Cat-derived superoxide dismutase as well as preparation method and application thereof

Abstract

The invention discloses feline-derived superoxide dismutase as well as a preparation method and application thereof, and particularly discloses a nucleic acid molecule, and the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1; or a nucleotide sequence which is obtained by substitution and / or deletion and / or addition of one or more modified base sequences on the nucleotide sequence as shown in SEQ ID NO: 1 and is consistent with the amino acid sequence obtained by translation of SEQ ID NO: 1. According to the preparation method provided by the invention, the cat superoxide dismutase gene subjected to codon optimization is transferred into pichia pastoris through two different plasmids by utilizing a technical means of genetic engineering, so that a pichia pastoris engineering strain is constructed; the cat superoxide dismutase can be efficiently and stably expressed by using different induction types of pichia pastoris engineering strains, so that the technical blank is filled, and a foundation is laid for developing the application of cat-derived SOD products in pet treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a cat-derived superoxide dismutase and a preparation method and application thereof. Background Art

[0002] Superoxide dismutase (SOD) is an important metalloenzyme that can scavenge free radicals and provide antioxidant protection. It is widely distributed in animals, plants, and microorganisms and plays a key role in the body's oxidation and antioxidant balance. Its catalytic mechanism is to catalyze the alternating redox reactions of metal ions, causing superoxide anion radicals to undergo dismutation reactions to produce hydrogen peroxide and oxygen. SOD can be divided into four major categories based on the metal cofactor: copper-zinc superoxide dismutase, manganese superoxide dismutase, iron superoxide dismutase, and nickel superoxide dismutase. Eukaryotic copper-zinc superoxide dismutases are divided into SOD1 and SOD3. Among them, SOD1 is mainly distributed in chloroplasts and cytoplasm and exists as a homodimer, while SOD3 is mainly distributed in the extracellular space of plants and mammals and exists as a tetramer. The feline superoxide dismutase (Felis catus superoxide dismutase, FSOD) of the present invention belongs to the copper-zinc superoxide dismutase.

[0003] Superoxide dismutase (SOD) is a vital component of the body's antioxidant defense system and is widely used in medicine, food, cosmetics, chemicals, and agriculture. Clinically, SOD can be used to treat intestinal diseases, prostate injuries, burns, corneal ulcers, and other conditions. It is also used to prevent and treat chronic diseases such as cardiovascular disease, cancer, asthma, neurological diseases, diabetes, and transplant rejection prevention and postoperative care. In cosmetics, SOD is used in products for anti-inflammatory and antioxidant effects, as well as to slow skin aging and damage. Furthermore, SOD has significant potential for application in veterinary products. Studies have shown that exogenous SOD can act as an inflammation suppressant, alleviating inflammation in various animal models. SOD can reduce oxidative stress caused by excessive reactive oxygen species production in animal cells, alleviate the damage and inflammation caused by oxidative stress, and promote growth. It is a candidate product to replace antioxidant enzyme preparations. Adding SOD enzyme preparations to feed can effectively offset the adverse effects of antibiotic withdrawal on farmed animals, making SOD an effective alternative to veterinary antibiotics.

[0004] Currently, SOD is primarily produced by extracting it from the serum or liver of animals such as cattle, pigs, and chickens, but it can also be extracted from seeds, grains, vegetables, and fruits. However, the low concentration, poor stability, and low extraction rate of natural SOD in plants and animals have hindered its industrialization. Among commonly used exogenous protein expression systems, the Escherichia coli expression system suffers from drawbacks such as the inability to glycosylate or perform complex assembly of the expressed protein; while mammalian cell expression systems suffer from high cell culture costs, stringent conditions, and low protein expression levels. Currently, there are no reports of exogenously expressing feline SOD in microorganisms. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a nucleic acid molecule that can be used to prepare FSOD, laying a theoretical foundation and providing technical support for the development of FSOD products for the treatment of pets.

[0006] The present invention also provides a biomaterial.

[0007] The invention also provides a method for preparing cat-derived superoxide dismutase.

[0008] The present invention also provides a cat-derived superoxide dismutase prepared by the above preparation method.

[0009] The present invention also provides the application of the cat-derived superoxide dismutase.

[0010] The invention also provides an antioxidant product.

[0011] According to the first aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleotide sequence of the nucleic acid molecule is:

[0012] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0013] (2) A nucleotide sequence obtained by replacing and / or deleting and / or adding one or more modified base sequences of the nucleotide sequence shown in SEQ ID NO: 1, and obtaining a nucleotide sequence that is consistent with the amino acid sequence obtained by translating the nucleotide sequence described in (1).

