CN112500472B

CN112500472B - Cat omega interferon mutant and preparation method and application thereof

Info

Publication number: CN112500472B
Application number: CN202011408856.4A
Authority: CN
Inventors: 刘昕; 赖强; 王弋; 谢汝祝; 郑飞; 彭小珍; 欧海航
Original assignee: Guangzhou Yuanbo Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Yuanbo Pharmaceutical Technology Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-09-23
Anticipated expiration: 2040-12-04
Also published as: CN112500472A

Abstract

The invention belongs to the technical field of interferon genetic engineering, and particularly relates to a cat omega interferon mutant and a preparation method and application thereof. The mutants include mature peptide feline omega interferon mutants; the mature peptide cat omega interferon mutant is obtained by performing site-specific mutagenesis on mature peptide cat omega interferon, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1-SEQ ID NO.6 in a sequence table; the cat omega interferon is subjected to mutation of amino acid sites to form a mutant with glycosylation sites, the cat omega interferon and the cat omega interferon mutant subjected to optimized modification of a pichia pastoris codon are expressed, proper glycosylation modification improves the biological activity of the cat interferon mutant and prolongs the half life period, and the problems of low content of cat interferon, low biological activity, short half life period, poor stability, difficulty in formation of disulfide bonds, complex production process, high purification preparation cost and the like prepared by the conventional expression system and related biological technologies are solved.

Description

Cat omega interferon mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of interferon genetic engineering, and particularly relates to a cat omega interferon mutant and a preparation method and application thereof.

Background

In 1985, interferon omega was first cloned from sendai virus-induced Namalwa cells. In 1992, the cat interferon gene is separated from Nakamura for the first time, and a recombinant interferon medicament based on related research is successfully developed and is marketed in Japan, and is mainly used for treating feline calicivirus and canine parvovirus diseases. Since the discovery of IFN-omega, it has been a hot spot of research at home and abroad due to its species specificity and more efficient antiviral activity. IFN-omega is present in animals such as humans, felines, pigs, horses, rabbits, and bats, and is not found in dogs and mice.

The cat omega interferon is the cat interferon preparation which is applied earliest and is the cat interferon with the strongest antiviral activity. The feline omega interferon has a broad-spectrum antiviral effect, binds to a cell surface receptor, induces cells to produce a plurality of antiviral proteins, so that the timbre virus replicates in the cells, is effective to both RNA and DNA viruses, and has 94% homology with FeIFN-alpha and FeIFN-omega, but the antiviral, antiproliferative and immunoregulatory activities of the FeIFN-alpha and the FeIFN-omega are greatly different although the homology between the FeIFN-alpha and the FeIFN-omega is high. Researchers have also conducted comparative studies on the antiviral activities of FeIFN-alpha and FeIFN-omega, and the antiviral activity of FeIFN-omega is 160 times and 4 times higher than that of FeIFN-alpha in respect of two viruses, H9N2 subtype Avian Influenza Virus (AIV) and Canine Distemper Virus (CDV).

The feline omega interferon has the effects of accelerating and strengthening the immunity of the vaccine, can shorten the time for generating the antibody and improve the antibody level of an organism when being used together with the vaccine, has excellent anti-tumor activity, and has the potential of being a first-choice therapeutic drug for the feline breast cancer, but FeIFN-omega has poor pharmacokinetics, short half-life period which is less than 2h and limits the application of the FeIFN-omega in clinic.

The problems of low expression quantity, low activity, short half-life period and the like exist in the related research of the cat interferon in the prior art. Exogenous expression of the feline 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 expression are faced, and complicated protein purification processes such as renaturation, endotoxin removal, heat source removal and the like exist; the expression by animal cells has the conditions of complicated industry, higher cost and difficult large-scale industrialized production.

Improves the pharmaceutical property of the IFN-omega, and has wide application prospect in developing safe, high-stability and long-acting IFN-omega to IFN-omega. In the research of long-acting modification, technologies such as PEG modification, serum albumin modification, FC protein and the like are all used for improving the half-life of interferon, the PEG modification and FC protein fusion expression technology prolongs the half-life, but the antiviral activity is lower than that of natural IFN-omega. The problems of breakage, enzyme degradation, influence on the activity of cat interferon and the like easily occur during the modification and fusion expression of serum albumin, and the selection of fusion protein in different expression systems is a technical problem.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a cat omega interferon mutant, a preparation method and an application thereof. The FeIFN-omega with glycosylation sites is formed by mutating amino acid sites of FeIFN-omega, the biological activity of the cat interferon mutant is improved, the half-life period is prolonged, and the problems of low 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 of the cat interferon prepared by the conventional expression system and related biotechnology are solved.

