CN112079912A - High-activity canine alpha interferon recombinant protein and preparation method and application thereof - Google Patents
High-activity canine alpha interferon recombinant protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological genetic engineering, and particularly relates to a high-activity canine alpha interferon recombinant protein, and a preparation method and application thereof. The preparation method comprises the steps of carrying out codon optimization on CaIFN-alpha before carrying out amino acid site mutation on the CaIFN-alpha to canine alpha interferon mutant CaIFN-alpha-Mut, CaIFN-alpha-Mut and protein label synthesis, then synthesizing on a plasmid vector, carrying out double enzyme digestion, cloning to an expression vector to obtain a recombinant plasmid, then carrying out enzyme digestion linearization, introducing into an expression host bacterium to obtain recombinant yeast, and then carrying out induced expression and chromatographic purification to obtain a target protein, namely the canine alpha interferon recombinant protein. Solves the problems of low content of the prior canine alpha interferon, low biological activity, short half-life period, poor stability, difficult formation of disulfide bonds, complex production process, high purification and preparation cost and the like, and can greatly reduce the production cost in large-scale production.
Description
Technical Field
The invention belongs to the technical field of biological genetic engineering, and particularly relates to a high-activity canine alpha interferon recombinant protein, and a preparation method and application thereof.
Background
Canine alpha interferon (CaIFN-alpha) is a protein with broad-spectrum biological activities of antivirus, immunoregulation, anti-tumor and the like, the antivirus activity is the main biological activity of the canine alpha interferon, and the canine alpha interferon is widely used for preventing and treating canine viral diseases and comprises the following components: canine distemper, canine parvovirus enteritis, canine adenovirus, canine parainfluenza, canine coronavirus infection, canine herpes virus infection, canine viral keratitis and other viral diseases. The action mechanism is that the canine alpha interferon stimulates an organism to generate an ISG (interferon stimulated gene) product through a series of signal transduction, and the ISG product comprises a plurality of antiviral proteins and can effectively inhibit the replication of viruses. Meanwhile, the antiviral effect can be realized by effective modes such as improving the immune effect of the organism, adjusting the transcription process of the virus and the like. The natural interferon including CaIFN-alpha has the characteristic of short half-life period, about 4h, and the related research of canine alpha interferon in the prior art has the problems of low expression level, low activity, short half-life period and the like.
In the prior art, the related research of the canine alpha interferon has the problems of low expression level, low activity, short half-life period and the like. The exogenous expression of canine alpha interferon is mainly expressed in three expression systems of escherichia coli, pichia pastoris and animal cells, and the escherichia coli expression system lacks a mechanism for effectively releasing and secreting protein, so that the problems of small yield and inclusion body in an expression form are faced, and complicated protein purification processes such as renaturation, endotoxin removal, heat source removal and the like exist; meanwhile, the Escherichia coli expression system cannot effectively form a disulfide bond, so that correct folding of the canine alpha interferon is not facilitated, the natural conformation and the stability of the canine alpha interferon are influenced, and the biological activity of the canine alpha interferon is further influenced; the expression by animal cells has the conditions of complicated industry, higher cost and difficult large-scale industrialized production, and the phenomenon of low bioactivity of the expressed canine alpha interferon commonly exists in the corresponding expression system due to the translation folding process of protein and the like.
In the research of long-acting modification, techniques such as serum albumin modification, ricin B chain, GST and the like are all used for fusing canine alpha interferon, the problems of breakage, enzyme degradation, influence on the activity of the canine alpha interferon and the like are easy to occur during fusion expression, and the selection of fusion protein in different expression systems is a technical problem.
In the aspect of preventing canine viral diseases, vaccines and antibodies are mostly applied in China, but because the interference of maternal antibodies and the antigen drift of strains can sometimes cause the immune failure, the development of safe and efficient interferon antiviral preparations has great significance. The canine alpha interferon has the biological activities of broad-spectrum antivirus, immunoregulation, anti-tumor and the like. But its own short half-life and prior art limit the clinical application of canine interferon-alpha. How to improve the biological activity of the canine alpha interferon, prolong the half-life period and explore an efficient expression system has wide prospect for the clinical application of the canine alpha interferon.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a high-activity canine alpha interferon recombinant protein, a preparation method and application thereof, and a canine alpha interferon mutant which has high biological activity, prolonged half-life period, good stability and difficult degradation is formed.
