CN108276486B - Cat omega 2 interferon mutant and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cat omega 2 interferon mutant and a preparation method and application thereof, belonging to the field of preparation and application of cat omega 2 interferon. The invention obtains the cat omega 2 interferon mutant by comparing the gene sequences and amino acid sequences of 13 subtypes of cat omega interferon and carrying out amino acid site-directed mutagenesis on the cat omega 2 interferon. The invention further discloses a method for preparing the cat omega 2 interferon mutant, which comprises the following steps: the cat omega 2 gene is subjected to point mutation, the mutant gene is optimized, the gene coding the cat omega 2 interferon mutant is cloned into a baculovirus transfer vector to be recombined with baculovirus to infect an insect host, an exogenous gene is expressed, and a cat omega 2 interferon protein expression product is obtained, the antiviral activity of cat omega 2 interferon can be improved by more than 45% through mutation, and the antiviral activity of cat omega 2 interferon can be improved by more than 96% through optimization and mutation of the gene. The method has simple process, can efficiently and stably obtain the safe cat omega 2 interferon, and obviously improves the antiviral activity.

Description

Cat omega 2 interferon mutant and preparation method and application thereof

The application is a divisional application of a parent application with the application number of 201510197296.5 and the application date of 2015 4-23, and the name of the parent application is 'cat omega interferon mutant and a preparation method and application thereof'.

Technical Field

The invention relates to a cat omega interferon, in particular to a cat omega 2 interferon mutant, and also relates to a preparation method of the cat omega 2 interferon mutant and application of the cat omega 2 interferon mutant in preparation of a medicament or a reagent for preventing or treating cat viral diseases, belonging to the field of preparation and application of cat omega 2 interferon.

Background

Interferon (IFN) is a glycoprotein with high biological activity produced by cells under the action of specific inducers, and has broad-spectrum antiviral activity in the animal cells. It is known that interferons do not directly kill viruses, but interfere with gene transcription of viruses or translation of viral protein components by inducing cells of the host itself to produce various enzymes. Interferons can be classified into type I and type II IFN according to different cell sources, physicochemical properties, biological activity and other aspects; type I IFN mainly includes IFN-alpha, beta, omega, delta, kappa, epsilon, zeta, tau, etc., and type II IFN only has one IFN-gamma (Cann AJ. principles of Molecular biology. Beijing: Science Press, 2006: 177-.

The species known to date to have omega interferon are human, cat, horse, sheep, cow, pig, rabbit, etc. Cats are bred in China as a traditional pet in a large amount, and infectious diseases of the cats, particularly viral diseases such as feline distemper, feline infectious rhinotracheitis and feline calicivirus infection, are getting more and more serious. At present, a specific medicine for treating viral diseases does not exist, high-quality and low-cost cat interferon products are urgently needed to be put on the market, but the expression quantity of the cat interferon is not high at present, and the antiviral activity is not strong.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a cat omega 2 interferon mutant and a cat omega 11 interferon mutant, wherein the cat omega interferon mutant has high antiviral activity;

the invention aims to solve another technical problem of providing a method for preparing the cat omega 2 interferon mutant or the cat omega 11 interferon mutant by using a bombyx mori baculovirus expression system;

the third technical problem to be solved by the invention is to provide the application of the cat omega 2 interferon mutant or the cat omega 11 interferon mutant in preparing a medicament or a reagent for preventing or treating cat viral diseases.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention firstly discloses a cat omega 2 interferon mutant, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO. 10. The nucleotide sequence of the gene for coding the cat omega 2 interferon mutant is shown as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.11 or SEQ ID NO. 12.

The invention also discloses a cat omega 11 interferon mutant, the amino acid sequence of which is shown in SEQ ID NO. 7. The nucleotide sequence of the gene for coding the cat omega 11 interferon mutant is shown as SEQ ID NO.8 or SEQ ID NO. 9.

The amino acid sequence of the cat omega 2 interferon before mutation is shown as SEQ ID NO.1, and the gene sequence thereof is shown as SEQ ID NO. 3; the amino acid sequence of the cat omega 11 interferon before mutation is shown as SEQ ID NO. 6.

By comparing gene sequences and amino acid sequences of 13 subtypes of cat omega interferon, the invention discovers that amino acid compositions of various subtypes have tiny mutation, such as amino acid changing from P to L, G to A, A to V and the like; the cat omega interferon 2 type and the cat omega interferon 4 type have higher antiviral activity, and compared with amino acid sequences, the amino acid sequences of the two types of omega interferon are found to have 7 more amino acids (RATGEGE) at the positions of 134-140, which indicates that the 7 amino acids have certain promotion effect on the antiviral expression of the cat omega interferon; type 11 and type 8 have lower antiviral activity, and the amino acid sequences are aligned to find that the amino acid at position 125 of the two types is mutated from L to V, which indicates that V possibly influences the antiviral activity of feline omega interferon. Based on the comparison result, in order to improve the antiviral activity of the cat omega interferon, the invention artificially corrects the changed site which is the same as omega 11 in the omega 2 type amino acid sequence, and introduces site-directed mutation of 4 amino acids by referring to the mutant site of omega 3: the obtained mutation sequences of 203 amino acids of R82Q, P105L, V148A and G201A are shown as SEQ ID NO. 2; the mutated sequence of the amino acid is reverse translated into a nucleotide sequence by DNAworks, and the nucleotide sequence is shown as SEQ ID NO. 4.

The invention further utilizes OptimumGene^TMThe technology optimizes cat omega 2 type interferon mutant genes, modifies gene sequences according to codon preference of a bioreactor silkworm, optimizes and designs various related parameters which influence gene transcription efficiency, translation efficiency, GC content of protein folding, CpG dinucleotide content, codon preference, secondary structure of mRNA, mRNA free energy stability, RNA instability motif, repetitive sequences and the like, is favorable for improving the transcription efficiency and the translation efficiency of the optimized genes in the silkworm, and keeps the protein sequences translated finally unchanged. In order to improve the translation initiation efficiency in a silkworm baculovirus eukaryotic expression system, a Kozak sequence AAC is added in front of a gene, and in order to improve the translation termination efficiency, a stop codon is changed into TAA. In addition, restriction sites such as BamHI, EcoRI, SmaI and the like in the gene sequence are removed, BamHI is added at the upstream of the gene, XbaI restriction sites are added at the downstream of the gene, and the sequence of the obtained cat omega 2 interferon mutation optimization gene is shown as SEQ ID NO. 5.

The invention also carries out multi-site mutation on the original cat omega 2 interferon, artificially corrects the changed sites in the omega 2 amino acid sequence which are the same as omega 11, and introduces site-directed mutation of 10 amino acids by referring to the mutant sites of omega 3, omega 4 and omega 13: A12V, P27L, P62L, A78T, R82Q, P103L, P105L, P142L, V148A and G201A, wherein the obtained 203-amino-acid multi-site mutation sequence is shown as SEQ ID NO. 10; the multi-site mutated sequence of amino acids was reverse translated into a nucleotide sequence by DNAworks, as shown in SEQ ID NO. 11. Using OptimumGene^TMTechnical couple cat omega 2 typeOptimizing the interferon multi-mutant gene, and modifying the gene sequence according to the codon preference of the bioreactor silkworm, wherein the nucleotide sequence after multi-site mutation and optimization is shown as SEQ ID NO. 12.

The invention mutates cat omega 11 interferon according to the same method as omega 2 interferon mutant, and introduces site-directed mutagenesis of 4 amino acids: R82Q, P105L, V141A and G194A, wherein the amino acid sequence of the mutated feline omega 11 interferon is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8. The invention further applies online software to analyze rare codons in mature cat omega 11 interferon mutant genes, then replaces all the rare codons in the mature cat omega 11 interferon genes with baculovirus preference codons by a baculovirus preference codon table, adds BamHI enzyme cutting sites and Kozak sequences at the 5 'end of the mature cat omega 11 interferon genes according to the same method, adds XbaI enzyme cutting sites at the 3' end of the mature cat omega 11 interferon genes, and optimizes the mutant gene sequences as shown in SEQ ID NO. 9.

The invention further discloses a method for preparing the cat omega 2 interferon mutant or the cat omega 11 interferon mutant, which comprises the following steps:

(1) respectively cloning a gene encoding the cat omega 2 interferon mutant or a gene encoding the cat omega 11 interferon mutant into a baculovirus transfer vector to construct a recombinant transfer vector;

(2) co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus;

(3) infecting the recombinant baculovirus into insect cells or insect hosts, culturing the infected insect cells or insect hosts to express corresponding protein, and purifying to obtain the recombinant baculovirus.

