dissolved oxygen was maintained above 20%; 3) Sugar-supplemented growth stage: The dissolved oxygen value was observed to decrease slowly at first and then suddenly increase. The rotation speed was increased to 1000 rpm. The dissolved oxygen rose to above 70%. A 50% glucose solution containing 12‰ PMT1 was added. The flow acceleration was 24 mL / L / min. The flow was continued for 8 h and the dissolved oxygen was maintained above 20%. 4) Methanol induction: After the addition of glucose, starve the culture for 0.5 h to 1 h, then start to add 100% methanol, gradually increase the flow rate from 1 mL / L / min in the first hour to 10 mL / L / min in the tenth hour, increase the rotation speed to 1100 rpm, increase the pH to 5.75, and control the dissolved oxygen to above 20% until the end of fermentation.

[0070] Figure 4 is the change of protein expression level of the recombinant strain at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 113 h of induced fermentation time. Figure 5 The curves of the changes in wet weight and total protein of the bacteria at different induction times.

[0071] Example 4 Purification of rFSA After preliminary screening, the hydrophobic column HiTrap Phenyl HP, the anion column HiTrap DEAE FF, and the cationic column HiPrep SP FF were unable to specifically bind to the target protein, and the purity of the product was less than 90% when used alone or in combination. HiTr Blue column material has the characteristic of specifically recognizing feline serum albumin, and the purification process is as follows: The HiTr Blue column was equilibrated with 20 mM phosphate buffer (A solution) at pH 5.0 for 3-5 column volumes before loading the sample. After the sample was injected, the impurity band was eluted with A solution first, and the target band was eluted with 2 M NaCl elution buffer (B solution) at pH 6.8. The elution peak was collected and the color was determined according to the UV. 280 nm Monitor the elution. Finally, wash the column with 0.1 M NaOH. The collected eluate is dialyzed against solution A for desalting, and after adjusting the pH to 5.0, a second purification is performed under the same conditions.

[0072] Figure 6 This is the SDS-PAGE image after rFSA purification. After purification by HiTr Blue chromatography column, no obvious impurity protein bands were observed. Figure 7 The RP-HPLC test for purified rFSA showed that the protein purity could reach 98%. The purified rFSA was then desalted through a 1 ku molecular weight dialysis bag, freeze-dried and stored for later use.

[0073] Example 5 Hemolytic activity of rFSA The rFSA lyophilized powder obtained in Example 4 was dissolved in sterile PBS (pH 7.4) to prepare a mother solution with a concentration of 160 μM (about 11.2 mg / mL), and the final concentration of 2-fold dilution was 1.25 μM (about 0.088 mg / mL). Take 6-8 week old SPF-grade ICR mice, collect blood from the eyeballs, and collect it into heparin sodium anticoagulation tubes. The collected blood was centrifuged at 4°C, 2000 rpm, and the red blood cells were washed repeatedly with PBS for 3 to 5 times until the supernatant was transparent and colorless. PBS was diluted to an 8% red blood cell suspension. The red blood cell suspension and rFSA of each concentration were mixed at 1:1 (v / v) and incubated at 37°C for 1 h. After centrifugation at 2000 rpm for 5 min, 100 μL of supernatant was taken from each well, added to a 96-well plate, and the absorbance at 540 nm was detected. At the same time, PBS and 0.1% Triton X-100 were 0% and 100% hemolysis control experiments, respectively.

[0074] The formula for calculating the degree of hemolysis is as follows (refer to Jung, Park et al., 2007): Hemolysis rate (%) = [(A rFSA -A PBS ) / (A 0.1%Triton X-100 -A PBS )]×100%. The result is Figure 8 As shown, the hemolysis rate of each concentration of rFSA was close to 0.

[0075] The results showed that rFSA in the concentration range of 1.25~160 μM hardly caused hemolysis of red blood cells.

[0076] Example 6 Determination of free thiol content of rFSA 125 μL L-Cys solution (5 μM~160 μM) and rFSA solution (40 μM, 2.8 mg / mL) were mixed with 25 μL DTNB solution (1 mg / mL) respectively, and the absorbance at 412 nm was measured after reacting at room temperature for 20 min. A standard curve of concentration and absorbance was established with L-Cys. The absorbance value of rFSA was substituted into the calculation. The results showed that the free thiol content of rFSA and natural FSA was 1.01 mol and 1.16 mol per mole, respectively, and the total free thiol content of the two was similar.