[0014] According to a second aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following (1) to (7):

[0015] (1) an expression cassette containing the nucleic acid molecule described in the first aspect of the present invention;

[0016] (2) a recombinant vector containing the nucleic acid molecule described in the first aspect of the present invention;

[0017] (3) a recombinant vector containing the expression cassette described in (1);

[0018] (4) a transgenic cell line containing the nucleic acid molecule described in the first aspect of the present invention;

[0019] (5) a transgenic cell line containing the expression cassette described in (1);

[0020] (6) A transgenic cell line containing the recombinant vector described in (2);

[0021] (7) A transgenic cell line containing the recombinant vector described in (3).

[0022] In some embodiments of the invention, the transgenic cell line does not comprise reproductive material.

[0023] According to a third aspect of the present invention, a method for preparing cat-derived superoxide dismutase is provided, comprising the following steps: transferring a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 1 into a host cell for inducing expression.

[0024] In some embodiments of the present invention, the nucleic acid molecule is codon-optimized for yeast preference based on the FSOD gene.

[0025] In some embodiments of the present invention, the vector backbones used for the transfer of the nucleic acid molecule include pPICZαA and pGAPZαA.

[0026] In some embodiments of the present invention, the host cells include Pichia pastoris X33 and Pichia pastoris SMD1168H.

[0027] In some embodiments of the present invention, the inducing agent used to induce expression contains methanol or glucose, and also includes sorbitol.

[0028] In some preferred embodiments of the present invention, the mixture of methanol and sorbitol is obtained by mixing equal volumes of methanol and 50% sorbitol.

[0029] In some preferred embodiments of the present invention, the mixture of glucose and sorbitol is obtained by mixing equal volumes of 20% glucose and 50% sorbitol.

[0030] In some preferred embodiments of the present invention, the inducer is added once in the morning and once in the evening.

[0031] In some embodiments of the present invention, the time for inducing expression is 4 to 8 days.

[0032] In some preferred embodiments of the present invention, the time for inducing expression in step S5 is 5 to 7 days.

[0033] According to a fourth aspect of the present invention, a cat-derived superoxide dismutase prepared by the preparation method described in the third aspect of the present invention is provided.

[0034] In some embodiments of the present invention, the amino acid sequence of the feline superoxide dismutase is shown in SEQ ID NO: 3.

[0035] According to a fifth aspect of the present invention, a use of the cat-derived superoxide dismutase as described in the fourth aspect of the present invention in the preparation of an antioxidant product is proposed.

[0036] According to a sixth aspect of the present invention, an antioxidant product is provided, wherein the antioxidant product is prepared by any one of (1) to (3):

[0037] (1) The nucleic acid molecule according to the first aspect of the present invention;

[0038] (2) The biomaterial according to the second aspect of the present invention;

[0039] (3) The cat-derived superoxide dismutase as described in the fourth aspect of the present invention.

[0040] In some embodiments of the present invention, the antioxidant product comprises a medicine or a feed additive.

[0041] In some preferred embodiments of the present invention, the dosage form of the drug includes at least one of injection, tablet, capsule, oral liquid, granule, ointment, suppository, and aerosol.

[0042] In some preferred embodiments of the present invention, the drug further comprises pharmaceutically acceptable excipients.

[0043] In some more preferred embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant or a preservative.

[0044] In some more preferred embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or alginic acid.

[0045] In some more preferred embodiments of the present invention, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose.

[0046] In some more preferred embodiments of the present invention, the lubricant is selected from at least one of micropowder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel.

[0047] In some more preferred embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone.

[0048] In some more preferred embodiments of the present invention, the wetting agent is sodium lauryl sulfate.

[0049] In some more preferred embodiments of the present invention, the flavoring agent is at least one of aspartame, stevioside, sucrose, maltitol or citric acid.

[0050] In some more preferred embodiments of the present invention, the suspending agent is selected from at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0051] In some more preferred embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate or glyceryl monostearate.

[0052] In some more preferred embodiments of the present invention, the preservative is selected from at least one of methylparaben and propylparaben.