Wherein the baculovirus transfer vector is selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, pAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIEl, pAcJPl, pAcUF 2, pAcMLF7, cMLLF 8, pAPlcM, pAcMP2, pAcRP23, pAcRP25, pAcRW4, pAMAG, pAcUWl, pAcUW21, pAcUW2A, pAcUW2B, pAcUcUpW 3, pAcUcWv 31, pApYNcVyNpYNpYNpYNpV 13972, pApYNcVpYNpVpVpV, pApPCV 13972, pApYNcVpPCV 36 42, pApYNcVpYNpPCV 3636 42, pApYVEpYwV, pApYwV 5972, pApYpYPV, pApYpYpYpYvEPV 2, pApYpYpVEpVEpVIV 3655, pApVEpVIL, pApVEpVEpVIV 3655, pAcVEpVIV, pApVIV 36pIII or pApVEpVEpVEpVEpVEpVEpIII; preferably pVL 1393;

the baculovirus is selected from bombyx mori baculovirus parent strain BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV; preferably bombyx mori baculovirus parent strain BmBacmid;

the insect host is selected from the group consisting of Bombyx mori (Bombyx mori), Bombyx mori (Bombyx mandarina), Ricinus communis (Philosamia cynthia ricim), Bombyx mori (Dictyoloca japonica), Ailanthus altissima (Philosamia cynthia pryeri), Antheraea pernyi (Antheraea pernyi), Antheraea japonica (Antheraea yamamai), Bombyx mori (Antheraea polyphylla), Autographa californica (Atogaria californica), Ectropicalis gigas (Ectropis obliqua), Trichoplusia (Mamestra brassicae), Spodoptera littoralis (Spodoptera littoralis), Spodoptera frugiperda (Spodoptera frugiperda), Trichoplusia ni (Spodoptera armyworm, Heliothis armyworm (Heliothis virens), Heliothis virtus, Helicosa (tobacco), Heliothis virens (tobacco), and Helicoverpa armigera (tobacco); preferably domestic silkworm (Bombyx mori);

the infection refers to that the recombinant baculovirus infects 1-5-year-old insect larvae or pupae bodies through swallowing or permeating epidermis; preferably, the recombinant silkworm baculovirus is used for infecting silkworm cells or inoculating silkworm larvae or pupae of 1-5 years old by puncture, and body fluid or tissue homogenate of the silkworm larvae or pupae containing various cat omega interferon genes is collected after infection for 3-6 days; wherein, the pupa is the early young pupa of 1-2 days optimally.

The invention also discloses an expression vector or a host cell containing the gene for coding the cat omega 2 interferon mutant or the gene for coding the cat omega 11 interferon mutant.

The transfer vector constructed by the invention comprises: vectors containing cat omega 2, 9 and 11 type interferon genes and omega 2 mutant gene original sequences, pVL 1393-FeIFN-omega 2, pVL 1393-FeIFN-omega 9, pVL 1393-FeIFN-omega 11, VL 1393-FeIFN-omega 11-M, pVL 1393-FeIFN-omega 2-M, pVL 1393-FeIFN-omega 2-MM; the vector pVL 1393-FeIFN-omega 2-O, VL 1393-FeIFN-omega 11-M-O, pVL 1393-FeIFN-omega 2-M-391O, VL 1393-FeIFN-omega 2-MM-O containing the codon optimized sequence of cat omega 2, 9 and 11 type interferon genes and omega 2 mutant genes.

The recombinant baculovirus obtained by the invention comprises: recombinant bombyx mori nuclear polyhedrosis virus rBmBacmid (FeIFN-omega 2, FeIFN-omega 9, FeIFN-omega 11-M, FeIFN-omega 2-M, FeIFN-omega 2-MM) and rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 11-M-O, FeIFN-omega 2-M-O, FeIFN-omega 2-MM-O).

The invention expresses original cat interferon omega 2 (shown in SEQ ID NO. 3), 9 and 11 genes and omega 2 mutant gene sequences (omega 2-M and shown in SEQ ID NO. 4) in a silkworm bioreactor, and detects the antiviral activity of cat omega-type interferon expressed by silkworm larvae on a CRFK/VSV GFP system by applying a micro cytopathy inhibition method. The results show that the cells added with the recombinant feline omega interferon protein have the capacity of resisting virus infection; the FeIFN-omega 2 expressed in silkworm larva has obvious antiviral activity and potency of 4.7X 10⁵U/mL, the antiviral activity of FeIFN-omega 11 is only 2.3X 10⁵U/mL; the antiviral potency of FeIFN-omega 2-M is obviously higher than that of FeIFN-omega 2 and is 6.8 multiplied by 10⁵U/mL, which indicates that it is feasible and effective to improve the antiviral activity of FeIFN-omega by gene point mutation.

The invention further expresses the optimized cat omega 2 interferon gene (omega 2-O, shown in SEQ ID NO. 13) and the cat omega 2 interferon mutation optimization gene (omega 2-M-O, shown in SEQ ID NO. 5) in a silkworm bioreactor, and Western blotting results show that a specific band with the size of 19kDa can be detected in the supernatant of a silkworm hemolymph sample after recombinant virus infection. The result of antiviral activity determination shows that the optimized cat omega 2 interferon gene and FeIFN-omega 2-O and FeIFN-omega 2-M-O expressed by mutant gene in silkworm larva have obvious antiviral activity, and the titer reaches 6.5 multiplied by 10 respectively⁵U/mL and 9.2X 10⁵U/mL, while the antiviral activity of FeIFN-omega 2 is only 4.7X 10⁵U/mL, the antiviral activity of FeIFN-omega 2 can be improved by more than 45% through mutation; the antiviral activity of the FeIFN-omega 2 can be improved by more than 38% through optimization; the antiviral activity of the FeIFN-omega 2-M can be improved by more than 35 percent by optimizing the mutant gene; the antiviral activity of the FeIFN-omega 2 can be improved by more than 96% through mutation and codon optimization, which shows that the mutation and optimization method is effective and practical and has a great effect on improving the antiviral activity of the FeIFN-omega 2.

The invention also expresses cat omega 2 interferon gene multi-site mutant gene (omega 2-MM shown in SEQ ID NO. 11) and multi-site mutant and optimized gene (omega 2-MM-O shown in SEQ ID NO. 12) in a silkworm bioreactor, and the antiviral activity determination result of the expression product shows that FeIFN-omega 2-MM and FeIFN-omega 2-MM-O expressed in silkworm larvae have obvious antiviral activity, and the titer reaches 6.3 multiplied by 10⁵U/mL and 8.2X 10⁵U/mL, but the improvement of the antiviral activity is not much of FeIFN-omega 2-M, which indicates that the antiviral activity of the FeIFN-omega 2 cannot be greatly improved by multi-site mutation, and the antiviral activity of the FeIFN-omega 2 can be improved only by selecting proper site mutation.

The invention expresses the mutant cat omega 11 interferon gene (omega 11-M, shown in SEQ ID NO. 8) and the optimized mutant cat omega 11 interferon gene (omega 11-M-O, shown in SEQ ID NO. 9) in a domestic silkworm bioreactor, and detects that the cat recombinant omega 11 interferon in silkworm haemolymph has antiviral activity on a CRFK cell/VSV GFP system by adopting a micro cytopathic effect inhibition method, and the titer of FeIFN-omega 11-M is 3.7 multiplied by 10⁵U/mL, FeIFN-. omega.11-M-O titer 4.5X 10⁵U/mL is greatly improved compared with FeIFN-omega 11, but is not as high as FeIFN-omega 2, which indicates that the same mutation on cat omega 11 interferon gene can also play a role in improving antiviral activity.

The cat omega 2 interferon mutant or the cat omega 11 interferon mutant can be applied to preparation of drugs or reagents for preventing or treating cat viral diseases. Wherein the feline viral disease comprises: feline distemper, feline infectious rhinotracheitis, feline calicivirus infection, feline leukemia virus infection, feline immunodeficiency virus infection, or vesicular stomatitis virus infection.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention introduces site-directed mutagenesis of 4 amino acids to the cat omega 2 interferon by comparing gene sequences and amino acid sequences of 13 subtypes of the cat omega interferon to obtain a cat omega 2 interferon mutant. The invention further optimizes the nucleotide sequence for coding the cat omega 2 interferon mutant, and expresses the cat omega 2 interferon mutant by using a silkworm baculovirus expression system, so that the antiviral activity of the expressed cat omega 2 interferon mutant is greatly improved. The invention also plays a role in improving antiviral activity by carrying out the same amino acid site-directed mutagenesis on the cat omega 11 interferon. The invention further introduces site-directed mutagenesis of 10 amino acids into the cat omega 2 interferon, optimizes a mutated gene sequence, and expresses the mutant in a domestic silkworm bioreactor, so that the obtained cat omega 2 interferon mutant has obvious antiviral activity, but the improvement of the antiviral activity is not improved by introducing site-directed mutagenesis of 4 amino acids, which indicates that the antiviral activity of the cat omega 2 interferon can be greatly improved only by selecting proper site mutagenesis.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell.

The term "transfection" refers to the process by which eukaryotic cells acquire a new genetic marker due to the incorporation of foreign DNA.