The technical content of the invention is as follows:

the invention provides a cat omega interferon mutant, which comprises a mature peptide cat omega interferon mutant;

the mature peptide cat omega interferon mutant (mFeIFN-omega) is obtained by performing site-specific mutagenesis on mature peptide cat omega interferon, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1-SEQ ID NO.6 of a sequence table;

the mature peptide cat omega interferon (mFeIFN-omega) is a protein obtained by adopting an RT-PCR and PCR method after induced stimulation is carried out on a cat, and the amino acid sequence of the mature peptide cat omega interferon is shown as a sequence table SEQ ID NO. 7;

the amino acid sites of the site-directed mutation comprise 22 th to 24 th sites, 69 th sites, 110 th sites, 165 th and 167 th sites, 169 th to 171 th sites and 181 th to 184 th sites of the mature peptide feline omega interferon, and the mutation of the amino acid sites of the mature peptide feline omega interferon is beneficial to forming mutants with glycosylation sites, improving the biological activity, improving the stability and shortening the half life of the interferon;

arginine (R), arginine (R) and leucine (L) at the 22 th to 24 th positions are respectively mutated into asparagine (N), leucine (L) and threonine (T) to form mFeIFN-omega-MutN 22 with glycosylation sites, and the amino acid sequence of the mFeIFN-omega-MutN 22 is shown in a sequence table SEQ ID NO. 1;

the 69 th amino acid leucine (L) is mutated into threonine (T) to form mFeIFN-omega-MutN 67 with glycosylation sites, and the amino acid sequence of the mFeIFN-omega-MutN 67 is shown in a sequence table SEQ ID NO. 2;

arginine (R) at the 110 th site is mutated into asparagine (N) to form mFeIFN-omega-MutN 110 with a glycosylation site, and the amino acid sequence of the mFeIFN-omega-MutN 110 is shown as a sequence table SEQ ID NO. 3;

the amino acids threonine (T) and glutamine (Q) at the 165 th and 167 th positions are mutated into asparagine (N) and threonine (T) to form mFeIFN-omega-MutN 165 with glycosylation sites, and the amino acid sequence of the mFeIFN-omega-MutN 165 is shown in a sequence table SEQ ID NO. 4;

the 169 th to 171 th amino acids serine (S), phenylalanine (F) and alanine (A) are mutated into asparagine (N), leucine (L) and threonine (T) to form mFeIFN-omega-MutN 169 with glycosylation sites, and the amino acid sequence of the mFeIFN-omega-MutN 169 is shown in a sequence table SEQ ID NO. 5;

asparagine (N), leucine (L), threonine (T) and serine (S) are introduced into the 181-184 th amino acid to form mFeIFN-omega-MutN 181 with glycosylation sites, and the amino acid sequence of the mFeIFN-omega-MutN 181 is shown in a sequence table SEQ ID NO. 6.

The invention also provides a preparation method of the cat omega interferon mutant, which comprises the following steps:

1) obtaining a mature peptide cat omega interferon sequence: after the cat is induced and stimulated, a mature peptide cat omega interferon (mFeIFN-omega) sequence is obtained by adopting an RT-PCR and PCR method;

2) mature peptide feline omega interferon recombinant plasmid: newly performing codon optimization on the mature peptide cat omega interferon and the protein tag, then synthesizing the mature peptide cat omega interferon and the protein tag on a plasmid vector, cloning the mature peptide cat omega interferon and the protein tag to a pichia pastoris expression vector after double enzyme digestion, and connecting and transforming to obtain a recombinant plasmid mFeIFN-omega-plasmid vector;

3) amino acid mutation: analyzing a tertiary structure according to the mFeIFN-omega sequence, avoiding IFNAR1 and IFNAR2 receptor binding sites, and carrying out site-directed mutagenesis on the obtained mature peptide cat omega interferon to form mFeIFN-omega-mut with glycosylation sites;

3) construction of mutant recombinant protein: the method comprises the steps of designing corresponding complementary primers by taking mFeIFN-omega-pPICZ alpha A as a template, purifying a product obtained by adopting circular PCR amplification, carrying out DpnI enzyme digestion, carrying out seamless cloning kit treatment on the purified digestion product, then converting DH5 alpha, carrying out PCR identification on a positive strain by using a bacterial liquid, extracting a plasmid, carrying out sequencing identification, carrying out enzyme digestion linearization on the positive plasmid, introducing the positive plasmid 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 cat omega interferon mutant recombinant protein.