[0053] The present invention has at least the following beneficial effects:

[0054] (1) The nucleic acid molecule provided by the present invention is obtained by codon-optimizing the FSOD gene, so that it can be well expressed in Pichia pastoris, avoiding translation barriers in yeast caused by rare codons in the cat gene;

[0055] (2) The present invention provides for the first time a method for preparing highly active cat-derived SOD using a Pichia pastoris expression system, and provides four combinations of expressing cat SOD using two promoters and two Pichia pastoris host bacteria. Cat SOD can be prepared by induction with methanol or by induction with glucose. Compared with the traditional method of extracting SOD from animal blood, which has problems such as limited raw material sources, individual differences, and viral contamination, this method can prepare highly active cat SOD in large quantities in a standardized manner;

[0056] (3) The preparation method provided by the present invention utilizes the Pichia pastoris eukaryotic expression system, which has the function of post-translational modification of recombinant protein expression. The structure of the expressed protein is similar to that of the protein in the animal body, and it has better compatibility for drug production. The present invention integrates the target gene into the yeast chromosome, the strain has good genetic stability, and is easy to ferment at high density. In addition, the cat SOD expressed by the yeast eukaryotic system does not contain endotoxins, which is more suitable for use in pet medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0058] Figure 1 This is a PCR amplification diagram of the feline SOD (FSOD) fragment in Example 1 of the present invention, wherein lanes 1 and 2 are all FSOD fragments;

[0059] Figure 2 This is a colony PCR verification diagram of the recombinant plasmid pPICZαA-FSOD in Example 1 of the present invention, wherein lanes 1 to 8 are all colony samples containing the recombinant plasmid pPICZαA-FSOD;

[0060] Figure 3 Schematic diagram of the expression of the recombinant plasmid pPICZαA-FSOD in Example 1 of the present invention;

[0061] Figure 4 This is a diagram showing the identification results of the recombinant plasmid pPICZαA-FSOD in Example 1 of the present invention, wherein lanes 1 and 2 are both pPICZαA-FSOD;

[0062] Figure 5 The figure shows the nucleic acid electrophoresis diagram of the recombinant plasmid pPICZαA-FSOD before and after linearization in Example 1 of the present invention, wherein lane 3 is the pPICZαA-FSOD linearized plasmid and lane 4 is the pPICZαA-FSOD non-linearized plasmid (control);

[0063] Figure 6 This is the electrophoresis diagram for positive identification of the recombinant yeast X33-pPICZαA-FSOD strain in Example 1 of the present invention, wherein lanes 1 to 24 are X33-pPICZαA-FSOD-1 to 24, respectively;

[0064] Figure 7 This is an SDS-PAGE electrophoresis diagram of the supernatant of the recombinant strain X33-pPICZαA-FSOD in Example 1 of the present invention, wherein lane 3 is the fermentation supernatant of the X33-pPICZαA-FSOD-21 strain;

[0065] Figure 8This is the electrophoresis diagram for positive identification of the recombinant yeast SMD1168H-pPICZαA-FSOD strain in Example 2 of the present invention, wherein lanes 1 to 24 are SMD1168H-pPICZαA-FSOD-1 to 24, respectively;

[0066] Figure 9 This is an SDS-PAGE electrophoresis diagram of the supernatant of the recombinant yeast SMD1168H-pPICZαA-FSOD strain in Example 2 of the present invention, wherein lane 5 is the fermentation supernatant of the SMD1168H-pPICZαA-FSOD-6 strain;

[0067] Figure 10 This is a colony PCR verification diagram of the recombinant plasmid pGAPZαA-FSOD in Example 3 of the present invention, wherein lanes 9 to 16 are colonies of the recombinant plasmid pGAPZαA-FSOD;

[0068] Figure 11 Schematic diagram of the expression of the recombinant plasmid pGAPZαA-FSOD in Example 3 of the present invention;

[0069] Figure 12 This is a diagram showing the identification results of the recombinant plasmid pGAPZαA-FSOD in Example 3 of the present invention, wherein lanes 3 to 4 are all pGAPZαA-FSOD;

[0070] Figure 13 The figure shows the nucleic acid electrophoresis diagram of the recombinant plasmid pGAPZαA-FSOD before and after linearization in Example 3 of the present invention, wherein lane 1 is the pGAPZαA-FSOD linearized plasmid and lane 2 is the pGAPZαA-FSOD non-linearized plasmid (control);

[0071] Figure 14 This is the electrophoresis diagram for positive identification of the recombinant yeast X33-pGAPZαA-FSOD strain in Example 3 of the present invention, wherein lanes 1 to 24 are X33-pGAPZαA-FSOD-1 to 24, respectively;