Drawings

FIG. 1 shows the double restriction enzyme identification of recombinant plasmid pVL-FeIFN- ω x; wherein, M: DNA molecular mass standard; ω 2: recombinant plasmid pVL-FeIFN-omega 2 double enzyme digestion product; ω 9: recombinant plasmid pVL-FeIFN-omega 9 double enzyme digestion product; ω 2-M: recombinant plasmid pVL-FeIFN-omega 2-M double enzyme digestion product; ω 11: recombinant plasmid pVL-FeIFN-omega 11 double enzyme digestion product; -is a negative control;

FIG. 2 shows the cells showing various ratios of fluorescence; wherein, A: interferon inhibits fluorescence exhibited by VSV virus; b: fluorescence exhibited by VSV virus infected controls; c: fluorescence exhibited by infection of a portion of the cells with VSV virus;

FIG. 3 shows the double restriction enzyme identification of recombinant plasmids pVL-FeIFN- ω 11-M and pVL-FeIFN- ω 11-M-O; wherein, M: DNA molecular mass standard; 1: recombinant plasmid pVL-FeIFN-omega 11-M double enzyme digestion product; 2: recombinant plasmid pVL-FeIFN-omega 11-M-O double enzyme digestion product;

FIG. 4 shows the double restriction enzyme digestion identification of recombinant plasmids pVL-FeIFN- ω 2-O and pVL-FeIFN- ω 2-M-O; wherein, M: DNA molecular mass standard; 1: recombinant plasmid pVL-FeIFN-omega 2-O double enzyme digestion product; 2: recombinant plasmid pVL-FeIFN-omega 2-M-O double enzyme digestion product;

FIG. 5 is a result of SDS-PAGE of recombinant feline omega 2 interferon; wherein, M: DNA molecular mass standard; 1: recombinant virus rBmBacmid (FeIFN-omega 2) expression product; 2: recombinant virus rBmBacmid (FeIFN-omega 2-O) expression product; 3: recombinant virus rBmBacmid (FeIFN-omega 2-M-O) expression product; 4: negative control;

FIG. 6 is a fluorescence plot corresponding to a cytopathic ratio; wherein, a, "-": (ii) cell-free lesions; b, "+/-": several cytopathies; c, "+": 20% -30% of cytopathic effect; d, "+ +": 50% -60% of cytopathic effect.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

1. Test materials and reagents

The silkworm variety JY1 is provided by the silkworm research institute of Jiangsu science and technology university, and the DNA of the parental virus Bm-Bacmid is constructed according to the method disclosed in the literature (patent application No. 201110142492.4, application publication No. CN 102286534A); VSV-GFP virus, transfer vector pVL1393, e.coli strain TOP10, bnn cells, CRFK cells, were all maintained and provided by the institute of biotechnology, chinese academy of agricultural sciences.

Restriction enzyme, T₄DNA ligase was purchased from Promega, liposomes from Invitrogen, DMEM cell culture medium, and fetal bovine serum was GIBCO.

Reference is made to the relevant tool book for the preparation of solutions and media (Joseph et al, third edition of the molecular cloning guidelines, 2002; Oseber, et al, eds. molecular biology guidelines, 1998); unless otherwise indicated, percentages and parts are by weight.

Example 1 expression and detection of cat interferon omega 2, 9, 11 and omega 2 mutant Gene sequences in silkworm bioreactors

1. Experimental methods

1.1 acquisition and Synthesis of feline omega-type Interferon Gene and construction of plasmid

1.1.1 acquisition of Gene

The gene sequences and amino acid sequences of FeIFN-. omega.2 (GenBank accession No. DQ420221), FeIFN-. omega.9 (GenBank accession No. DQ420228), and FeIFN-. omega.11 (GenBank accession No. DQ420230.1) were searched and obtained from NCBI.

Wherein the amino acid sequence of the cat omega 2 interferon is shown as SEQ ID NO.1, and the gene sequence thereof is shown as SEQ ID NO. 3; the amino acid sequence of the cat omega 11 interferon is shown as SEQ ID NO. 6.

Comparing gene sequences and amino acid sequences of 13 subtypes of cat omega interferon, finding that the amino acid composition of each subtype has tiny mutation, such as amino acid changing from P to L and G to A; the cat omega interferon 2 type and the cat omega interferon 4 type have higher antiviral activity, and compared with amino acid sequences, the amino acid sequences of the two types of omega interferon have 7 more amino acids (RATGEGE) at the position of 134-140, which shows that the 7 amino acids have certain promotion effect on the antiviral activity of the cat omega interferon; type 11 and type 8 have lower antiviral activity, and the amino acid sequences are aligned to find that the amino acid at position 125 of the two types is mutated from L to V, which indicates that V possibly influences the antiviral activity of feline omega interferon. Based on the comparison result, the same change site as omega 11 in the omega 2 type amino acid sequence is specially and artificially corrected, and the site-directed mutation of 4 amino acids is introduced by referring to the mutation site of omega 3: the obtained 203 amino acid mutation sequences of R82Q, P105L, V148A and G201A are shown as SEQ ID NO.2 and marked as FeIFN-omega 2-M, and the gene sequence is shown as SEQ ID NO. 4.

1.1.2 Synthesis of Gene sequences and plasmid construction

Restriction enzyme cutting sites were analyzed by DNAman software, and restriction enzyme cutting sites not present in the sequence of the target gene were added to both ends of the target gene based on the analysis results of the restriction enzyme cutting sites and the multiple cloning sites on the transfer vectors pVL1393 and pUC 57. According to the analysis result, a BamHI restriction site and a Kozak sequence are added at the 5 'end of the plasmid, an XbaI restriction site is added at the 3' end of the plasmid, the determined gene sequence is synthesized by Nanjing Kingsry Biotechnology GmbH, and a pUC57 vector is inserted to form plasmids pUC 57-FeIFN-omega 2, pUC 57-FeIFN-omega 9, pUC 57-FeIFN-omega 11 and pUC 57-FeIFN-omega 2-M.

1.2 construction of recombinant baculovirus transfer vectors

The synthesized plasmids pUC 57-FeIFN-. omega.2, pUC 57-FeIFN-. omega.9, pUC 57-FeIFN-. omega.11 and pUC 57-FeIFN-. omega.2-M were double-digested with BamHI and XbaI, and the target fragment recovered by the glass milk method was ligated with the inactivated baculovirus transfer vector pVL1393 double-digested with BamHI and XbaI using T4DNA ligase, and reacted at 16 ℃ for 8 hours or more. The ligation product is transformed into escherichia coli competent cell TOP10, colonies are selected and cultured, plasmids are upgraded, positive clones are identified by BamHI and XbaI double enzyme digestion, correctly identified recombinant plasmids are sent to Beijing engine biotechnology Limited for sequencing, and correctly sequenced plasmids are named as pVL-FeIFN-omega 2, pVL-FeIFN-omega 9, pVL-FeIFN-omega 11 and pVL-FeIFN-omega 2-M.

1.3 Co-transfection of recombinant plasmids with parental viruses

The recovery and passage of the BmN cells and the screening of the recombinant viruses are carried out according to the methods reported in the literature. When the BmN cells are cultured until the cell monolayer reaches about 80%, the old culture medium is poured out, washed three times by the serum-free TC-100 culture medium, and then the serum-free culture medium is added. And embedding the recombinant plasmids pVL-FeIFN-omega 2, pVL-FeIFN-omega 9, pVL-FeIFN-omega 11 and pVL-FeIFN-omega 2-M and the parental virus Bm-Bacmid DNA by using a liposome for 20min, and then co-transfecting the recovered BmN cells to perform intracellular homologous recombination. Culturing at 27 ℃ for about 5 days until the cells shed and float, collecting cell culture solution, obtaining recombinant viruses Bm-Bacmid (FeIFN-omega 2), Bm-Bacmid (FeIFN-omega 9), Bm-Bacmid (FeIFN-omega 11) and Bm-Bacmid (FeIFN-omega 2-M) containing target genes, and purifying and amplifying the viruses to be used as seed viruses.

1.4 expression of cat omega interferon in silkworm

Recombinant virus culture solution is added according to the formula 10⁵PFU/head dose is injected into 5-year-old silkworm, the silkworm is cultured under the condition of 27 ℃ and 70% -80% humidity, the growth late stage of the silkworm larva, and FeIFN-omega is efficiently expressed under the action of a polyhedrosis gene promoter. Inoculating and infecting for about 3.5-4 days, observing the symptoms of swelling, abnormal behavior, decreased appetite, etc. of silkworm larva, collecting hemolymph when the larva has obviously reduced volume, and storing at-20 deg.CAnd (5) standby.

1.5 detection of antiviral Activity of feline omega-type Interferon protein

The antiviral activity of feline omega-type interferon in silkworm haemolymph was tested on a CRFK/VSV-GFP system using a microcytopathy inhibition method. The CRFK cells in good condition were cultured at 3.0X 10⁵The density of each individual/ml was inoculated in a 96-well culture plate. Preparing the silkworm hemolymph with ultrasonic disruption and filter sterilization into solution with different dilutions by DMEM culture solution containing 70mL/L fetal bovine serum, inoculating diluted sample into culture wells full of CRFK cells at 100 μ L/well, setting at least 4 duplicate wells for each dilution and control silkworm blood, setting cell control group without silkworm hemolymph and VSV GFP and virus control group with VSV GFP, and culturing at 37 deg.C and 5% CO₂Culturing for 18-24 h under the condition. Diluting to 100TCID₅₀The VSV GFP virus of (1) was added to the culture well from which the supernatant had been aspirated at 100. mu.L/well, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing under the condition. When a large number of cells in each hole of the virus control group generate fluorescence and the cells in the cell control group still completely grow well and no fluorescence appears, the contrast system is completely qualified, and comprehensive observation can be carried out.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vectors pVL-FeIFN-omega 2, pVL-FeIFN-omega 9, pVL-FeIFN-omega 11 and pVL-FeIFN-omega 2-M are subjected to double enzyme digestion by BamHI and XbaI, 2 fragments are separated by electrophoresis in 1% agarose gel, the small fragments are respectively 612bp, 591bp and 612bp, and the large fragments are consistent with pVL1393 in size and are all 9607 bp. The electrophoresis results are shown in FIG. 1. The plasmid with correct enzyme restriction identification is sent to Beijing Optimalaceae New Biotechnology Limited for nucleic acid sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, which shows that omega-type interferon genes omega 2, 9, 11 and omega 2-M of the cat are successfully inserted between BamHI and XbaI in the pVL1393 transfer vector.