[0077] Example 7 Secondary structure analysis of rFSA The secondary structure of rFSA and FSA was analyzed using a Pistar π-180 circular dichroism spectrometer. The protein sample was dissolved in PBS to a final concentration of 400 μg / mL. The protein solution was added to a 1 mm diameter quartz cuvette for circular dichroism scanning. The scanning range was 190 nm~260 nm, and the scan was performed twice, with PBS as a blank control. The collected data was processed using the software Pro-Data Viewer and CDNN. The results are shown in Fig. 9 And as shown in Table 3.

[0078] Table 3 Proportion of each secondary structure element

[0079] The results of secondary structure analysis showed that the circular dichroism spectra of rFSA and FSA were almost identical, and the characteristic negative peaks of the two α-helices appeared near 208 nm and 222 nm. The proportions of each secondary structure element were consistent, with the helical structure accounting for a larger proportion, indicating that rFSA was folded correctly.

[0080] Example 8 Analysis of pH, temperature and plasma stability of rFSA In the temperature stability test, the rFSA solution was incubated at 25°C, 40°C, 55°C, 70°C, 85°C, and 100°C for 1 h. In the pH stability test, the rFSA solution was mixed with phosphate buffers of pH 1, 3, 5, 7, 9, 11, and 13 at a ratio of 1:9 and incubated at 25°C for 3 h. In the plasma stability test, the rFSA solution was mixed with mouse plasma at a ratio of 3:1 and incubated for 15 min, 30 min, 60 min, 120 min, 240 min, and 480 min. The rFSA retained after treatment under different conditions was relatively quantified by SDS-PAGE and ImageJ.Js tools, with the untreated rFSA as 100% retention rate. The results are shown in Figure 2. Figures 10-12 shown.

[0081] The results of the stability experiment showed that compared with naturally extracted FSA, rFSA showed similar stability at different temperatures and pH. Within 25℃~70℃, the protein retention rate was above 80%. When the temperature reached 85℃, the protein showed obvious degradation, and only about 10% remained at 100℃. Within pH 1~11, the protein retention rate was high, and it was most stable at pH 7, while in a highly alkaline environment of pH13, the protein retention rate was only 50%. For plasma stability, rFSA and FSA still did not show any obvious degradation after long-term incubation.

[0082] Example 9 Analysis of the in vitro antioxidant activity of rFSA 9.1 DPPH scavenging ability Add 125 μL of DPPH solution (0.2 mM) and 125 μL of rFSA solution with different concentration gradients (16 μM~0.5 μM) to a 96-well plate, mix, incubate at room temperature for 30 min, and measure the absorbance at a wavelength of 517 nm. The results are as follows: Fig.11 .

[0083] 9.2 About ABTS + • Clearing ability ABTS solution (7.4 mM) and K2S2O8 solution (2.6 mM) were mixed in a ratio of 1:1, reacted in the dark for 12 h, and then diluted about 50 times to obtain ABTS working solution (OD 405 nm ≈0.70±0.02). 160 μL of working solution and 40 μL of rFSA solution with different concentration gradients (16 μM~0.5 μM) were added to a 96-well plate, mixed, and allowed to stand in the dark for 5 min. The absorbance was detected at a wavelength of 405 nm.

[0084] 9.3 OH· Scavenging Capacity 35 μL FeSO4 (3 mM), 35 μL H2O2 (3 mM), 10 μL TMB (6 mg / mL) and 20 μL rFSA solution (16 μM~0.5 μM) were added to a 96-well plate in sequence and reacted for 20 min. 200 μL 10% acetic acid solution was added to terminate the reaction and the absorbance at 450 nm was immediately measured.

[0085] 9.4 Pairs • O2 — Clearing ability In a 96-well plate, add 60 μL rFSA solution (16 μM~0.5 μM), 40 μL NBT (0.8 mM), 40 μL xanthine (0.8 mM) and 10 μL xanthine oxidase (10 U / mL) in sequence, mix, react at 37°C for 30 min, and then measure the absorbance at 560 nm.

[0086] The scavenging rate of the four free radicals was calculated according to the following formula: Scavenging rate (%) = (1-(Asample-Asamplecontrol) / Acontrol) × 100, where Asample is the absorbance of the sample solution and each reagent solution; Asamplecontrol is the absorbance of the sample solution itself; and Acontrol is the absorbance of the sample solvent and each reagent solution. The results are as follows: Figures 13-16 shown.