The technical content of the invention is as follows:
the invention provides a high-activity canine alpha interferon recombinant protein, wherein a fusion fragment of the canine alpha interferon recombinant protein comprises canine alpha interferon or a canine alpha interferon mutant, and an amino acid sequence of the canine alpha interferon recombinant protein is shown in a sequence table SEQ ID NO. 1-4;
the canine alpha interferon mutant is CaIFN-alpha-Mut formed by carrying out amino acid site mutation on CaIFN-alpha (the amino acid sequence of which is shown in a sequence table SEQ ID NO. 5); the canine alpha interferon mutant is CaIFN-alpha-Mut formed by carrying out amino acid site mutation on CaIFN-alpha, the mutated amino acid comprises 1 or 2 amino acids of 150 th to 164 th sites of the canine alpha interferon, the biological activity can be enhanced by mutating the amino acid site of the canine alpha interferon, and the mutated site is obtained by analyzing amino acid selection of the canine alpha interferon;
the mutated amino acid comprises that the 160 th amino acid of the canine alpha interferon is mutated into valine, and the amino acid sequence of the obtained mutant CaIFN-alpha-Mut 1 is shown in a sequence table SEQ ID NO. 6; or mutated into leucine, and the amino acid sequence of the obtained mutant CaIFN-alpha-Mut 2 is shown in the sequence table SEQ ID NO.7
The mutated amino acids comprise 163 th amino acid and 164 th amino acid of canine alpha interferon which are respectively mutated into lysine and glutamic acid, and the amino acid sequence of the obtained mutant CaIFN-alpha-Mut 3 is shown in a sequence table SEQ ID NO. 8;
the canine alpha interferon recombinant protein also comprises a His-Tag fragment, the amino acid sequence of which is shown in a sequence table SEQ ID NO.9, and a His-Tag protein label is preferably selected, so that the purification of a target protein is facilitated;
before the fusion of CaIFN-alpha, CaIFN-alpha-Mut 1, CaIFN-alpha-Mut 2, CaIFN-alpha-Mut 3 and His-Tag, codon optimization is carried out, and the optimized base sequences are respectively shown in SEQ ID NO. 10-14;
the invention also provides a preparation method of the high-activity canine alpha interferon recombinant protein, which comprises the following steps:
carrying out codon optimization on CaIFN-alpha before amino acid site mutation into canine alpha interferon mutant CaIFN-alpha-Mut, CaIFN-alpha-Mut and protein label synthesis, then synthesizing on a plasmid vector, cloning to a yeast expression vector to obtain a recombinant plasmid after double enzyme digestion, then carrying out enzyme digestion linearization, introducing into an expression host bacterium to obtain recombinant yeast, and then carrying out induction expression and chromatographic purification to obtain the target protein, namely the canine alpha interferon recombinant protein.
The yeast expression vector comprises pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pGAPZ alpha A, pPIC9K, pPIC9, pHIL-S1 and pYAM 75P;
the expression host bacteria comprise pichia pastoris host bacteria or saccharomyces cerevisiae, and the pichia pastoris host bacteria comprise X33, GS115, KM71 and SMD1168, so that formation of disulfide bonds is facilitated;
the induction expression comprises the steps of adopting a methanol induction technology, selecting a high-efficiency pichia pastoris methanol induction secretion expression system, utilizing the methanol induction technology to efficiently induce protein expression, effectively improving the expression quantity of target protein to gram level, greatly reducing the production cost in large-scale production, and simultaneously being beneficial to the formation of self disulfide bonds of canine alpha interferon by the expression system;
the CaIFN-alpha and the mutant thereof modified by proper glycosylation of the yeast improve the biological activity and prolong the half-life period. The half-life of the mutant modified by the yeast self-glycosylation is improved by 2.5 times.
The invention also provides a high-activity canine alpha interferon recombinant protein for preparing antiviral drugs.