2.2 obtaining of recombinant viruses of feline Interferon and detection of recombinant products

Detection of silkworm larva on CRFK/VSV GFP system by using micro cytopathy inhibition methodExpressed feline omega-type interferon antiviral activity. The growth state of the cells in the cell control group is good and no fluorescence appears when the cells are observed under an inverted fluorescence microscope; cells in the virus-infected control group developed lesions, almost 100% of the cells showed fluorescence, and cells added with recombinant feline omega interferon protein had the ability to resist virus infection (fig. 2). Observing the pathological degree of cells according to the protective effect of cat omega-type interferon on CRFK cells, marking the cells in the hole as "+" when green fluorescent cells appear, and calculating the titer of the interferon according to a Reed-Muench method. The detection results are shown in Table 1, and the results of the antiviral activity determination show that the FeIFN-omega 2 expressed in silkworm larva bodies has more obvious antiviral activity, and the potency reaches 4.7 multiplied by 10⁵U/mL, the antiviral activity of FeIFN-omega 11 is only 2.3X 10⁵U/mL, the antiviral activity of FeIFN-omega 9 is between the two, 3.4X 10⁵U/mL, the result trend is the same as the reported result; the antiviral potency of FeIFN-omega 2-M is obviously higher than that of FeIFN-omega 2 and is 6.8 multiplied by 10⁵U/mL, the expected effect is achieved, and the fact that the FeIFN-omega antiviral activity is improved through gene point mutation is feasible and effective is shown.

TABLE 1 results of the detection of the antiviral activity of recombinant feline omega interferon

Example 2 expression and detection of a mutated and optimized feline interferon omega 11 Gene in a silkworm bioreactor

Based on the alignment results of example 1, the feline omega 2 interferon gene mutation contributes to the improvement of antiviral activity, and for this reason, the feline omega 11 type interferon gene was mutated in the same manner as omega 2-M.

1. Experimental methods

1.1 mutation, optimization and Synthesis of Cat omega 11 type Interferon Gene

FeIFN-. omega.11 (GenBank accession number: DQ420230.1) gene sequence and amino acid sequence were obtained from NCBI and introduced with site-directed mutagenesis of 4 amino acids according to the method of example 1: the obtained 196 amino acid mutant sequences of R82Q, P105L, V141A and G194A are shown in SEQ ID NO.7, and the deduced mutant gene sequence is shown in SEQ ID NO. 8. The mutant gene sequence was analyzed for restriction enzyme sites using DNAman software, and restriction enzyme sites not present in the target gene sequence were added to both ends of the target gene based on the results of the analysis of the restriction enzyme sites and the multiple cloning sites on the transfer vectors pVL1393 and pUC 57. According to the analysis result, a BamHI restriction site and a Kozak sequence are added to the 5 'end of the plasmid, an XbaI restriction site is added to the 3' end of the plasmid, a gene sequence is synthesized by Nanjing Kingsler Biotech limited, and a pUC57 vector is inserted to form a plasmid pUC 57-FeIFN-omega 11-M.

On-line software http:// gcua. schoedl. de/is used for analyzing rare codons in mature cat omega 11 interferon mutant genes, then all the rare codons in the mature cat omega 11 interferon genes are synonymously replaced by baculovirus preference codons through a baculovirus preference codon table, optimized cat omega 11-M interferon genes are obtained through synthesis, BamHI enzyme cutting sites and Kozak sequences are added to the 5 'ends of the optimized cat omega 11-M interferon genes according to the same method, XbaI enzyme cutting sites are added to the 3' ends of the optimized cat omega 11-M interferon genes, the gene sequences are synthesized by Nanjing Kingsry Biotech limited, and a pUC57 vector is inserted to form plasmids pUC 57-FeIFN-omega 11-M-O. The sequence of the optimized mutant gene of the cat omega 11 is shown as SEQ ID NO. 9.

1.2 construction of recombinant baculovirus transfer vectors

The synthesized plasmids pUC 57-FeIFN-omega 11-M and pUC 57-FeIFN-omega 11-M-O were digested with BamHI and XbaI, and the target fragment recovered by the glass milk method was ligated with BamHI and XbaI digested and inactivated baculovirus transfer vector pVL1393 with T4DNA ligase, and reacted at 16 ℃ for 8 hours or more. The ligation product is transformed into escherichia coli competent cell TOP10, colonies are selected and cultured, plasmids are upgraded, positive clones are identified by BamHI and XbaI double enzyme digestion, correctly identified recombinant plasmids are sent to Beijing Optimalaceae biotechnology Limited for sequencing, and correctly sequenced plasmids are named as pVL-FeIFN-omega 11-M and pVL-FeIFN-omega 11-M-O.

1.3 Co-transfection of recombinant plasmids with parental viruses

The recovery and passage of the BmN cells and the screening of the recombinant viruses are carried out according to the methods reported in the literature. When the BmN cells are cultured until the cell monolayer reaches about 80%, the old culture medium is poured out, washed three times by the serum-free TC-100 culture medium, and then the serum-free culture medium is added. Embedding the recombinant plasmids pVL-FeIFN-omega 11-M and pVL-FeIFN-omega 11-M-O and the parental virus Bm-Bacmid DNA with liposome for 20min, and then co-transfecting the recovered BmN cells to perform intracellular homologous recombination. Culturing at 27 deg.C for about 5 days until cell shedding and floating, collecting cell culture solution to obtain recombinant virus containing target gene, and purifying and amplifying virus to obtain seed virus.

1.4 expression of cat omega interferon in silkworm

Recombinant virus culture solution is added according to the formula 10⁵Injecting 5 th instar silkworm with PFU/head dosage, feeding the silkworm at 27 ℃ under the condition of 70-80% relative humidity, and efficiently expressing Bm-Bacmid (FeIFN-omega 11-M) and Bm-Bacmid (FeIFN-omega 11-M-O) at the late growth stage of silkworm larva under the action of a polyhedron gene promoter. The inoculation infection is about 3.5-4 days, the symptoms of swelling of the body node, abnormal behavior, appetite decrease and the like of the silkworm larva can be observed, when the obvious reduction of the larva volume is observed, hemolymph is collected and stored at-20 ℃ for later use.

1.5 detection of antiviral Activity of feline omega-type Interferon protein

The antiviral activity of feline omega interferon in silkworm haemolymph was tested on a CRFK/VSV-GFP system using the microcytopathy inhibition method, which was performed as described in example 1.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vectors pVL-FeIFN-omega 11-M and pVL-FeIFN-omega 11-M-O are identified by BamHI and XbaI double digestion, and small fragments separated by 1% agarose gel electrophoresis are 591bp, and large fragments are over 9000bp and are consistent with pVL1393(9607 bp). The electrophoresis results are shown in FIG. 3. The correct plasmid is identified by enzyme digestion, nucleic acid sequencing is carried out, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, which indicates that the omega 11-M type interferon gene and the optimized gene of the cat are successfully inserted between BamHI and XbaI in the pVL1393 transfer vector. The plasmid DNA with correct enzyme digestion detection is sent to Beijing Optimalaceae New Biotechnology Limited company for sequencing, the result is shown in SEQ ID NO.8 and SEQ ID NO.9, and the obtained recombinant plasmids are named as recombinant transfer vectors pVL-FeIFN-omega 11-M and pVL-FeIFN-omega 11-M-O.

2.2 obtaining of recombinant viruses of feline Interferon and expression of recombinant products

Inoculation of about 1X 10⁶Cells at 15cm²After the cells were attached to the wall in the flask, the medium containing Fetal Bovine Serum (FBS) was removed, washed three times with FBS-free medium, and 1.5ml FBS-free medium was added. Sequentially adding 1 mu g of bombyx mori baculovirus parent strain BmBacmid DNA, 2 mu g of recombinant transfer plasmid pVL-FeIFN-omega 11-M, pVL-FeIFN-omega 11-M-O and 5 mu l of liposome into a sterilizing tube, complementing the volume to 60 mu l by using sterile double distilled water, gently mixing the components uniformly, standing the mixture for 15min, and then dropwise adding the mixture into a culture bottle for cotransfection. After 4h incubation at 27 ℃ 1.5ml serum free medium and 300. mu.l FBS were supplemented. Culturing at the constant temperature of 27 ℃ for 4-5 days, and collecting supernatant for screening recombinant virus rBmBacmid (FeIFN-omega 11-M, FeIFN-omega 11-M-O). Inoculating a proper amount of cells (about 70-80%) in a small 35mm dish, sucking out the culture medium after the cells adhere to the wall, diluting the co-transfection supernatant at different concentrations, and adding 1ml of co-transfection solution into the adherent cells for uniform distribution. After infection for 1h at 27 ℃, absorbing infection liquid, melting 2% low melting point agarose gel in water bath at 60 ℃, cooling to 40 ℃, uniformly mixing with 2 XTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, adding 4ml of the gel into each dish, sealing with Parafilm after solidification, carrying out inverted culture at 27 ℃ for 3-5 d, and observing by using a microscope. And (3) selecting the plaques which do not contain the polyhedron, repeating the steps, and carrying out 2-3 rounds of purification to obtain the pure recombinant silkworm baculovirus rBmBacmid (FeIFN-omega 11-M, FeIFN-omega 11-M-O).

Infecting the recombinant bombyx mori baculovirus rBmBacmid (FeIFN-omega 11-M, FeIFN-omega 11-M-O) with the normally growing BmN cells, culturing for 3 days, collecting supernatant, and collecting the supernatant to obtain the supernatant containing a large amount of the recombinant virus rBmBacmid (FeIFN-omega 11-M, FeIFN-omega 11-M-O).