[0072] Figure 15 This is an SDS-PAGE electrophoresis diagram of the supernatant of the recombinant yeast X33-pGAPZαA-FSOD strain in Example 3 of the present invention, wherein lane 2 is the fermentation supernatant of the X33-pGAPZαA-FSOD-3 strain;

[0073] Figure 16 This is the electrophoresis diagram for positive identification of the recombinant yeast SMD1168-pGAPZαA-FSOD strain in Example 4 of the present invention, wherein lanes 1 to 24 are SMD1168-pGAPZαA-FSOD-1 to 24, respectively;

[0074] Figure 17This is an SDS-PAGE electrophoresis diagram of the supernatant of the recombinant yeast SMD1168-pGAPZαA-FSOD strain in Example 4 of the present invention, wherein lane 4 is the fermentation supernatant of the SMD1168-pGAPZαA-FSOD-18 strain;

[0075] Figure 18 These are enzyme activity trend graphs of the culture supernatant samples of the four recombinant yeast strains provided in Examples 1 to 4 in the test examples of the present invention; wherein, Figure A is a FSOD enzyme activity trend graph of the culture supernatant of recombinant yeast X33-pPICZαA-FSOD-21 in Example 1; Figure B is a FSOD enzyme activity trend graph of the culture supernatant of recombinant yeast SMD1168H-pPICZαA-FSOD-6 in Example 2; Figure C is a FSOD enzyme activity trend graph of the culture supernatant of recombinant yeast X33-pGAPZαA-FSOD-3 in Example 3; and Figure D is a FSOD enzyme activity trend graph of the culture supernatant of recombinant yeast SMD1168H-pGAPZαA-FSOD-18 in Example 4. DETAILED DESCRIPTION

[0076] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0077] Example 1

[0078] This embodiment provides a method for preparing FSOD and preparing highly active FSOD, the preparation method specifically comprising the following steps:

[0079] 1) Optimization of the cDNA sequence of the FSOD gene:

[0080] The amino acid sequence of feline SOD (XP_023109018) was searched in the NCBI database. Based on the restriction site information in the pPICZαA vector's multiple cloning site, an XhoI restriction site and a Lys-Arg sequence (Kex2 restriction site) corresponding to the gene were added to the 5' end of the cDNA fragment of the target gene, and an XbaI restriction site was added to the 3' end. After 3D modeling and codon optimization (the XhoI and XbaI restriction sites were not optimized), the cDNA fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis of the feline SOD gene. Parameters for optimizing the cDNA sequence of the target gene included yeast codon preference, DNA repeat sequences, and GC content. After optimization, the codon adaptation index increased to between 0.8 and 1, and the GC content was optimized to within 40% to 60%. The feline SOD gene fragment was then transformed into Escherichia coli and stored in a bacterial culture. The codon-optimized FSOD nucleotide sequence is as follows:

[0081] 5'-CTCGAGAAGAGGATGGAAATGAAGGCCGTCTGCGTCTTGAAAGGTCAAGG TCCAGTTGAGGGTACTATCCACTTCGTCCAAAAAGGTAACGGTCCTGTTTGTTGTCTCCGGTACTATCACTGGTTTGACTGAAGGTGAACACGGTTTCCACGTTCACCAATTCGGTGACAATACTCAGGGTTGTACTTCTGCTGGTCCACACTTCAACCCACTGTCTAAGAAACATGGTGGTCCAAAGGACCAAGAGAGACACGTTGGAGATTTGGGTA ATGTTACTGCCGGTAAGGACGGTGTGCTAACTTTCTATGGAAGATTCCTTGATCGCCTTGTCTGGTGACCACTCCATTATCGGTAGAACCATGGTCGTTCACGAGAAGAGATGACCTTGGTAAAGGTGGTAACGAAGAGTCCACTCAGACTGGTAATGCTGGTTCCAGATTGGCCTGTGGTGTTATCGGTATCGCTAAGTAATCTAGA-3'(SEQ ID NO:1).