The invention has the following beneficial effects:
compared with pure canine alpha interferon, the high-activity canine alpha interferon recombinant protein has the advantages of high stability, high biological activity and prolonged half-life period, solves the problems of low canine alpha interferon content, low biological activity, short half-life period, poor stability, difficult formation of disulfide bonds, complex production process, high purification preparation cost and the like in the conventional expression system and the related biological technology, can greatly reduce the production cost of the canine alpha interferon in large-scale production, and can effectively realize that the canine alpha interferon is used in antiviral drugs;
in the preparation process of the recombinant protein, a high-efficiency pichia pastoris methanol induction secretion expression system is selected, the CaIFN-alpha-Mut with higher biological activity is obtained by mutating the amino acid site of the canine alpha interferon, and the stability and half-life period of the CaIFN-alpha-Mut are improved, so that the exogenous expression of the canine alpha interferon has the advantages of high expression content, moderate glycosylation, easy formation of disulfide bonds, good biological activity of an expression product, less background protein, simplicity and convenience in operation, easiness in industrial production and the like.
Drawings
FIG. 1 is a schematic diagram of the construction of a protein of interest;
FIG. 2 shows the PCR identification result of the bacterial liquid;
FIG. 3 shows the PCR identification result of the recombinant yeast solution;
FIG. 4 is a graph comparing the deglycosylation treatment of the induced supernatant.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Examples
Preparation and related detection of high-activity canine alpha interferon recombinant protein:
1. carrying out amino acid site mutation on the CaIFN-alpha to obtain canine alpha interferon mutant CaIFN-alpha-Mut, carrying out pichia pastoris codon optimization before synthesis of the CaIFN-alpha, the CaIFN-alpha-Mut and a protein Tag His-Tag, then synthesizing the mixture on a pUC57 plasmid vector, introducing the His-Tag and a stop codon TAA into the C end of a target gene, carrying out EcoRI and XbaI double enzyme digestion, and cloning the target gene to an expression vector to obtain a recombinant plasmid;
the expression vector comprises pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pGAPZ alpha A, pPIC9K, pPIC9, pHIL-S1 and pYAM75P, the expression vector is selectively constructed on pPICZ alpha A, T4 ligase connection and transformation competence DH5 alpha are carried out, and the construction schematic diagram of the obtained recombinant plasmid is shown in figure 1;
the obtained recombinant plasmid CaIFN-alpha-pPICZ alpha A, CaIFN-alpha-Mut 1-pPICZ alpha A, CaIFN-alpha-Mut 2-pPICZ alpha A, CaIFN-alpha-Mut 3-pPICZ alpha A is subjected to positive transformation identification, identification primers are respectively synthesized by alpha-factor and 3' Aox1, Guangzhou Jinzhi Biotech limited company, a PCR identification system is adopted and is shown in Table 1, and identification programs are shown in Table 2:
TABLE 1 PCR identification System
TABLE 2 PCR identification procedure
| Procedure for measuring the movement of a moving object | Temperature of | Time of day | Number of cycles |
| Pre-denaturation | 94℃ | 4min | / |
| Denaturation of the material | 94℃ | 30s | 30 |
| Annealing | 55℃ | 30s | |
| Extension of | 72℃ | 30s | |
| Re-extension of | 72℃ | 4min | / |
| Preservation of | 16℃ | / | / |
And performing 1% agarose gel electrophoresis on the PCR product, selecting the PCR product to identify positive bacteria, performing plasmid extraction and sequencing identification, wherein the result is shown in figure 2, the figure shows that all groups of CaIFN-alpha-pPICZ alpha A, CaIFN-alpha-Mut 1-pPICZ alpha A, CaIFN-alpha-Mut 2-pPICZ alpha A, CaIFN-alpha-Mut 3-pPICZ alpha A are positive, and the plasmid sequencing result shows that all groups of bacteria are sequenced correctly as shown in SEQ ID NO. 1-4.
2. The recombinant plasmid is linearized by digestion
Referring to the enzyme digestion test manual of TAKARA company, Sac I is used for single enzyme digestion of each recombinant plasmid, agarose gel electrophoresis is carried out to detect complete linearization, a linearization product is purified and recovered, and the purification and recovery method refers to the kit use instruction.