The amino acid sequence of the mature peptide cat omega interferon in the step 1) is shown in a sequence table SEQ ID NO. 7;

the protein Tag His-Tag is fused at the C end of the mutant, the amino acid sequence of the protein Tag His-Tag is shown in a sequence table SEQ ID NO.9, and the His-Tag protein Tag is convenient for the purification of target protein;

before the mature peptide cat omega interferon and the cat omega interferon mutant are fused with His-tag, pichia pastoris codon optimization is carried out, and the optimized nucleic acid sequences are respectively shown as SEQ ID NO. 8-SEQ ID NO.15 of a sequence table;

amino acid sequences of the mature peptide cat omega interferon, the cat omega interferon mutant and the His-tag after fusion are respectively shown in sequence tables SEQ ID NO. 16-SEQ ID NO. 22;

the pichia pastoris expression vector in the step 3) comprises one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pGAPZ alpha A, pPIC9K, pPIC9, pHIL-S1 and pYAM75P, the pichia pastoris has the characteristics of fast prokaryotic organism growth and easy operation, and simultaneously has the modification function of a eukaryotic cell expression system, the post-translational processing and modification modes of the expressed protein are similar to those of higher eukaryotes, and the pichia pastoris secretion expression system is a high-yield expression system;

the host bacteria in the step 3) comprise one of pichia pastoris host bacteria X33, GS115, KM71 and SMD1168, and are more favorable for forming disulfide bonds;

the nucleic acid sequence of the primer in the step 4) is shown in a sequence table SEQ ID NO. 23-SEQ ID NO. 34.

The invention also provides a cat omega interferon mutant used for preparing antiviral drugs.

The invention has the following beneficial effects:

the cat omega interferon mutant provided by the invention is characterized in that a mutant with a glycosylation site is formed by mutating an amino acid site of mature peptide cat omega interferon, a high-efficiency pichia pastoris methanol induced secretion expression system is utilized to express the mature peptide cat omega interferon subjected to pichia pastoris codon optimization modification and the cat omega interferon mutant, proper glycosylation modification improves the biological activity of the cat interferon mutant and prolongs the half-life period, and the problems of low cat interferon content, low biological activity, short half-life period, poor stability, difficulty in forming disulfide bonds, complex production process, high purification preparation cost and the like in the conventional expression system and related biotechnology are solved. The mature peptide feline omega interferon and the mutant thereof are integrated on the chromosome of pichia pastoris, and have the advantages of high protein expression content, moderate glycosylation, easy formation of disulfide bonds, good biological activity of an expression product, less background protein, simple and convenient operation, easy industrial production and the like.

Drawings

FIG. 1 is a schematic diagram of the construction of a mature peptide feline omega interferon recombinant protein;

FIG. 2 shows the result of PCR identification of mFeIFN- ω -pPICZ α A-DH5 α;

FIG. 3 shows the result of loop PCR amplification of the mFeIFN- ω -mut mutant;

FIG. 4 shows the result of PCR identification after the mFeIFN- ω -mut mutant was transformed;

FIG. 5 is a diagram showing the results of PCR identification of the recombinant yeast solutions after electrotransformation of the mFeIFN- ω and mFeIFN- ω -mut mutant groups;

FIG. 6 is a SDS-PAGE result of the supernatant of the recombinant yeast induced expression.

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 of a cat omega interferon mutant-recombinant protein:

1. synthesis of whole gene fragment and construction of recombinant plasmid

After stimulating domestic cats with interferon inducer polymyosome (poly I: C) and mitogen Phytohemagglutinin (PHA), extracting RNA of spleen tissues of cats to obtain a cat interferon FeIFN-omega sequence by using RT-PCR (reverse transcription-polymerase chain reaction) and PCR (polymerase chain reaction), carrying out TA (polymerase chain reaction) cloning on the sequence, carrying out signal peptide analysis on the sequence after sequencing, and removing the signal peptide to obtain a mature peptide mFeIFN-omega sequence;

according to the sequence of mFeIFN-omega and the map of an expression vector pPICZ alpha A, introducing a histidine tag and a termination codon TAA at the C end of a target gene, introducing an EcoRI enzyme digestion site at the upstream, and introducing XbaI at the downstream;

after the related sequence is optimized by a pichia pastoris codon, the whole gene is synthesized to a plasmid of pUC57 to obtain mFeIFN-omega-pUC 57;

cloning a target fragment obtained by enzyme digestion of EcoRI and XbaI on mFeIFN-omega-pUC 57 to pPICZ alpha A subjected to same double enzyme digestion, and carrying out T4 ligase connection and transformation competence DH5 alpha to obtain mFeIFN-omega-pPICZ alpha A-DH5 alpha;

the construction scheme of the recombinant plasmid is shown in FIG. 1.