2.3 detection of expression products

The antiviral activity of cat omega-type interferon expressed by silkworm larvae is detected on a CRFK/VSV-GFP system by using a micro cytopathic inhibition method. The growth state of the cells in the cell control group is good and no fluorescence appears when observed under an inverted fluorescence microscope, and the virus infects the control groupThe cells in (1) are diseased, almost 100% of the cells show fluorescence, and the cells added with the recombinant feline omega interferon protein have the capacity of resisting virus infection. According to the protective effect of cat omega-type interferon on CRFK cells, the pathological degree of the cells is observed, when green fluorescent cells appear, the cells in the holes are marked as "+", and the titer of the interferon is calculated according to the Reed-Muench method, and the result is shown in Table 2. The results show that the FeIFN-omega 11-M and the FeIFN-omega 11-M-O expressed in the silkworm larva bodies have obvious antiviral activity, and the potency reaches 3.7 multiplied by 10 respectively⁵U/mL and 4.5X 10⁵U/mL, the antiviral activity of FeIFN-omega 11 is only 2.3X 10⁵U/mL, the antiviral activity of FeIFN-omega 11 expression can be improved by 61% through gene mutation, the antiviral activity of a product expressed by the mutant gene can be improved by 21% through optimizing the mutant gene, the activity of expressed interferon can be improved by more than 96% through mutation and optimization compared with FeIFN-omega 11, the effect generated by mutation is higher than that of optimization, and the antiviral activity of FeIFN-omega 11 can be greatly improved through combination of mutation and optimization.

TABLE 2 results of the measurement of the antiviral activity of recombinant feline omega interferon

Example 3 mutation and optimization of Cat interferon omega 2 Gene and expression and detection in silkworm bioreactor

Based on the results of examples 1 and 2, the feline omega interferon gene mutation and optimization facilitated the improvement of antiviral activity, for which the feline omega 2-type interferon gene and mutant gene were optimized for omega 2 and omega 2-M genes according to the optimization method of example 2, as follows:

1. experimental methods

1.1 construction of cat omega 2 type interferon mutant gene and optimization and synthesis of omega 2 interferon gene and mutant gene sequence

1.1.1 construction of Cat omega 2 type Interferon mutant Gene

Based on the comparison result of 13 subtype gene sequences and amino acid sequences of cat omega interferon in example 1, in order to improve the expression amount of cat omega interferon in a baculovirus expression system, the invention artificially corrects the variation site in the omega 2 type amino acid sequence, which is the same as omega 11, and introduces site-directed mutation of 4 amino acids by referring to the mutation site of omega 3: the obtained mutation sequences of 203 amino acids of R82Q, P105L, V148A and G201A are shown in SEQ ID NO. 2. The mutated sequence of the amino acid is reverse translated into a nucleotide sequence by DNAworks, and the nucleotide sequence is shown as SEQ ID NO. 4.

1.1.2 optimization and Synthesis of Cat omega 2 type Interferon Gene and mutant Gene sequences

Using OptimumGene^TMThe technology optimizes cat omega 2 type interferon genes and mutant genes, modifies gene sequences according to codon preference of a bioreactor silkworm, optimizes and designs various related parameters which influence gene transcription efficiency, translation efficiency, GC content of protein folding, CpG dinucleotide content, codon preference, secondary structure of mRNA, mRNA free energy stability, RNA instability motif, repetitive sequences and the like, is favorable for improving the transcription efficiency recording and translation efficiency of the optimized genes in the silkworm, and keeps the finally translated protein sequences unchanged.

In order to improve the translation initiation efficiency in a silkworm baculovirus eukaryotic expression system, a Kozak sequence AAC is added in front of a gene, and in order to improve the translation termination efficiency, a stop codon is changed into TAA. In addition, restriction sites for BamHI, EcoRI, SmaI, etc. within the gene sequence were removed, BamHI was added upstream of the gene and XbaI restriction sites were added downstream of the gene for subsequent cloning into the eukaryotic transfer vector pVL 1393. The gene sequence is artificially synthesized by a biotechnology company, the cat omega 2 interferon mutation optimizes the gene sequence, namely the gene sequence of omega 2-M-O is shown as SEQ ID NO.5, the gene sequence of omega 2 type interferon after optimization is shown as SEQ ID NO.13, and the gene sequence of omega 2-O is respectively inserted into a pUC57 vector to form plasmids pUC 57-FeIFN-omega which are named as pUCS-FeIFN-omega 2-O and pUCS-FeIFN-omega 2-M-O.

1.2 construction of recombinant baculovirus transfer vectors

The synthesized plasmids pUC 57-FeIFN-omega 2-O and pUC 57-FeIFN-omega 2-M-O were double digested with BamHI and XbaI, the target fragment recovered by the glass milk method was ligated with the inactivated baculovirus transfer vector pVL1393 double digested with BamHI and XbaI using T4DNA ligase, and reacted at 16 ℃ for 8 hours or more. The ligation product is transformed into escherichia coli competent cell TOP10, colonies are selected for culturing, plasmids are upgraded, positive clones are identified by BamHI and XbaI double enzyme digestion, correctly identified recombinant plasmids are sent to Beijing Optimalaceae biotechnology Limited for sequencing, and correctly sequenced plasmids are named as pVL-FeIFN-omega 2-O and pVL-FeIFN-omega 2-M-O.

1.3 Co-transfection of recombinant plasmids with parental viruses

The recovery and passage of the BmN cells and the screening of the recombinant viruses are carried out according to the methods reported in the literature. When the BmN cells are cultured until the cell monolayer reaches about 80%, the old culture medium is poured out, washed three times by the serum-free TC-100 culture medium, and then the serum-free culture medium is added. Embedding the recombinant plasmids pVL-FeIFN-omega 2-O and pVL-FeIFN-omega 2-M-O and the parental virus Bm-Bacmid DNA with liposome for 20min, and then co-transfecting the recovered BmN cells to perform intracellular homologous recombination. Culturing at 27 deg.C for about 5 days until cell shedding and floating, collecting cell culture solution to obtain recombinant virus Bm-Bacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O) containing target gene, and purifying and amplifying virus to obtain seed virus.

1.4 expression of cat omega interferon in silkworm

Recombinant virus culture solution is added according to the formula 10⁵Injecting 5 th instar silkworm with PFU/head dosage, feeding the silkworm at 27 ℃ under the condition of 70-80% relative humidity, and efficiently expressing Bm-Bacmid (FeIFN-omega 2-O) and Bm-Bacmid (FeIFN-omega 2-M-O) at the later growth stage of silkworm larva under the action of a polyhedron gene promoter. The inoculation infection is about 3.5-4 days, the symptoms of swelling of the body node, abnormal behavior, appetite decrease and the like of the silkworm larva can be observed, when the obvious reduction of the larva volume is observed, hemolymph is collected and stored at-20 ℃ for later use.

1.5 detection of antiviral Activity of feline omega-type Interferon protein

The antiviral activity of feline omega-type interferon in silkworm haemolymph was tested on a CRFK/VSV-GFP system using a microcytopathy inhibition method. The procedure was as in example 1.

1.6 Western blotting detection

Diluting 10 times of ultrasonic waves by PBS (pH 7.4) for silkworm hemolymph infected by recombinant virus, carrying out SDS-PAGE gel electrophoresis, carrying out gel concentration of 5% and separation gel concentration of 15%, transferring proteins onto a polyvinylidene fluoride (PVDF) membrane by a semi-dry transfer method, preparing 3% BSA (bovine serum albumin) by PBST for blocking, using serum after a prokaryotic expression His-FeIFN-omega 2 protein immune mouse as a primary antibody (1:1000 dilution), using HRP-labeled goat anti-mouse IgG as a secondary antibody (1:5000 dilution), finally developing by DAB (diaminobenzidine), terminating by deionized water, and detecting a result.

2 results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vectors pVL-FeIFN-omega 2-O and pVL-FeIFN-omega 2-M-O are identified by BamHI and XbaI double digestion, and small fragments separated by 1% agarose gel electrophoresis are 591bp, and large fragments are over 9000bp and are consistent with pVL1393(9607 bp). The electrophoresis results are shown in FIG. 4. The correct plasmid was identified by digestion and nucleic acid sequencing was performed, and the MegaAlign alignment showed that the sequence was identical to the originally designed sequence, indicating that the optimized omega 2 interferon gene and the optimized mutant gene of cat had been successfully inserted between BamHI and XbaI in pVL1393 transfer vector.