3. Introducing the linearized product into an expression host bacterium
3.1 preparation of Pichia competent cells
1) Inoculating yeast recipient strain (including X33, GS115, KM71 and SMD1168, in this example KM71) to YPD plate, and culturing at 30 deg.C for 2 days;
2) selecting single colony on the plate, inoculating the single colony in 10mLYPD liquid culture medium, shaking the single colony in a shaking table at 30 ℃ overnight;
3) after overnight culture, inoculating the strain into a 100mLYPD culture medium according to the inoculation amount of about 1%, and performing shake culture until the OD value is 1.2-1.5;
4) centrifuging at 4 ℃ and 5000rpm for 5min, collecting precipitated thalli, and re-suspending the thalli by using 100mL of precooled sterile water;
5) centrifuging at 4 ℃ and 5000rpm for 10min, collecting precipitated thalli, and re-suspending the thalli by using 100mL of precooled sterile water;
6) centrifuging at 4 ℃ and 5000rpm for 10min again to collect precipitated thalli, and re-suspending the thalli by using 100mL of precooled sterile water;
7)20ml of 1mol/L sorbitol is washed for 1 time;
8) the cells were dissolved in 1mL of 1M pre-cooled sorbitol without glycerol and left at-80 ℃ for several hours to be transformed.
3.2 electrotransformation of Pichia competent cells with linearized product
1) Preparing 80 mu L of KM71 yeast competence (KM 71 is adopted in the embodiment, and the use modes of other host bacteria are the same) to be mixed with 1-5 mu g of linearized plasmid (precooling for 15min on ice), quickly putting into an electric shock cup of 0.2cm (precooling sterilization on ice in the electric shock cup), and electrically shocking;
the electric transfer parameter is Voltage: 1500V; capacitance: 25 muF; resistance: 200 omega; cuvette (mm): 2 mm;
2) and (3) after electric shock is finished, quickly adding 1mL of sorbitol (1M), standing for 15min on ice, then, standing and culturing for 1h in a 30 ℃ incubator, adding 1mLYPD liquid culture medium, performing shaking culture at 30 ℃ and 200r/min for 1h, centrifuging at the normal temperature and 4000r/min, collecting thalli, and coating the thalli in a YPDS plate containing 100 mu g/mu L of YPDS for standing and culturing for 3d at 30 ℃ to obtain the recombinant yeast.
3.3 identification of recombinant Yeast and screening of high-copy
Using a sterilized gun head to finely pick a single colony with Zeocin resistance growing on a YPDS plate, inoculating the single colony into 2mL YPD liquid culture medium (containing 150 mu g/mLzeocin), and carrying out shaking culture at 30 ℃ and 200r/min for overnight;
the P.pastoris transformants were analyzed by PCR using the bacterial liquid, the PCR identification system is as in Table 1, and the PCR identification program is as shown in Table 3:
TABLE 3 PCR identification procedure for recombinant yeast solutions
And (3) carrying out 1% agarose gel electrophoresis on the PCR product to identify clonidine of a target band amplified by the primer as a positive transformant, wherein the PCR identification result is shown in figure 3, and each group has a positive recombinant yeast strain, which indicates that the linearized product is successfully electrically transformed and introduced into the host bacterium KM 71.
High copy selection requires a combination of banding intensity in PCR identification and high resistance YPD plate (200. mu.g/mLzeocin) assay results.
4. High copy recombinant yeast induced expression
1) Selecting a single colony with Zeocin resistance growing on a YPD plate by using a sterilized gun head, selecting the single colony in a BMGY liquid culture medium of 30mL for activated culture, oscillating at 30 ℃ at 200r/min overnight until OD600 is 2-6, and then enabling the cell to be in a logarithmic phase;
2) centrifuging at 3000r/min at room temperature for 5min, collecting precipitate, suspending in 15mL BMMY, wrapping with four layers of clean gauze and two layers of newspaper, and performing shake culture in a 250mL triangular conical flask;
3) adding 100% methanol every 24h to the final concentration of 1%, and performing induction culture;
4) after culturing for 96h, samples are collected, centrifuged, and the supernatant is immediately subjected to SDS-PAGE or stored at-80 ℃.