And (3) carrying out bacteria liquid PCR identification on the mFeIFN-omega-pPICZ alpha A-DH5 alpha, selecting the PCR to identify positive bacteria, carrying out plasmid extraction and sequencing identification, wherein the identification system of the PCR is shown in a table 1, and the identification program is shown in a table 2.

TABLE 1 PCR identification System

Name of reagent	Dosage (mu L)
		2×Rapid Taq Master PCR Mix	12.5
α-factor(25μmol/L)	0.5
		3’Aox1(25μmol/L)	0.5
Bacterial liquid	2.0
		ddH ₂ O	9.5

TABLE 2 PCR identification procedure

As shown in FIG. 2, the result of PCR identification was positive, indicating that the recombinant plasmid was constructed correctly.

Site-directed mutagenesis of glycosylation sites of mFeIFN- -Mut

Analyzing a tertiary structure according to an mFeIFN-omega sequence, avoiding IFNAR1 and IFNAR2 receptor binding sites, carrying out site-directed mutation on the mFeIFN-omega to obtain mFeIFN-omega-Mut with glycosylation sites, designing corresponding complementary primers (the primer sequences are shown in table 3) by taking the mFeIFN-omega-pPICZ alpha A as a template, carrying out DpnI enzyme digestion after purifying a product obtained by adopting loop PCR amplification, and converting the purified digestion product into DH5 alpha after carrying out seamless cloning kit treatment.

The site of the site-directed mutation of the mFeIFN-omega amino acid comprises:

respectively mutating arginine (R), arginine (R) and leucine (L) at positions 22-24 into asparagine (N), leucine (L) and threonine (T) to form mFeIFN-omega-MutN 22 with glycosylation sites;

mutating leucine (L) at the 69 th amino acid to threonine (T) to form mFeIFN-omega-MutN 67 with glycosylation sites;

mutating arginine (R) at position 110 to asparagine (N) to form mFeIFN-omega-MutN 110 with glycosylation sites;

mutating amino acids threonine (T) and glutamine (Q) at positions 165 and 167 into asparagine (N) and threonine (T) to form mFeIFN-omega-MutN 165 with glycosylation sites;

mutating amino acids including serine (S), phenylalanine (F) and alanine (A) at positions 169-171 into asparagine (N), leucine (L) and threonine (T) to form mFeIFN-omega-MutN 169 with glycosylation sites;

introducing amino acids 181-184 into asparagine (N), leucine (L), threonine (T) and serine (S) to form mFeIFN-omega-MutN 181 with glycosylation sites.

TABLE 3 Loop PCR amplification primers

The results of the loop PCR are shown in FIG. 3, and the amplification of the loop PCR of each group is positive, which indicates that each mutant plasmid is successfully amplified.

3. PCR identification of recombinant positive transformants

The primers were identified as α -factor and 3' Aox1 (synthesized by jinzhi biotechnology, guangzhou, respectively), the PCR system and procedure are shown in tables 1 and 2, and the PCR products were subjected to 1% agarose gel electrophoresis.

The PCR is selected and identified as positive bacteria to carry out plasmid extraction and sequencing identification, and the identification result is shown in figure 4: all groups of bacterial fluid are positive, and the sequencing result of the plasmid shows that all groups of bacteria are sequenced correctly.

4. Enzyme digestion linearization and purification recovery of recombinant plasmid

Referring to the enzyme digestion test manual of TAKARA company, Sac I is used for single enzyme digestion of each recombinant plasmid, and agarose gel electrophoresis detection is carried out to complete linearization;

and (4) purifying and recovering the linearized product, wherein the purification and recovery method refers to a kit use instruction.

5. Preparation of Pichia competent cells

1) Inoculating yeast recipient bacteria (including X33, GS115, KM71 and SMD1168, in this example, X33) to YPD plate, and culturing at 30 deg.C for 2 days;

2) selecting a single colony on a plate, inoculating the single colony in a 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.