2.2 construction and acquisition of recombinant Bombyx mori baculovirus rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O)

Inoculation of about 1X 10⁶Cells at 15cm²After the cells were attached to the wall in the flask, the medium containing Fetal Bovine Serum (FBS) was removed, washed three times with FBS-free medium, and 1.5ml FBS-free medium was added. Sequentially adding 1 mu g of bombyx mori baculovirus parent strain BmBacmid DNA, 2 mu g of recombinant transfer plasmid pVL-FeIFN-omega 2-O, pVL-FeIFN-omega 2-M-O and 5 mu l of liposome into a sterilizing tube, complementing the volume to 60 mu l by using sterile double distilled water, gently mixing the components uniformly, standing the mixture for 15min, and then dropwise adding the mixture into a culture bottle for cotransfection. After 4h incubation at 27 ℃ 1.5ml serum free medium and 300. mu.l FBS were supplemented. Culturing at the constant temperature of 27 ℃ for 4-5 days, and collecting supernatant for screening recombinant virus rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O). Inoculating a proper amount of cells (about 70-80%) in a small 35mm dish, after the cells adhere to the wall, sucking off the culture medium, and mixingThe transfection supernatant was diluted to different concentrations and 1ml of co-transfection solution was added to adherent cells and distributed evenly. After infection for 1h at 27 ℃, absorbing infection liquid, melting 2% low melting point agarose gel in water bath at 60 ℃, cooling to 40 ℃, uniformly mixing with 2 XTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, adding 4ml of the gel into each dish, sealing with Parafilm after solidification, carrying out inverted culture at 27 ℃ for 3-5 d, and observing by using a microscope. And (3) selecting the plaques which do not contain the polyhedron, repeating the steps, and carrying out 2-3 rounds of purification to obtain the pure recombinant silkworm baculovirus rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O).

2.3 amplification of recombinant Virus rBmBacmid (FeIFN-. omega.2-O, FeIFN-. omega.2-M-O) in Bombyx mori cells

Infecting the recombinant bombyx mori baculovirus rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O) with the normally growing BmN cells, culturing for 3 days, collecting supernatant, and collecting the supernatant to obtain the supernatant containing a large amount of the recombinant virus rBmBacmid (FeIFN-omega 2-O, FeIFN-omega 2-M-O).

2.4 Western blotting detection results

Western blotting results (FIG. 5) showed that a specific band of 19kDa size was detectable in the supernatant of silkworm hemolymph samples after recombinant virus infection.

2.5 measurement of antiviral Activity of expression product

The antiviral activity of the expression products was determined on the Vero-VSV GFP system using cytopathic inhibition (see FIG. 6), the procedure and procedure being as in example 1. According to the protective effect of cat omega-type interferon on CRFK cells, the pathological degree of the cells is observed, when green fluorescent cells appear, the cells in the holes are marked as "+", and the titer of the interferon is calculated according to a Reed-Muench method, and the result is shown in a table 3. The result shows that the optimized FeIFN-omega 2-O and FeIFN-omega 2-M-O expressed by the cat interferon omega 2 gene and the mutant gene in silkworm larvae have obvious antiviral activity, and the titer respectively reaches 6.5 multiplied by 10⁵U/mL and 9.2X 10⁵U/mL, while the antiviral activity of FeIFN-omega 2 is only 4.7X 10⁵U/mL, the antiviral activity of FeIFN-omega 2 can be improved by more than 45% through mutation; the antiviral activity of the FeIFN-omega 2 can be improved by more than 38% through optimization; optimization of the mutant Gene to enable FeIFN-omega 2-MThe antiviral activity is improved by more than 35 percent; the antiviral activity of the FeIFN-omega 2 can be improved by more than 96% through mutation and codon optimization, which shows that the mutation and optimization method is effective and practical and has a great effect on improving the antiviral activity of the FeIFN-omega 2.

TABLE 3 results of the measurement of the antiviral activity of recombinant feline omega interferon

Example 4 Cat interferon omega 2 Gene Multi-site mutation and expression and detection in silkworm bioreactor

Based on the results of examples 1, 2 and 3, the optimization can improve the antiviral activity of cat omega interferon by 20-40%, the mutation can improve the antiviral activity by more than 40%, and the mutation and optimization can improve the antiviral activity by 90%, so that the cat omega 2 type interferon gene is subjected to multi-site mutation by the following specific method:

1. experimental methods

1.1 construction of cat omega 2 type interferon mutant gene and optimization and synthesis of omega 2 interferon gene and mutant gene sequence

1.1.1 construction of cat omega 2 type interferon multiple mutant genes

Based on the comparison result of 13 subtype gene sequences and amino acid sequences of cat omega interferon in example 1, in order to improve the expression amount of cat omega interferon in a baculovirus expression system, the invention artificially corrects the variation site in the omega 2 type amino acid sequence, which is the same as omega 11, and introduces 10 amino acid site-directed mutations by referring to the mutation sites of omega 3, omega 4 and omega 13: the obtained 203 amino acid multi-site mutation sequence of A12V, P27L, P62L, A78T, R82Q, P103L, P105L, P142L, V148A and G201A is shown as SEQ ID NO. 10. The multi-site mutation sequence of the amino acid is reversely translated into a nucleotide sequence by DNAworks, and the nucleotide sequence is shown as SEQ ID NO. 11.

1.1.2 optimization and Synthesis of Cat omega 2 type Interferon Multi-mutant Gene sequences

Using OptimumGene^TMTechnical pair catThe omega 2 type interferon multi-mutant gene is optimized, a gene sequence is modified according to the codon preference of a bioreactor silkworm, and various related parameters influencing the gene transcription efficiency, the translation efficiency, the protein folding GC content, the CpG dinucleotide content, the codon preference, the secondary structure of mRNA, the mRNA free energy stability, the RNA instability motif, the repetitive sequence and the like are optimized and designed, so that the improvement of the transcription efficiency recording and translation efficiency of the optimized gene in the silkworm is facilitated, and the final translated protein sequence is kept unchanged.

In order to improve the translation initiation efficiency in a silkworm baculovirus eukaryotic expression system, a Kozak sequence AAC is added in front of a gene, and in order to improve the translation termination efficiency, a stop codon is changed into TAA. In addition, restriction sites for BamHI, EcoRI, SmaI, etc. within the gene sequence were removed, BamHI was added upstream of the gene and XbaI restriction sites were added downstream of the gene for subsequent cloning into the eukaryotic transfer vector pVL 1393. The gene sequence is artificially synthesized, the nucleotide sequence of multi-site mutation of the cat omega 2 gene is shown as SEQ ID No.11, the nucleotide sequence of multi-site mutation and optimization of the cat omega 2 gene is shown as SEQ ID No.12, and the gene is inserted into a pUC57 vector to form a plasmid pUC 57-FeIFN-omega which is named as pUCS-FeIFN-omega 2-MM and pUCS-FeIFN-omega 2-MM-O.

1.2 construction of recombinant baculovirus transfer vectors

The synthesized plasmids pUC 57-FeIFN-omega 2-MM and pUC 57-FeIFN-omega 2-MM-O are subjected to double digestion by BamHI and XbaI, the target fragment recovered by the glass milk method is connected with a baculovirus transfer vector pVL1393 subjected to double digestion inactivation by BamHI and XbaI by T4DNA ligase, and the reaction is carried out at 16 ℃ for more than 8 hours. The ligation product is transformed into escherichia coli competent cell TOP10, colonies are selected and cultured, quality-improved grains are obtained, positive clones are identified by BamHI and XbaI double enzyme digestion, correctly identified recombinant plasmids are sent to Beijing engine biotechnology Limited for sequencing, and correctly sequenced plasmids are named as pVL-FeIFN-omega 2-MM and pVL-FeIFN-omega 2-MM-O.

1.3 Co-transfection of recombinant plasmids with parental viruses

The recovery and passage of the BmN cells and the screening of the recombinant viruses are carried out according to the methods reported in the literature. When the BmN cells are cultured until the cell monolayer reaches about 80%, the old culture medium is poured out, washed three times by the serum-free TC-100 culture medium, and then the serum-free culture medium is added. Embedding the recombinant plasmids pVL-FeIFN-omega 2-MM and pVL-FeIFN-omega 2-MM-O and the parental virus Bm-Bacmid DNA with liposome for 20min, then co-transfecting the recovered BmN cells, and carrying out intracellular homologous recombination. Culturing at 27 deg.C for about 5 days until cell shedding and floating, collecting cell culture solution to obtain recombinant virus Bm-Bacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O) containing target gene, and purifying and amplifying virus to obtain seed virus.

1.4 expression of cat omega interferon in silkworm

Recombinant virus culture solution is added according to the formula 10⁵PFU/head dose is injected into 5-year-old silkworm, and the silkworm is raised at 27 ℃ and 70-80% relative humidity, and FeIFN-omega 2-MM-O are efficiently expressed under the action of polyhedron gene promoter in the later growth stage of silkworm larva. The inoculation infection is about 3.5-4 days, the symptoms of swelling of the body node, abnormal behavior, appetite decrease and the like of the silkworm larva can be observed, when the obvious reduction of the larva volume is observed, hemolymph is collected and stored at-20 ℃ for later use.

1.5 detection of antiviral Activity of feline omega-type Interferon protein

The antiviral activity of feline omega-type interferon in silkworm haemolymph was tested on a CRFK/VSV-GFP system using a microcytopathy inhibition method. The procedure was as in example 1.

1.6 Western blotting detection

Diluting 10 times of ultrasonic waves by PBS (pH 7.4) for silkworm hemolymph infected by recombinant virus, carrying out SDS-PAGE gel electrophoresis, carrying out gel concentration of 5% and separation gel concentration of 15%, transferring proteins onto a polyvinylidene fluoride (PVDF) membrane by a semi-dry transfer method, preparing 3% BSA (bovine serum albumin) by PBST for blocking, using serum after a prokaryotic expression His-FeIFN-omega 2 protein immune mouse as a primary antibody (1:1000 dilution), using HRP-labeled goat anti-mouse IgG as a secondary antibody (1:5000 dilution), finally developing by DAB (diaminobenzidine), terminating by deionized water, and detecting a result.