[0082] The FSOD nucleotide sequence before codon optimization is as follows:

[0083] 5’-CTCGAGAAAAGAATGCTGGCGCCGGCGCTGCTGTGTGCCTACCTGCTGCTG GCGGCCCGCGCCTCGCGCGCCTGGTCCGACCCGGACCCGGAGGAGCCCGGCTCCAGCACGGCGGCGCAGATCCGCGACATGCACGAGAAAGTGACGGCGATCTGGCAGGAGATGACGCAGCGGCAGGCGGCGGGCGACCGCCCGGACGCCGCGCTCCACGCCGCCTGCCGGGTGCAGCCGTCGGCCACGCTGGACGCGGCGCAGCCCCGGGTGAGCGGCCTCGTGCTCTTCCGGCAGCAGGTGCCCGGCGCCCGGCTCGAGGCCTTCTTCGACCTGGAGGGCTTCCCGGCCGAGCCCAACAACTCCAGCCGCGCCATCCACGTGCACCAGTTCGGGGACCTGAGCCAGGGCTGCGACTCCACCGGCGCGCACTACAACCCGCGGGCCGTGCCGCACCCGCAGCACCCGGGCGACTTCGGCAACTTCGCCGTGCGCGACGGCCGCCTCTGGAAGTACCGCGGCGGCCTGGCCGCCTCGCTCTTCGGCCCGCACTCGATCGTGGGCCGCGCCGTGGTGGTCCACGCGGGCGAGGACGACCTGGGCCGCGGCGGCAACGCGGCCAGCGTGGAGAACGGCAACGCGGGGCCCCGGCTCGCCTGCTGCGTGGTGGGCGTGTGCGGGCCGCTGCCCTGGGCGCGCCAGGCGCAGGAGCACGCCGAGCGCCGAAAGCGGCGGCGGGACAGCGAGTGCAAGGCCGTCTGATCTAGA-3’(SEQ ID NO:2).

[0084] The amino acid sequence of the FSOD is specifically as follows:

[0085] LEKRMLAPALLCAYLLLAARASRAWSDPDPEEPGSSTAAQIRDMHEKVTAIWQEMTQRQAAGDRPDAALHAACRVQPSATLDAAQPRVSGLVLFRQQVPGARLEAFFDLEGFPAEPNNSSRAIHVHQ FGDLSQGCDSTGAHYNPRAVPHPQHPGDFGNFAVRDGRLWKYRGGLAASLFGPHSIVGRAVVVHAGEDDLGRGGNAASVENGNAGPRLACCVVGVCGPLPWARQAQEHAERRKRRRDSECKAV*(SEQ ID NO:3).

[0086] 2) Cloning of the target gene FSOD:

[0087] Based on the restriction site information in the multiple cloning site of the Pichia pastoris expression vector, a homologous sequence to the restriction site XhoI was added to the 5' end of the target gene cDNA fragment, and a homologous sequence to the restriction site XbaI was added to the 3' end. After optimization, the FSOD gene cDNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. After synthesis of the target gene cDNA, the fragment was amplified by PCR using a high-fidelity enzyme. The PCR amplification system is shown in Table 1, and the reaction procedure is shown in Table 2. The specific sequences of the amplification primers used are as follows:

[0088] Upstream primer: 5′-GAAGAAGGGGTATCTCTCGAGAAGAGGATGGAAATGAAGGCCG TC-3′ (SEQ ID NO: 4);

[0089] Downstream primer: 5′-CTGAGATGAGTTTTTGTTCTAGATTACTTAGCGATACCGATAACA CCAC-3′ (SEQ ID NO: 5).

[0090] Table 1 PCR amplification reaction system

[0091]

[0092]

[0093] Table 2 Target gene PCR amplification program

[0094]

[0095] After PCR amplification, the amplified product was detected by 1% agarose gel electrophoresis. When the product met the expected fragment size, the gel was cut and recovered. The test results were as follows: Figure 1As shown. The theoretical molecular weight of FSOD is 15.8 kDa, and the cDNA length of the target gene is 462 bp. The detection product is expected to be recombinant, and the target fragment is cut from the gel and recovered for later use. To facilitate the subsequent identification of positive recombinant bacteria, a signal peptide gene covering a certain length is used. After adding the identification primer length, the PCR length for positive identification is 858 bp (covering the target gene). The target band is located between 750 bp and 1000 bp on the electrophoretogram.

[0096] 3) Construction of recombinant vector pPICZαA-FSOD:

[0097] ① Obtaining the expression plasmid: Escherichia coli containing the empty vector pPICZαA was cultured and the expression plasmid was extracted using the Nanjing Novozymes Plasmid Extraction Kit. After chloroform extraction and purification, it was double-digested with XbaI and XhoI enzymes, and then extracted and purified again with chloroform.