6. Electrical transformation of pichia competent cell by linear expression plasmid

1) Preparing 80 mu L of yeast competence (X33 is adopted in the embodiment, and the using mode of other host bacteria is the same) to be mixed with 1-5 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) after the electric shock was terminated, 1mL of sorbitol (1M) was rapidly added, and the mixture was allowed to stand on ice for 15min, followed by standing culture in an incubator at 30 ℃ for 1 h. Adding 1mLYPD liquid culture medium, shaking and culturing at 30 deg.C and 200r/min for 1 hr, centrifuging at 4000r/min at normal temperature, collecting thallus, and spreading onto YPDS plate containing 100 μ g/μ L for standing culture at 30 deg.C for 3 d.

7. Identification of recombinant yeast and screening of high copy number

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 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 4:

TABLE 4 PCR identification procedure for recombinant yeast solutions

The PCR product was subjected to 1% agarose gel electrophoresis to identify transformants in which the primers amplified the band of interest. High copy selection requires a combination of banding intensity in PCR identification and high resistance YPD plate (200. mu.g/mLzeocin) assay results.

As shown in FIG. 5, the PCR identification result of the recombinant yeast solution shows that: all identified strains in each group were positive recombinant yeast strains, indicating that the electrotransformation of X33 was successful.

YPD (containing 100. mu.g/mLzeocin) plate streaking can be carried out on the corresponding strain for subsequent induced expression.

8. Inducible expression of high-copy recombinant yeast

(1) Selecting a single colony with Zeocin resistance growing on a YPD plate by using a sterilized gun head, selecting the single colony in 20mL BMGY liquid culture medium for activated culture, oscillating at 30 ℃ at 200r/min overnight until OD600 is 2-6, and then enabling the cell to be in an exponential phase;

(2) centrifuging at 3000r/min at room temperature for 5min, collecting precipitate, suspending in 1mL BMMY, wrapping with four layers of clean gauze and two layers of newspaper, and performing shake culture in 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 ℃.

9. SDS-PAGE of recombinant yeast induced expression supernatant

And (3) performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on the supernatant of the recombinant yeast induced expression 4d, setting a corresponding empty plasmid pPICZ alpha A-X33 control group, and comparing the control group with the mFeIFN-omega group to observe the glycosylation modification condition of the target protein by the expression system.

The protein Loading Buffer solution is 5 multiplied by Loading Buffer, and the Loading amount is 12L;

as shown in FIG. 6, except that there is no significant band in group N67, mFeIFN- ω and corresponding glycosylation site modification mutants mFeIFN- ω -MutN22, mFeIFN- ω -MutN110, mFeIFN- ω -MutN165, mFeIFN- ω -MutN169, and mFeIFN- ω -MutN181 were all expressed efficiently in yeast expression systems, and the band size matched the expected 21kDa due to the absence of the mFeIFN- ω glycosylation site. As can be seen from comparison of the deglycosylation treatment group, the non-deglycosylation treatment histone is slightly larger than the deglycosylation treatment group, which indicates that the yeast expression system carries out moderate glycosylation modification on mFeIFN-omega-MutN 22, mFeIFN-omega-MutN 110, mFeIFN-omega-MutN 165, mFeIFN-omega-MutN 169 and mFeIFN-omega-MutN 181. No band is obvious in the mFeIFN-omega-MutN 67 group, the expression level is low, a band can be observed after purification, and the result is not shown.

10. Purifying, recovering, purifying and recovering the expression product,

in order to research the influence of pichia pastoris glycosylation modification on the activity of cat omega interferon, a deglycosylation treatment group (a deglycosylation reagent with the final concentration of 1 percent and incubation for 3h at 37 ℃) and a non-deglycosylation treatment group are arranged, the two groups of purified expressed supernatants are combined with His Tag to carry out nickel column affinity chromatography for protein adsorption, elution and purification, and the concentration is measured after imidazole is removed by a dialysis method. The concentrations of the purified samples of each group are shown in table 5 below, and the samples of each group are subjected to freeze-drying preservation for later use.

TABLE 5 sample concentrations after purification

Detection of biological activity and half-life of recombinant proteins:

1. determination of biological Activity-Microlesion inhibition method

Detecting the activity of a target protein by using a CRFK-VSV (CRFK-VSV) micro-lesion inhibition method, paving a cell culture plate with 96 holes for digested CRFK cells, adding 100 mu L of purified cat omega interference (a deglycosylation treatment group and a non-deglycosylation treatment group) diluted by 4 times into each hole after the cells are completely attached to the wall, neutralizing the cells with 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 viruses;

the inhibition of cytopathic effect was observed after 48h, with the highest interferon dilution inhibiting 50% of the cytopathic CPE50 being 1 activity unit.