2. Results of the experiment

2.1 identification of recombinant transfer vectors

The recombinant transfer vectors pVL-FeIFN-omega 2-MM and pVL-FeIFN-omega 2-MM-O are identified by BamHI and XbaI through double enzyme digestion, and small fragments separated by 1% agarose gel electrophoresis are 591bp, and large fragments are over 9000bp and are consistent with pVL1393(9607 bp). The correct plasmid is identified by enzyme digestion and subjected to nucleic acid sequencing, and the MegaAlign comparison result shows that the sequence is consistent with the originally designed sequence, so that the omega 2-MM type interferon gene and the optimized gene of the cat are successfully inserted between BamHI and XbaI in the pVL1393 transfer vector.

2.2 construction and acquisition of recombinant Bombyx mori baculovirus rBmBacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O)

Inoculation of about 1X 10⁶Cells at 15cm²After the cells were attached to the wall in the flask, the medium containing Fetal Bovine Serum (FBS) was removed, washed three times with FBS-free medium, and 1.5ml FBS-free medium was added. Adding 1 mu g of bombyx mori baculovirus parent strain BmBacmid DNA, 2 mu g of recombinant transfer plasmid PVLFeIFN-omega 2-MM, PVLFeIFN-omega 2-MM-O and 5 mu l of liposome into a sterilizing tube in sequence, complementing the volume to 60 mu l by using sterile double distilled water, gently mixing uniformly, standing for 15min, and then dropwise adding into a culture bottle for cotransfection. After 4h incubation at 27 ℃ 1.5ml serum free medium and 300. mu.l FBS were supplemented. Culturing at the constant temperature of 27 ℃ for 4-5 days, and collecting supernatant for screening recombinant viruses rBmBacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O). Inoculating a proper amount of cells (about 70-80%) in a small 35mm dish, sucking out the culture medium after the cells adhere to the wall, diluting the co-transfection supernatant at different concentrations, and adding 1ml of co-transfection solution into the adherent cells for uniform distribution. After infection for 1h at 27 ℃, absorbing infection liquid, melting 2% low melting point agarose gel in water bath at 60 ℃, cooling to 40 ℃, uniformly mixing with 2 XTC-100 culture medium (containing 20% FBS) preheated at 40 ℃, adding 4ml of the gel into each dish, sealing with Parafilm after solidification, carrying out inverted culture at 27 ℃ for 3-5 d, and observing by using a microscope. And (3) selecting the plaques which do not contain the polyhedron, repeating the steps, and carrying out 2-3 rounds of purification to obtain the pure recombinant silkworm baculovirus rBmBacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O).

2.3 amplification of recombinant viruses rBmBacmid (FeIFN-. omega.2-MM, FeIFN-. omega.2-MM-O) in Bombyx mori cells

Infecting the recombinant silkworm baculovirus rBmBacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O) with the normally growing BmN cells, culturing for 3 days, and collecting supernatant, wherein the supernatant contains a large amount of the recombinant virus rBmBacmid (FeIFN-omega 2-MM, FeIFN-omega 2-MM-O).

2.4 Western blotting detection results

Western blotting results showed that a specific band of 19kDa was detectable in the supernatant of a silkworm hemolymph sample after infection with the recombinant virus.

2.5 measurement of antiviral Activity of expression product

The antiviral activity of the expression product was measured on a Vero-VSV GFP system using cytopathic inhibition, the procedure and procedure were as in example 1, and the extent of the pathological changes of the cells was observed on the basis of the protective effect of feline omega-type interferon on CRFK cells, and the cells in the well were marked as "+" when green fluorescent cells appeared, and the potency of interferon was calculated according to the Reed-Muench method, and the results are shown in Table 4. The result shows that the optimized FeIFN-omega 2-MM and FeIFN-omega 2-MM-O expressed by the cat interferon omega 2 gene and the mutant gene in silkworm larvae have obvious antiviral activity, and the titer respectively reaches 6.3 multiplied by 10⁵U/mL and 8.2X 10⁵U/mL, but the improvement of the antiviral activity is not much of FeIFN-omega 2-M, which indicates that the antiviral activity of the FeIFN-omega 2 cannot be greatly improved by multi-site mutation, and the antiviral activity of the FeIFN-omega 2 can be improved only by selecting proper site mutation.

TABLE 4 results of the measurement of the antiviral activity of recombinant feline omega interferon

SEQUENCE LISTING

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> cat omega 2 interferon mutant and preparation method and application thereof

<130> BJ-2002-1150306F1

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 203

<212> PRT

<213> artifical sequence

<400> 1

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

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

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Ala Val

65 70 75 80

Leu Arg Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

Ser Ser Ala Ala Trp Ser Pro Ala Pro Leu His Gly Leu Arg Ser Gly

100 105 110

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

115 120 125

Gly Glu Gly Glu Arg Ala Thr Gly Glu Gly Glu Arg Ala Pro Gly Met

130 135 140

His Gly Pro Val Leu Ala Ile Lys Arg Tyr Phe Gln Asp Ile Arg Val

145 150 155 160

Tyr Leu Glu Asp Glu Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg

165 170 175

Leu Glu Ile Met Arg Ala Leu Ser Ser Ser Ala Thr Leu Gln Asp Ser

180 185 190

Leu Ala Ile Lys Asp Gly Asp Leu Gly Ser Ser

195 200

<210> 2

<211> 203

<212> PRT

<213> artifical sequence

<400> 2

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

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

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Ala Val

65 70 75 80

Leu Gln Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

Ser Ser Ala Ala Trp Ser Pro Ala Leu Leu His Gly Leu Arg Ser Gly

100 105 110

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

115 120 125

Gly Glu Gly Glu Arg Ala Thr Gly Glu Gly Glu Arg Ala Pro Gly Met

130 135 140

His Gly Pro Ala Leu Ala Ile Lys Arg Tyr Phe Gln Asp Ile Arg Val

145 150 155 160

Tyr Leu Glu Asp Glu Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg

165 170 175

Leu Glu Ile Met Arg Ala Leu Ser Ser Ser Ala Thr Leu Gln Asp Ser

180 185 190

Leu Ala Ile Lys Asp Gly Asp Leu Ala Ser Ser

195 200

<210> 3

<211> 612

<212> DNA

<213> artifical sequence

<400> 3

atggccctcc tgctccctct actgaccgcc ctggcgctgc tcacctgccg ccctggaggc 60

tctctgggct gtgccctgcc tgggagccac gcgcaggtta gcagggacaa cttggtgctc 120

ctgggccaga tgcggagact gtcccctttc ttgtgcctgc gggccagaaa agacttccgc 180

ttcccccggg agatgctgga gggcggccag ctccgggagg cccaggccgc cgccgccgtc 240

ctgcgggagc tgctccagca gaccttcaac ctgttgcaca cggagcgctc ctcggcggcc 300

tggagccccg cgccgctgca cggactccgc tctggcctcc accggcagct ggaagccctg 360

gacgcctgct tgctgcaggc cacgggcgag ggagagcgcg ccacgggcga gggagagcgc 420

gccccgggga tgcacggccc tgtcctggcc atcaagaggt acttccagga catccgcgtc 480

tacctggagg acgagggata cagtgactgc gcctgggaaa ttgtcaggct ggaaatcatg 540

agagccttgt cctcctcggc gaccttgcaa gacagcttgg ccatcaagga tggagacctg 600

ggctcatctt ga 612

<210> 4

<211> 612

<212> DNA

<213> artifical sequence

<400> 4

atggccctcc tgctccctct actgaccgcc ctggcgctgc tcacctgccg ccctggaggc 60

tctctgggct gtgccctgcc tgggagccac gcgcaggtta gcagggacaa cttggtgctc 120

ctgggccaga tgcggagact gtcccctttc ttgtgcctgc gggccagaaa agacttccgc 180

ttcccccggg agatgctgga gggcggccag ctccgggagg cccaggccgc cgccgccgtc 240

ctgcaggagc tgctccagca gaccttcaac ctgttgcaca cggagcgctc ctcggcggcc 300

tggagccccg tgccgctgca cggactccgc tctggcctcc accggcagct ggaagccctg 360

gacgcctgct tgctgcaggc cacgggcgag ggagagcgcg ccacgggcga gggagagcgc 420

gccccgggga tgcacggccc tgccctggcc atcaagaggt acttccagga catccgcgtc 480

tacctggagg acgagggata cagtgactgc gcctgggaaa ttgtcagact ggaaatcatg 540

agagccttgt cctcctcggc gaccttgcaa gacagcttgg ccatcaagga tggagacctg 600

gcgtcatctt ga 612

<210> 5

<211> 609

<212> DNA

<213> artifical sequence

<400> 5

atggccctgt tgctcccgtt acttacagca ttggcgctgt tgacttgccg tccgggtggt 60

tcactgggtt gtgcactccc cggatctcac gcgcaagtga gcagggacaa cttagttctc 120

ttgggtcaga tgagacgcct cagccctttc ctctgcttac gtgccaggaa agatttcaga 180

tttccacgcg aaatgctcga gggcggtcaa ttacgcgaag ctcaggctgc cgcagcggtt 240

ctgcaagagc ttctgcaaca gacctttaat ttgctccaca cggaaagatc atctgctgcc 300

tggtccccag ccttacttca cggattacgt tcgggccttc ataggcaact tgaagcactg 360

gacgcgtgcc tgttgcaggc tacaggagaa ggcgagagag caaccggtga aggagagagg 420

gcacctggaa tgcatggtcc agctttggcc ataaaacgtt acttccaaga catcagggtc 480

tacctggaag acgagggcta tagtgattgt gcttgggaaa tcgtaagact tgagattatg 540

cgcgctctca gctcctcggc cacactgcaa gattcattgg cgatcaagga cggtgatttg 600

gcaagttca 609

<210> 6

<211> 196

<212> PRT

<213> artifical sequence

<400> 6

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

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

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Ala Val

65 70 75 80

Leu Arg Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

Ser Ser Ala Ala Trp Ser Pro Ala Pro Leu His Gly Leu Arg Ser Gly

100 105 110

Leu His Arg Gln Leu Glu Ala Leu Asp Ala Cys Trp Leu Gln Ala Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Leu Gly Ser Ser