[0098] ② Connection between vector and target fragment: Seamless cloning kit (Novozyme, The FSOD fragment amplified and recovered in step 2) was ligated with the double-enzyme-digested expression vector pPICZαA using the MLtra One Step Cloning Kit and transformed into Escherichia coli TOP10. LB medium was added and cultured at 37°C for 1 hour, followed by centrifugation and coating on an LB plate (containing 100 μg / mL of Zeocin). The seamless cloning system is shown in Table 3; the seamless ligation PCR reaction procedure is shown in Table 4; the molar ratio of the plasmid vector to the insert DNA fragment in the ligation reaction was approximately 1:1, and the final DNA concentration was 10 μg / mL.

[0099] Table 3 Seamless cloning connection system Reagent volume (μL)

[0100]

[0101] Table 4 Seamless PCR reaction program

[0102]

[0103] ③ Identification of positive colonies of E. coli containing the recombinant plasmid pPICZαA-FSOD:

[0104] Single colonies were picked from the plates and inoculated into LB medium containing 100 μg / mL Zeocin. Cultured on a shaker at 37°C. After the cells became turbid, PCR positive identification was performed. The PCR positive identification system is shown in Table 5, the PCR program is shown in Table 6, and the PCR primer sequences for positive identification of E. coli containing the recombinant plasmid are as follows:

[0105] Upstream primer: 5′-GCATCCTCCGCATTAGCTGCTCCAGTCAACAC-3′ (SEQ ID NO: 6);

[0106] Downstream primer: 5'-CTCTCAGGCAAATGGCATTCTGACATCCTCTTG-3' (SEQ ID NO: 7).

[0107] Table 5 Recombinant vector PCR positive identification system (10 μL)

[0108]

[0109] Table 6 Recombinant vector PCR identification reaction procedures

[0110]

[0111]

[0112] ④After PCR, 1% agarose gel electrophoresis was used to determine whether the transformation was successful and whether the selected colony was the target colony. The results of the identification of the recombinant plasmid pPICZαA-FSOD Escherichia coli colony were as follows: Figure 2 As shown, the recombinant plasmid was extracted and double enzyme digestion was performed for verification. The plasmid with successful double enzyme digestion was sent for sequencing, and the positive strain containing the recombinant plasmid with correct sequencing was saved for future use. The plasmid map of pPICZαA-FSOD is shown in Figure 3 shown.

[0113] 4) Linearization and electroporation of the recombinant vector pPICZαA-FSOD:

[0114] The E. coli TOP10 containing the recombinant vector pPICZαA-FSOD obtained in step 3) was expanded and cultured, and the plasmid was extracted. The band size was detected by 1% agarose gel electrophoresis. The detection results are as follows: Figure 4 Then, the plasmid was purified by chloroform extraction and linearized with SacI enzyme. The linearization enzyme system is shown in Table 7. Then, the plasmid was dephosphorylated and the linearized recombinant plasmid was extracted with chloroform. 2 μL of the linearized product was subjected to 1% agarose gel electrophoresis. The results are shown in Table 7. Figure 5 shown.

[0115] Table 7 Linearization system of recombinant vector pPICZαA-FSOD (500 μL)

[0116]

[0117] The linearized recombinant vector was electroporated into Pichia pastoris X33, and the cells were resuspended with 1 mol / L sorbitol. After standing in a 30°C water bath for 1 hour, they were transferred to a shaking incubator and cultured at 30°C and 200 rpm for 2 hours. After centrifugation at 4500g for 5 minutes, 100 μL of supernatant was retained and the rest of the supernatant was discarded. After resuspending the remaining supernatant and precipitating, the bacterial solution was spread on a YPD plate (containing 300 μg / mL of bleomycin) and cultured at 30°C until a single colony grew.

[0118] 5) Positive identification of X33-pPICZαA-FSOD strain:

[0119] Step 4) After a single colony grows on the coated plate, pick the single colony from the plate, inoculate it into a 2 mL EP tube and culture it for about 8 hours. Use liquid nitrogen quick freezing and ultrasonic disruption to lyse the X33-pPICZαA-FSOD bacterial solution, and identify it by PCR and agarose gel electrophoresis. The identification results are as follows: Figure 6 The positive recombinants were named X33-pPICZαA-FSOD (abbreviated as XP-FSOD). A total of 24 positive recombinant clones were obtained, designated X33-pPICZαA-FSOD-1 to X33-pPICZαA-FSOD-24. The primers for positive PCR identification of recombinant yeast solutions are shown in SEQ ID NOs: 5 to 6. The amplification system for PCR identification of recombinant yeast solutions is shown in Table 5, and the PCR program is shown in Table 6.