TABLE 6 Activity of purified samples

Group of	Deglycosylation treatment group	Group not subjected to deglycosylation
			mFeIFN-omega group	-	1.21×10 ⁷ U/mg
mFeIFN-omega-MutN 22 group	1.24×10 ⁷ U/mg	2.35×10 ⁷ U/mg
			mFeIFN-omega-MutN 67 group	1.18×10 ⁷ U/mg	1.54×10 ⁷ U/mg
mFeIFN-omega-MutN 110 group	1.26×10 ⁷ U/mg	2.78×10 ⁷ U/mg
			mFeIFN-omega-MutN 165 group	1.22×10 ⁷ U/mg	3.74×10 ⁷ U/mg
mFeIFN-omega-MutN 169 group	1.19×10 ⁷ U/mg	4.07×10 ⁷ U/mg
			mFeIFN-omega-MutN 181 group	1.25×10 ⁷ U/mg	3.98×10 ⁷ U/mg

As can be seen from Table 6, mFeIFN-omega-MutN 22, mFeIFN-omega-MutN 110, mFeIFN-omega-MutN 165, mFeIFN-omega-MutN 169 and mFeIFN-omega-MutN 181 in the deglycosylated group and the non-deglycosylated group have higher activities, and the activity of the non-deglycosylated group is higher than that of the deglycosylated group, indicating that moderate glycosylation can effectively improve the activity of feline omega interferon. The comparison of the activities of the mFeIFN-omega group and the mutant shows that the activity of each group of mFeIFN-omega-MutN 169 > mFeIFN-omega-MutN 181 > mFeIFN-omega-MutN 165 > mFeIFN-omega-MutN 110> mFeIFN-omega-MutN 22 > mFeIFN-omega-MutN 67 > mFeIFN-omega, and especially the activity of the mFeIFN-omega-MutN 169 is improved by 3.36 times compared with that of the natural mFeIFN-omega, which indicates that the mutation of an appropriate amino acid site is favorable for improving the activity of the feline omega interferon.

2. Determination of half-life of feline omega interferon

And selecting a deglycosylation treatment group and a non-deglycosylation treatment group of the mFeIFN-omega-Mut to measure the half life of the cat omega interferon, wherein the relation between the blood concentration of the cat omega interferon and the time is measured by adopting a cytopathic inhibition method.

6 adult domestic cats with weight close to 4kg are respectively half male and female.

Lyophilized mFeIFN-omega and mFeIFN-omega-Mut were injected subcutaneously in the neck at a dose of 1 mg/mL/dose. Blood was collected intravenously at 1h, 2h, 4h, 8h, 16h, 24h, 48h, 72h after injection, respectively. After blood sample is coagulated at 4 ℃ and centrifuged at 3000r/min for 5min, the upper serum is obtained. And (3) determining the concentration of the cat omega interferon in serum at different time points by adopting a cytopathic effect inhibition method, performing curve fitting by using DAS (data acquisition system) pharmacokinetic software, and calculating related parameters.

TABLE 7 Activity of purified samples

The half-life of the deglycosylated group of the mFeIFN-omega-Mut of each group is about 1.4h, which is equivalent to that of the natural mFeIFN-omega group, the half-life of the non-deglycosylated group of the mFeIFN-omega-Mut 22, the mFeIFN-omega-Mut N110, the mFeIFN-omega-Mut N165, the mFeIFN-omega-Mut N169 and the mFeIFN-omega-Mut N181 is obviously improved compared with that of the deglycosylated group, and the half-life of the mFeIFN-omega-Mut N169 group is improved by 2.8 times compared with that of the mFeIFN-omega group.

The mature peptide cat omega interferon glycosylation modification mutant in a yeast expression system can obviously prolong the half-life period, simplifies the long-acting modification process of cat omega interferon, and has wide application prospect.