[0087] The results showed that rFSA and FSA had a dose-effect relationship in scavenging the four free radicals, and the overall trend was consistent with only partial differences. + • and • O2 — The inhibition rate of rFSA on DPPH• and OH• was higher than that of FSA. At a higher concentration of 16 μM, the inhibition rate of rFSA on DPPH• and OH• was higher than that of FSA.

[0088] In summary, the present invention successfully constructed the pPIC9K-rFSA recombinant expression vector. Pme After linearization, I was successfully transformed into Pichia pastoris GS115 to obtain a recombinant yeast strain, and the fermentation broth was obtained through high-density fermentation, and rFSA was obtained through purification. rFSA has the same secondary structure, stability and antioxidant activity as natural FSA, so it has good application prospects in production practice.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An optimized nucleic acid sequence, characterized in that It is shown as SEQ ID NO.

2.

2. A biological material containing the nucleic acid sequence according to claim 1.

3. The biomaterial according to claim 2, characterized in that The biological material is recombinant DNA, expression cassette, transposon, plasmid vector or virus vector.

4. A genetically engineered Pichia pastoris, characterized in that: It contains the nucleic acid sequence according to claim 1.

5. The method for constructing the genetically engineered Pichia pastoris according to claim 4, characterized in that: include: The nucleic acid sequence of claim 1 is connected to the pPIC9K vector to obtain a recombinant yeast expression vector; and then the recombinant yeast expression vector is linearized and transferred into Pichia pastoris.

6. Use of the genetically engineered Pichia pastoris bacteria according to claim 4 or the genetically engineered Pichia pastoris bacteria obtained by the construction method according to claim 5 in the preparation of feline serum albumin.

7. A method for preparing cat serum albumin, characterized in that: include: The feline serum albumin is produced by fermentation using the genetically engineered Pichia pastoris bacteria described in claim 4 or the genetically engineered Pichia pastoris bacteria obtained by the construction method described in claim 5.

8. The preparation method according to claim 7, characterized in that: The fermentation production steps include: inoculating the Pichia pastoris genetically engineered bacteria into a YPD liquid culture medium, and culturing in a shaking table at 28°C to 32°C to obtain a seed solution; inoculating the seed solution into a basal salt culture medium, adjusting the pH value to 4.8 to 5.2, and maintaining the dissolved oxygen at more than 20% after adding PMT1 for fermentation culture; when the dissolved oxygen rises to more than 70%, adding a glucose solution containing PMT1, and then adding methanol for induction, adjusting the pH to 5.75, and controlling the dissolved oxygen to more than 20% until the fermentation ends; The basic salt culture medium includes: 40-50 g / L glucose, 45-55 g / L NH4H2PO4, 15-25 g / L K2SO4, 10-20 g / L MgSO4·7H2O, 2-8 g / L KH2PO4, 0.1-0.6 g / L CaSO4 and 0.5-3 g / L KOH; Preferably, the fermentation production step further includes purifying the fermentation broth after the fermentation is completed, and the purification step is as follows: equilibrate the HiTr Blue chromatography column with a phosphate buffer of pH 4.8-5.2 for 3-5 column volumes and then load the sample; after the loading is completed, first elute the impurity band with a phosphate buffer, and then elute the target band with a NaCl solution of pH 6.5-7.0, and collect the eluate; dialyze the eluate in a phosphate buffer for desalination, and adjust the pH to 4.8-5.2, and then purify it more than 2 times.

9. Cat serum albumin prepared by the preparation method according to claim 7 or 8; preferably, the hemolysis rate of the cat serum albumin at a concentration below 160 μM is <1%; and / or, the free thiol content per mole of cat serum albumin is 1.00-1.20 mol; and / or, the protein retention rate of the recombinant cat serum albumin at 25° C.-70° C. is >80%; the protein retention rate at pH 1-11 is >75%; the protein retention rate of the recombinant cat serum albumin after incubation in plasma for 480 minutes is >95%; and / or, the recombinant cat serum albumin has the ability to scavenge free radicals.

10. The use of cat serum albumin according to claim 9 in at least one of the following aspects: (1) preparing cat food; (2) Preparation of cat immunomodulators, immunoenhancers, nutritional supplements for illness, postpartum nutritional supplements or postoperative nutritional supplements; (3) Preparation of nanocarrier targeted drugs; (4) Preparation of vaccine adjuvants; (5) Prepare cell culture medium.

Images