[0120] 6) Inducible expression of X33-pPICZαA-FSOD series strains:

[0121] In a clean bench, 100 μL of the positive strain identified by PCR in step 5) was taken and inoculated into 100 mL of BMMY medium. The culture was shaken at 30°C and 200 rpm until the bacteria grew. When the bacterial concentration OD 600 = 1, and then induce with an equal volume mixture of methanol and 50% sorbitol. 600 Calculate the amount of inducer to be added. Add the inducer once in the morning and evening. The amount to be added is calculated as follows: Amount of inducer added (μL) = OD 600 × dilution factor before detection × culture medium volume × 0.05 × 10.

[0122] 7) Identification of the expressed product by SDS-PAGE electrophoresis of the X33-pPICZαA-FSOD strain:

[0123] Step 6) After the induction culture is completed, take 20 μL of the culture supernatant, add 5 μL of 5× Loding Buffer, and boil in boiling water for 2 minutes. Prepare SDS-PAGE protein gel (15% separation gel and 5% stacking gel). Load 16 μL of sample into each well and run electrophoresis at 120V for about 1 hour. Then, stain, decolorize, and photograph the protein gel. The protein electrophoresis results are as follows: Figure 7 shown.

[0124] According to the SDS-PAGE protein electrophoresis diagram and protein marker, the bands of the X33-pPICZαA-FSOD strain are between 17 and 25 kDa. The theoretical value of the target protein is 15.8 kDa. The target protein on the gel image is around 17 kDa. Because the yeast glycosylates the target protein, the molecular weight of the target protein increases slightly. The size of the target protein on the gel image is consistent with expectations.

[0125] Example 2

[0126] This example provides a method for preparing FSOD and preparing highly active FSOD. The specific steps of the preparation method differ from those of Example 1 only in that Pichia pastoris X33 is replaced with an equal amount of Pichia pastoris SMD1168H, thereby obtaining the SMD1168H-pPICZαA-FSOD recombinant strain (abbreviated as SP-FSOD).

[0127] Among them, the PCR positive identification results of the SMD1168H-pPICZαA-FSOD recombinant strain are as follows Figure 8 As shown in Figure 2, there are 24 positive single clones, which are named SMD1168H-pPICZαA-FSOD-1 to SMD1168H-pPICZαA-FSOD-24. The SDS-PAGE identification results of the SMD1168H-pPICZαA-FSOD recombinant strain are shown in Figure 2. Figure 9 The theoretical molecular weight of the target protein in the SMD1168H-pPICZαA-FSOD strain is 15.8 kDa. According to the SDS-PAGE protein electrophoresis diagram and protein marker, the band of the SMD1168H-pPICZαA-FSOD strain is around 17 kDa. This is due to the glycosylation modification of the target protein by yeast. The protein band is slightly larger than the theoretical molecular weight. The size of the target protein on the gel image is consistent with the expectation.

[0128] Example 3

[0129] This example provides a method for preparing FSOD and preparing highly active FSOD. The specific steps of the preparation method differ from those of Example 1 only in that the pPICZαA vector is replaced with a pGAPZαA vector, and the inducer is replaced from a methanol-50% sorbitol mixture with a 20% glucose-50% sorbitol mixture (mixed in equal volumes).

[0130] Among them, the enzyme linearization system of the recombinant plasmid pGAPZαA-FSOD is shown in Table 8; the positive identification results of the recombinant plasmid pGAPZαA-FSOD Escherichia coli are shown in Figure 10 The plasmid map of pGAPZαA-FSOD is shown in Figure 11 As shown; the identification results of the pGAPZαA-FSOD plasmid obtained by expanded culture and extraction are shown as follows Figure 12 As shown; the electrophoresis results of the recombinant plasmid pGAPZαA-FSOD after linear enzyme digestion are shown Figure 13 As shown; the positive identification results of the X33-pGAPZαA-FSOD (abbreviated as XG-FSOD) strain are as follows Figure 14 As shown in Figure 2, there were 24 positive recombinant clones, named X33-pGAPZαA-FSOD-1 to X33-pGAPZαA-FSOD-24. The SDS-PAGE protein electrophoresis results of the X33-pGAPZαA-FSOD strain were shown in Figure 2. Figure 15 As shown, Figure 15 The target protein is clearly shown as a band.