5. SDS-PAGE of recombinant yeast induced expression supernatant
Carrying out deglycosylation treatment on the supernatant of the recombinant yeast induced expression 4d, setting the final concentration of a deglycosylation reagent to be 1%, incubating for 3h at 37 ℃, setting corresponding KM71 and an empty plasmid pPICZ alpha A-KM71 control group, setting a non-deglycosylation reagent group at the same time, observing the glycosylation modification condition of the expression system on target protein, wherein the protein Loading Buffer solution is 5 multiplied by Loading Buffer, and the Loading amount is 12 mu L;
as shown in FIG. 4, CaIFN-alpha and the corresponding mutants CaIFN-alpha-Mut 1, CaIFN-alpha-Mut 2 and CaIFN-alpha-Mut 3 were all expressed efficiently in yeast expression system, with a band size of 20kDa, consistent with the expectation. As can be seen from comparison of the deglycosylated group and the non-deglycosylated group, the non-deglycosylated histone is slightly larger than the deglycosylated group, which indicates that the yeast expression system carries out proper glycosylation modification on CaIFN-alpha, CaIFN-alpha-Mut 1, CaIFN-alpha-Mut 2 and CaIFN-alpha-Mut 3.
6. Purification and recovery of expression product
In order to research the influence of pichia pastoris glycosylation modification on the activity of canine alpha interferon, a deglycosylation treatment group and a non-deglycosylation treatment group are arranged, two groups of purified expressed supernatant combined with His Tag are subjected to protein adsorption and elution purification by a nickel column affinity chromatography method, the concentration is determined after imidazole is removed by a dialysis method, and the concentration of each group of purified samples is shown in the following table 4.
TABLE 4 concentration of each group of samples after purification
7. Detection of biological Activity of protein of interest
Detecting the activity of a target protein by adopting an MDCK-VSV (modified Golay-Virus) micro-lesion inhibition method, paving digested MDCK cells on a 96-hole cell culture plate, adding 100 mu L of purified canine alpha interferon (a deglycosylated treatment group and a non-deglycosylated treatment group) diluted by 4 times into each hole after the cells are completely attached to the wall, neutralizing the virus by using 100TCID50VSV in each hole after incubating for 24 hours at 37 ℃, and simultaneously setting a normal cell control group and a virus control group only added with virus;
the inhibition of cytopathic effects was examined after 48h, with the highest interferon dilution inhibiting 50% of the cytopathic CPE50 being 1 activity unit. The inhibition of cytopathic effect was observed after 48h, with the highest interferon dilution inhibiting 50% of the cytopathic CPE50 being 1 activity unit, and the results are shown in table 5. The results are shown in Table 5.
TABLE 5 Activity of various groups of proteins of interest
The results show that: CaIFN-alpha-His, CaIFN-alpha-Mut 1-His, CaIFN-alpha-Mut 2-His and CaIFN-alpha-Mut 3-His in the deglycosylation treatment group and the non-deglycosylation treatment group have higher activity, and the activity of the non-deglycosylation treatment group is higher than that of the deglycosylation treatment group, which shows that the activity of the canine alpha interferon can be effectively improved by proper glycosylation;
compared with the activity of the mutant, the activity of each group of active CaIFN-alpha-Mut 2-His > CaIFN-alpha-Mut 3-His > CaIFN-alpha-Mut 1-His > CaIFN-alpha-His, especially the activity of CaIFN-alpha-Mut 2 is far higher than that of the natural CaIFN-alpha, and the mutation of an appropriate amino acid site is favorable for improving the activity of canine alpha interferon.
8. Determination of half-life of recombinant canine alpha interferon protein
Selecting a deglycosylated group and an unglycosylated group of CaIFN-alpha-His, CaIFN-alpha-Mut 1-His, CaIFN-alpha-Mut 2-His, CaIFN-alpha-Mut 3-HisCaIFN-alpha-Mut 2-His of each group to determine the half life of the canine alpha interferon, wherein the determination method adopts a cytopathic inhibition method to determine the relation between the blood concentration of the canine alpha interferon and time;
taking adult beagle dogs (6 beagle dogs per group) with the weight close to 10kg, injecting freeze-dried samples into the neck part subcutaneously for half each female dog and half male dog according to the dose of 1mg/mL, respectively sampling blood at veins of 1h, 2h, 4h, 8h, 16h, 24h, 48h and 72h after injection, and centrifuging at the low temperature of 3000r/min for 5min after blood sample is solidified at the low temperature of 4 ℃ to obtain upper serum;
the cytopathic inhibition method is adopted to measure the concentration of the canine alpha interferon in serum at different time points, DAS pharmacokinetic software is utilized to carry out curve fitting and calculate related parameters, and the results are shown in the following table:
TABLE 6 determination of half-life
The half-life period of the deglycosylation treatment group of each group is 4.2h, is equivalent to the half-life period of 4h of natural CaIFN-alpha-His reported in documents, and the half-life period of the non-deglycosylation treatment group is 10.2h-10.6h, which is improved by about 2.5 times compared with the deglycosylation treatment group.