Sequence listing

<110> Guangzhou Yuanbo medicine science and technology Co., Ltd

<120> cat omega interferon mutant and preparation method and application thereof

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Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Asn Leu Thr Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

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Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Thr His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 3

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 3

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Asn Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 4

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 4

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Asn Leu Thr Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 5

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 5

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Asn Leu Thr Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 6

<211> 184

<212> PRT

<213> Artificial Sequence

<400> 6

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser Asn Leu Thr Ser

180

<210> 7

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 7

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 8

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 8

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agattctttt gctattaaag atggagattt ggcttcttct 540

<210> 9

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 9

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgaatttga cttctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agattctttt gctattaaag atggagattt ggcttcttct 540

<210> 10

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 10

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgactcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agattctttt gctattaaag atggagattt ggcttcttct 540

<210> 11

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 11

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaat gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agattctttt gctattaaag atggagattt ggcttcttct 540

<210> 12

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 12

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctaatttgac tgattctttt gctattaaag atggagattt ggcttcttct 540

<210> 13

<211> 540

<212> DNA

<213> Artificial Sequence

<400> 13

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agataatttg actattaaag atggagattt ggcttcttct 540

<210> 14

<211> 552

<212> DNA

<213> Artificial Sequence

<400> 14

tgtgctttgc caggatctga tgctcaagtt agtagagata atttggtttt gttgggacaa 60

atgagaagat tgtctccatt tttgtgtttg agagctagaa aagattttag atttccaaga 120

gaaatgttgg aaagaggaca attgagagaa gctcaagctg ctgctccagt tttgagagaa 180

ttgttgcaac aaacttttaa tttgttgcat actgaaagat cttctgctgc ttggtctcca 240

gctccattgc atggattgag atctggattg catagacaat tggaagcttt ggatgcttgt 300

ttgttgcaag ctactaggga aggcgaaaga gcgacgggag aaggtgaaag ggctccaggg 360

atgcatggac cagttttggc tatcaagaga tattttcagg atattagagt ttatttggaa 420

gatgaaggat attctgattg tgcttgggaa attgttagat tggaaattat gagagctttg 480

ttttcttctg ctactttgca agattctttt gctattaaag atggagattt ggcttcttct 540

aatttgacta gt 552

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 15

catcatcatc atcatcat 18

<210> 16

<211> 186

<212> PRT

<213> Artificial Sequence

<400> 16

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser His His His His His His

180 185

<210> 17

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 17

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Asn Leu Thr Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser

180

<210> 18

<211> 186

<212> PRT

<213> Artificial Sequence

<400> 18

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Thr His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser His His His His His His

180 185

<210> 19

<211> 186

<212> PRT

<213> Artificial Sequence

<400> 19

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Asn Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser His His His His His His

180 185

<210> 20

<211> 186

<212> PRT

<213> Artificial Sequence

<400> 20

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Asn Leu Thr Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser His His His His His His

180 185

<210> 21

<211> 186

<212> PRT

<213> Artificial Sequence

<400> 21

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Asn Leu Thr Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser His His His His His His