[0131] Table 8 Enzyme linearization system of recombinant plasmid pGAPZαA-FSOD (500 μL)

[0132]

[0133] Example 4

[0134] This example provides a method for preparing FSOD and preparing highly active FSOD. The specific steps of the preparation method differ from those of Example 3 only in that Pichia pastoris X33 is replaced with an equal amount of Pichia pastoris SMD1168H, thereby obtaining the SMD1168H-pGAPZαA-FSOD recombinant strain (abbreviated as SG-FSOD).

[0135] Among them, the PCR positive identification results of the SMD1168H-pGAPZαA-FSOD recombinant strain are as follows Figure 16 As shown in Figure 2, there were 24 positive single clones, which were named SMD1168H-pGAPZαA-FSOD-1 to 24 respectively. The SDS-PAGE identification results of the SMD1168H-pGAPZαA-FSOD recombinant strain were shown in Figure 2. Figure 17 As shown, Figure 17 The target protein is clearly shown as a band.

[0136] Test example

[0137] This test example tested the enzymatic activity of cat-derived superoxide dismutase prepared in Examples 1 to 4. The specific test steps and results are as follows:

[0138] During the induction culture process, the supernatant was centrifuged every day for enzyme activity detection (using Nanjing Jiancheng Superoxide Dismutase Assay Kit, Product No. A001-3). The FSOD enzyme activity detection operation table is shown in Table 9. The results are shown in Table 9. Figure 18 shown.

[0139] Table 9 FSOD enzyme activity detection operation table

[0140]

[0141] Place in a 37°C constant temperature water bath or incubator and incubate for 40 minutes

[0142]

[0143] FSOD enzyme activity calculation formula:

[0144]

[0145] Depend on Figure 18 It can be seen that among the four combinations, when the host bacteria is SMD1168H and the pGAPZαA-FSOD recombinant plasmid is used, the prepared FSOD enzyme activity is the highest, reaching 2298.16 U / mL; and the FSOD enzyme activity prepared by the four combinations all reached above 1200 U / mL.

[0146] In summary, the method for preparing feline superoxide dismutase provided by the present invention utilizes genetic engineering techniques to transfer the codon-optimized feline superoxide dismutase gene into Pichia pastoris via two different plasmids to construct an engineered Pichia pastoris strain. This method can efficiently and stably express feline superoxide dismutase using different induction types of Pichia pastoris engineered strains, filling a technological gap and laying the foundation for the development of feline SOD products for pet treatment.

[0147] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is: (1) the nucleotide sequence shown in SEQ ID NO: 1; or (2) A nucleotide sequence obtained by replacing and / or deleting and / or adding one or more modified base sequences of the nucleotide sequence shown in SEQ ID NO: 1, and obtaining a nucleotide sequence that is consistent with the amino acid sequence obtained by translating the nucleotide sequence described in (1).

2. Biomaterial, characterized in that The biological material is any one of the following (1) to (7): (1) An expression cassette containing the nucleic acid molecule of claim 1; (2) a recombinant vector containing the nucleic acid molecule of claim 1; (3) a recombinant vector containing the expression cassette described in (1); (4) A transgenic cell line containing the nucleic acid molecule of claim 1; (5) a transgenic cell line containing the expression cassette described in (1); (6) A transgenic cell line containing the recombinant vector described in (2); (7) A transgenic cell line containing the recombinant vector described in (3).

3. A method for preparing cat-derived superoxide dismutase, characterized in that: The preparation method comprises the following steps: transferring a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 1 into a host cell and inducing expression to obtain the product.

4. The preparation method according to claim 3, characterized in that The vector backbones used when the nucleic acid molecule is transferred include pPICZαA and pGAPZαA.

5. The preparation method according to claim 3, characterized in that The host cells include Pichia pastoris X33 and Pichia pastoris SMD1168H.

6. The preparation method according to claim 3, characterized in that The inducing agent used for inducing expression contains methanol or glucose, and also includes sorbitol.

7. A cat-derived superoxide dismutase prepared by the method according to any one of claims 3 to 6.

8. The cat-derived superoxide dismutase according to claim 7, wherein The amino acid sequence of the cat-derived superoxide dismutase is shown in SEQ ID NO:

3.

9. Use of the cat-derived superoxide dismutase according to claim 7 or 8 in the preparation of an antioxidant product.

10. An antioxidant product, characterized in that The antioxidant product includes any one of (1) to (3): (1) The nucleic acid molecule according to claim 1; (2) The biomaterial according to claim 2; (3) The cat-derived superoxide dismutase according to claim 7 or 8; Preferably, the antioxidant product comprises a medicine or a feed additive.

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