The glycosylation modification of the canine alpha interferon in a yeast expression system can obviously prolong the half-life period, simplify the long-acting modification process of the canine alpha interferon, and has wide application prospect.
Sequence listing
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<120> high-activity canine alpha interferon recombinant protein, preparation method and application thereof
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Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Leu
145 150 155 160
Arg Arg Arg Lys His His His His His His
165 170
<210> 4
<211> 170
<212> PRT
<213> Artificial Sequence
<400> 4
Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
1 5 10 15
Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr
20 25 30
Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Ile
145 150 155 160
Arg Arg Lys Glu His His His His His His
165 170
<210> 5
<211> 164
<212> PRT
<213> Artificial Sequence
<400> 5
Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
1 5 10 15
Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr
20 25 30
Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Ile
145 150 155 160
Arg Arg Arg Lys
<210> 6
<211> 164
<212> PRT
<213> Artificial Sequence
<400> 6
Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
1 5 10 15
Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr
20 25 30
Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Val
145 150 155 160
Arg Arg Arg Lys
<210> 7
<211> 164
<212> PRT
<213> Artificial Sequence
<400> 7
Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
1 5 10 15
Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr
20 25 30
Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Leu
145 150 155 160
Arg Arg Arg Lys
<210> 8
<211> 164
<212> PRT
<213> Artificial Sequence
<400> 8
Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr
1 5 10 15
Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr
20 25 30
Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu
35 40 45
Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val
50 55 60
Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr
65 70 75 80
Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu
85 90 95
Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met
100 105 110
His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr
115 120 125
Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala
130 135 140
Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Ile
145 150 155 160
Arg Arg Lys Glu
<210> 9
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 9
His His His His His His
1 5
<210> 10
<211> 492
<212> DNA
<213> Artificial Sequence
<400> 10
tgtcatttgc cagataccca tggtttacga aactggaggg tgttaacttt gcttggtcaa 60
atgagacgtc tgtcagctgg ttcttgtgat cattacacta atgactttgc tttccccaaa 120
gaactgtttg acggacagcg tttgcaagaa gctcaagctt tatctgtggt ccacgttatg 180
acacaaaagg tattccattt attttgtcct gatacctcaa gtgctccatg gaacatgacc 240
ctattagaag agctatgtag tggtttgtct gaacagctgg atgacctgga ggcatgtccc 300
ttgcaagaag ccggtttggc tgagaccccc ttgatgcacg aggattctac tttgagaact 360
tacttccaac gtatttcatt atatttgcag gatagaaacc actccccttg cgcttgggaa 420
atggttagag ccgaaattgg aagaagtttc tttagttcta ctatcctgca ggaacgtatt 480
agacgtagaa aa 492
<210> 11
<211> 492
<212> DNA
<213> Artificial Sequence
<400> 11
tgtcatttgc cagataccca tggtttacga aactggaggg tgttaacttt gcttggtcaa 60
atgagacgtc tgtcagctgg ttcttgtgat cattacacta atgactttgc tttccccaaa 120
gaactgtttg acggacagcg tttgcaagaa gctcaagctt tatctgtggt ccacgttatg 180
acacaaaagg tattccattt attttgtcct gatacctcaa gtgctccatg gaacatgacc 240
ctattagaag agctatgtag tggtttgtct gaacagctgg atgacctgga ggcatgtccc 300
ttgcaagaag ccggtttggc tgagaccccc ttgatgcacg aggattctac tttgagaact 360
tacttccaac gtatttcatt atatttgcag gatagaaacc actccccttg cgcttgggaa 420
atggttagag ccgaaattgg aagaagtttc tttagttcta ctatcctgca ggaacgtgtt 480
agacgtagaa aa 492
<210> 12
<211> 492
<212> DNA
<213> Artificial Sequence