180 185

<210> 22

<211> 190

<212> PRT

<213> Artificial Sequence

<400> 22

Cys Ala Leu Pro Gly Ser Asp Ala Gln Val Ser Arg Asp Asn Leu Val

1 5 10 15

Leu Leu Gly Gln Met Arg Arg Leu Ser Pro Phe Leu Cys Leu Arg Ala

20 25 30

Arg Lys Asp Phe Arg Phe Pro Arg Glu Met Leu Glu Arg Gly Gln Leu

35 40 45

Arg Glu Ala Gln Ala Ala Ala Pro Val Leu Arg Glu Leu Leu Gln Gln

50 55 60

Thr Phe Asn Leu Leu His Thr Glu Arg Ser Ser Ala Ala Trp Ser Pro

65 70 75 80

Ala Pro Leu His Gly Leu Arg Ser Gly Leu His Arg Gln Leu Glu Ala

85 90 95

Leu Asp Ala Cys Leu Leu Gln Ala Thr Arg Glu Gly Glu Arg Ala Thr

100 105 110

Gly Glu Gly Glu Arg Ala Pro Gly Met His Gly Pro Val Leu Ala Ile

115 120 125

Lys Arg Tyr Phe Gln Asp Ile Arg Val Tyr Leu Glu Asp Glu Gly Tyr

130 135 140

Ser Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ala Leu

145 150 155 160

Phe Ser Ser Ala Thr Leu Gln Asp Ser Phe Ala Ile Lys Asp Gly Asp

165 170 175

Leu Ala Ser Ser Asn Leu Thr Ser His His His His His His

180 185 190

<210> 23

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 23

atgaatttga cttctccatt tttgtgtttg agagctag 38

<210> 24

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 24

atggagaagt caaattcatt tgtcccaaca aaaccaaatt atc 43

<210> 25

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 25

acttttaatt tgactcatac tgaaagatct tctgctgctt g 41

<210> 26

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 26

gtatgagtca aattaaaagt ttgttgcaac aattctctc 39

<210> 27

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 27

gcgaaaatgc gacgggagaa ggtgaaaggg c 31

<210> 28

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 28

tctcccgtcg cattttcgcc ttccctagta gcttgcaac 39

<210> 29

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 29

ctgctaattt gactgattct tttgctatta aagatggaga t 41

<210> 30

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 30

gaatcagtca aattagcaga agaaaacaaa gctctcataa t 41

<210> 31

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 31

aagataattt gactattaaa gatggagatt tggcttcttc 40

<210> 32

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 32

ctttaatagt caaattatct tgcaaagtag cagaagaaaa c 41

<210> 33

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 33

tctaatttga ctagtcatca tcatcatcat cattaatcta gaac 44

<210> 34

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 34

atgatgacta gtcaaattag aagaagccaa atctccatct tt 42

Claims

1. The cat omega interferon mutant is characterized by being obtained by performing site-specific mutagenesis on mature peptide cat omega interferon, and the amino acid sequence of the mutant is shown in a sequence table SEQ ID NO. 1-SEQ ID NO. 6.

2. The cat omega interferon mutant of claim 1, wherein the site-directed mutation has an amino acid position in which a new amino acid is introduced at positions 22 to 24, 69, 110, 165, 167, 169 to 171, or 181 to 184 of the mature peptide cat omega interferon.

3. The cat omega interferon mutant of claim 2, wherein the 22 nd to 24 th arginine, arginine and leucine are mutated into asparagine, leucine and threonine, respectively;

the leucine at position 69 is mutated to threonine;

the 110 th arginine is mutated to asparagine;

the threonine and the glutamine at the 165 th and the 167 th positions are mutated into asparagine and threonine;

the 169 th to 171 th serine, phenylalanine and alanine are mutated into asparagine, leucine and threonine;

the 181-184 th sites are introduced with asparagine, leucine, threonine and serine.

4. A method of making a cat omega interferon mutant according to claim 1, comprising the steps of:

1) mature peptide feline omega interferon recombinant plasmid: newly performing codon optimization on the mature peptide cat omega interferon and the protein tag, then synthesizing the mature peptide cat omega interferon and the protein tag on a plasmid vector, cloning the mature peptide cat omega interferon and the protein tag to a pichia pastoris expression vector after double enzyme digestion, and connecting and transforming to obtain a recombinant plasmid mFeIFN-omega-plasmid vector;

the amino acid sequence of the mature peptide cat omega interferon is shown in a sequence table SEQ ID NO. 7;

2) amino acid mutation: performing site-directed mutagenesis on the obtained mature peptide cat omega interferon to form a mature peptide cat omega interferon mutant mFeIFN-omega-mut with glycosylation sites;

3) mutant expression: the method comprises the steps of designing a corresponding complementary primer by taking an mFeIFN-omega-plasmid vector as a template, purifying a product obtained by adopting circular PCR amplification, carrying out DpnI enzyme digestion, carrying out seamless cloning kit treatment on the purified digestion product, then converting DH5 alpha, identifying a positive strain by bacterial liquid PCR, extracting a plasmid, carrying out sequencing identification, carrying out enzyme digestion linearization on the positive plasmid, introducing the positive plasmid into an expression host bacterium to obtain recombinant saccharomycetes, and carrying out induced expression and chromatographic purification to obtain a target protein, namely the cat omega interferon mutant recombinant protein.

5. The method for preparing the cat omega interferon mutant according to claim 4, wherein the nucleic acid sequence of the mature peptide cat omega interferon in the step 1) is shown as the sequence table SEQ ID NO. 8.

6. The method for preparing the cat omega interferon mutant recombinant protein according to claim 4, wherein the pichia pastoris expression vector in the step 2) comprises one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pGAPZ alpha A, pPIC9K, pPIC9, hil-S1 and pYAM 75P.

7. The method for preparing the feline omega interferon mutant recombinant protein according to claim 4, wherein the host bacteria of the step 2) comprise one of pichia host bacteria X33, GS115, KM71 and SMD 1168.

8. The preparation method of the cat omega interferon mutant recombinant protein as claimed in claim 4, wherein the nucleic acid sequence of the primer in the step 3) is shown as SEQ ID No. 23-SEQ ID No.34 of the sequence table.

9. A feline omega interferon mutant of claim 1 for use in preparing an antiviral medicament.