<400> 12
tgtcatttgc cagataccca tggtttacga aactggaggg tgttaacttt gcttggtcaa 60
atgagacgtc tgtcagctgg ttcttgtgat cattacacta atgactttgc tttccccaaa 120
gaactgtttg acggacagcg tttgcaagaa gctcaagctt tatctgtggt ccacgttatg 180
acacaaaagg tattccattt attttgtcct gatacctcaa gtgctccatg gaacatgacc 240
ctattagaag agctatgtag tggtttgtct gaacagctgg atgacctgga ggcatgtccc 300
ttgcaagaag ccggtttggc tgagaccccc ttgatgcacg aggattctac tttgagaact 360
tacttccaac gtatttcatt atatttgcag gatagaaacc actccccttg cgcttgggaa 420
atggttagag ccgaaattgg aagaagtttc tttagttcta ctatcctgca ggaacgtctg 480
agacgtagaa aa 492
<210> 13
<211> 492
<212> DNA
<213> Artificial Sequence
<400> 13
tgtcatttgc cagataccca tggtttacga aactggaggg tgttaacttt gcttggtcaa 60
atgagacgtc tgtcagctgg ttcttgtgat cattacacta atgactttgc tttccccaaa 120
gaactgtttg acggacagcg tttgcaagaa gctcaagctt tatctgtggt ccacgttatg 180
acacaaaagg tattccattt attttgtcct gatacctcaa gtgctccatg gaacatgacc 240
ctattagaag agctatgtag tggtttgtct gaacagctgg atgacctgga ggcatgtccc 300
ttgcaagaag ccggtttggc tgagaccccc ttgatgcacg aggattctac tttgagaact 360
tacttccaac gtatttcatt atatttgcag gatagaaacc actccccttg cgcttgggaa 420
atggttagag ccgaaattgg aagaagtttc tttagttcta ctatcctgca ggaacgtatt 480
agacgtaaag aa 492
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 14
catcatcatc atcatcat 18
Claims (6)
1. The high-activity canine alpha interferon recombinant protein is characterized in that a fusion fragment of the canine alpha interferon recombinant protein comprises canine alpha interferon or a canine alpha interferon mutant, and the amino acid sequence of the canine alpha interferon recombinant protein is shown in a sequence table SEQ ID NO. 1-4.
2. The recombinant canine interferon-alpha protein of claim 1, wherein the mutant canine interferon-alpha is CaIFN-alpha-Mut formed by mutating CaIFN-alpha at amino acid sites, and the mutated amino acid comprises 1 or 2 amino acids at the 150-164 th site of canine interferon-alpha.
3. A method for preparing the highly active recombinant canine interferon-alpha protein of claim 1 or 2, comprising the steps of:
carrying out codon optimization on CaIFN-alpha before carrying out amino acid site mutation on the CaIFN-alpha to obtain canine alpha interferon mutant CaIFN-alpha-Mut, CaIFN-alpha-Mut and protein label synthesis, then synthesizing on a plasmid vector, cloning to a yeast expression vector to obtain a recombinant plasmid after double enzyme digestion, then carrying out enzyme digestion linearization, introducing into an expression host bacterium to obtain recombinant yeast, and then carrying out induction expression and chromatographic purification to obtain a target protein, namely the canine alpha interferon recombinant protein.
4. The canine interferon-alpha recombinant protein of claim 3, wherein the yeast expression vector comprises pPICZ α A, pPICZ α B, pPICZ α C, pGAPZ α A, pPIC9K, pPIC9, pHIL-S1, and pYAM 75P.
5. The canine interferon-alpha recombinant protein of claim 3, wherein the expression host bacterium comprises a Pichia host bacterium or Saccharomyces cerevisiae.
6. A highly active recombinant canine interferon-alpha protein as defined in claim 1 or 2 for use in the preparation of antiviral drugs.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234171A (en) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | CaIFN-alpha & T alpha 1 fusion protein, vector, recombinant strain and application thereof |
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| CN108486127A (en) * | 2018-01-12 | 2018-09-04 | 中国农业科学院北京畜牧兽医研究所 | 6 α of dog interferon-α, 7 recombinant proteins and the preparation method and application thereof |
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| CN113234171A (en) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | CaIFN-alpha & T alpha 1 fusion protein, vector, recombinant strain and application thereof |
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