195

<210> 7

<211> 196

<212> PRT

<213> artifical sequence

<400> 7

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Ala Leu Leu Thr Cys

1 5 10 15

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

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Pro Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Ala Ala Val

65 70 75 80

Leu Gln Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

Ser Ser Ala Ala Trp Ser Pro Ala Leu Leu His Gly Leu Arg Ser Gly

100 105 110

Leu His Arg Gln Leu Glu Ala Leu Asp Ala Cys Trp Leu Gln Ala Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Leu Ala Ser Ser

195

<210> 8

<211> 591

<212> DNA

<213> artifical sequence

<400> 8

atggccctcc tgctccctct gctgaccgcc ctggcgctgc tcacctgccg ccctggaggc 60

tctctgggct gtgccctgcc tgggagccac gcgcaggtta gcagggacaa cttggtgctc 120

ctgggccaga tgcggagact gtcccctttc ttgtgcctgc gggccagaaa agacttccgc 180

ttcccccggg agatgctgga gggcggccag ctccgggagg cccaggccgc cgccgccgtc 240

ctgcgggagc tgctccagca gaccttcaac ctgttgcaca cggagcgctc ctcggcggcc 300

tggagccccg cgccgctgca cggactccgc tctggcctcc accggcagct ggaagccctg 360

gacgcctgct ggctgcaggc cacgggcgag ggagagcgcg ccccggggat gcacggccct 420

gtcctggcca tcaagaggta cttccaggac atccgcgtct acctggagga cgagggatac 480

agtgactgcg cctgggaaat tgtcaggctg gaaatcatga gagccttgtc ctcctcggcg 540

accttacaag acagcttggc catcaaggat ggagacctgg gctcatcttg a 591

<210> 9

<211> 588

<212> DNA

<213> artifical sequence

<400> 9

atggctttat tgcttcctct cctaactgcc ctggcattat tgacctgtcg tcccggtggc 60

tctcttggat gcgcgctccc agggtcccat gctcaagttt cacgcgataa tctagtcctg 120

ttaggtcaga tgcgacggtt gtcgccgttt ctttgtctca gagccaggaa agacttccgt 180

tttcctcgcg aaatgctaga gggcggacaa ctgcgagaag cacaggcggc tgccgcagta 240

ttacaagagt tgcttcagca aacattcaac ctcctacaca cggaacggag tagcgcggct 300

tggtctcccg ccctgttaca tgggttgaga tccggtcttc acaggcagct cgaggcacta 360

gatgcgtgct ggctgcaagc tactggcgaa ggagagcgtg ccccagggat gcatggtccg 420

gcattagcga ttaagcgcta ttttcaggac atccgagtgt acttggaaga tgagggctat 480

tcagactgtg cttgggaaat agttcggctt gagattatga gagccctctc gagtagcgca 540

accctacaag attctctggc gatcaaagac ggagatttag cttcctca 588

<210> 10

<211> 203

<212> PRT

<213> artifical sequence

<400> 10

Met Ala Leu Leu Leu Pro Leu Leu Thr Ala Leu Val Leu Leu Thr Cys

1 5 10 15

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

20 25 30

Val Ser Arg Asp Asn Leu Val Leu Leu Gly Gln Met Arg Arg Leu Ser

35 40 45

Pro Phe Leu Cys Leu Arg Ala Arg Lys Asp Phe Arg Phe Leu Arg Glu

50 55 60

Met Leu Glu Gly Gly Gln Leu Arg Glu Ala Gln Ala Ala Thr Ala Val

65 70 75 80

Leu Gln Glu Leu Leu Gln Gln Thr Phe Asn Leu Leu His Thr Glu Arg

85 90 95

Ser Ser Ala Ala Trp Ser Leu Ala Leu Leu His Gly Leu Arg Ser Gly

100 105 110

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

115 120 125

Gly Glu Gly Glu Arg Ala Thr Gly Glu Gly Glu Arg Ala Leu Gly Met

130 135 140

His Gly Pro Ala Leu Ala Ile Lys Arg Tyr Phe Gln Asp Ile Arg Val

145 150 155 160

Tyr Leu Glu Asp Glu Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg

165 170 175

Leu Glu Ile Met Arg Ala Leu Ser Ser Ser Ala Thr Leu Gln Asp Ser

180 185 190

Leu Ala Ile Lys Asp Gly Asp Leu Ala Ser Ser

195 200

<210> 11

<211> 609

<212> DNA

<213> artifical sequence

<400> 11

atggctttat tgcttcctct cctaactgcc ctggttttat tgacctgtcg tcccggtggc 60

tctcttggat gcgcactcct agggtcccat gcgcaagtct cacgcgataa tctggtatta 120

ttgggtcaga tgcgacggct ttcgccattt ctctgtctaa gagctaggaa agacttccgt 180

tttctgcgcg aaatgttaga gggcggacaa ttgcgagaag cccaggcagc gacagctgtg 240

cttcaagagc tcctacagca aacgttcaac ctgttacaca ctgaacggag tagcgccgca 300

tggtctttgg cgcttctcca tgggctaaga tccggtctgc acaggcagtt agaggctttg 360

gatgcctgcc ttctccaagc aaccggcgaa ggagagcgtg cgacagggga aggtgagcgc 420

gctctaggca tgcatggacc ggccctggca attaagcgat attttcagga catccgggtt 480

tacttagaag atgaggggta ttcagactgt gcgtgggaaa tagtcagatt ggagattatg 540

agggctcttt cgagtagcgc cacgctccaa gattctctag caatcaaaga cggtgatctg 600

gcgtcctca 609

<210> 12

<211> 609

<212> DNA

<213> artifical sequence

<400> 12

atggccctgt tgctcccttt acttacagct ttggtgctgt tgacttgccg tccaggtggt 60

tcactgggtt gtgctctctt aggatctcac gcccaagtga gcagggacaa cttagttctt 120

ctgggtcaga tgagacgcct tagcccgttc ctttgcctgc gtgctaggaa agatttcaga 180

tttttacgcg aaatgcttga gggcggtcaa ttaagggaag ctcaggctgc aaccgccgtc 240

ctgcaagagt tgctccaaca gacctttaat ttacttcaca cggaaagatc atcagctgct 300

tggtccctcg ccctgttgca cggactgcgt tcgggcttgc ataggcaatt ggaagcactc 360

gacgcgtgcc tcttacaggc aacaggagaa ggcgagagag cgactggtga aggagagcgc 420

gctttgggca tgcatggtcc tgctctcgcc ataaaacgtt acttccaaga catcagggtc 480

tacctggaag acgagggcta tagtgattgt gcttgggaaa tcgtaagact tgagattatg 540

cgcgcactga gctcctcggc gactctccag gattcattag ccatcaagga cggtgatttg 600

gcaagttca 609

<210> 13

<211> 609

<212> DNA

<213> artifical sequence

<400> 13

atggcgttac ttttacccct gctgactgcg ttggcattac tcacttgtag acctggtggc 60

tctctgggat gtgctttacc cggttcacac gctcaagtgt cgcgcgacaa cttagttctg 120

ttgggacaga tgagacgcct tagtcctttc ttatgccttc gtgccaggaa agatttcaga 180

tttccgcgcg aaatgcttga gggtggacaa ctgcgtgaag cacaggctgc cgcagcggtg 240

ctcagggagc tcttacaaca gacatttaat cttctgcaca ctgaaagatc atctgctgcc 300

tggagccctg ctccactgca cggattgcgt tccggtctgc ataggcaact cgaagcatta 360

gacgcgtgct tgctccaggc taccggcgaa ggagagagag caacaggaga aggagagagg 420

gcacctggta tgcatggacc agtcctggcc ataaagcgtt acttccaaga catcagggta 480

tacttggaag acgagggcta ttcagattgt gcgtgggaaa tcgtcagatt ggagattatg 540

cgcgctctca gctcttcagc cacacttcag gatagtttag ccatcaagga cggagattta 600

ggttcatcg 609

Claims (7)

1. The cat omega 2 interferon mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 10.

2. The gene encoding the feline omega 2 interferon mutant of claim 1 having a nucleotide sequence set forth in SEQ ID No.11 or SEQ ID No. 12.

3. An expression vector comprising the gene of claim 2.

4. A method of making a cat omega 2 interferon mutant according to claim 1, comprising the steps of:

(1) cloning the gene of claim 2 into a baculovirus transfer vector to construct a recombinant transfer vector;

(2) co-transfecting the recombinant transfer vector and baculovirus DNA into an insect cell to obtain recombinant baculovirus;

(3) infecting the recombinant baculovirus into insect cells or insect hosts, culturing the infected insect cells or insect hosts to express corresponding protein, and purifying to obtain the recombinant baculovirus.

5. The method of claim 4, wherein: the baculovirus transfer vector is selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ, BacPAK6, Bac to Pac, Bacmid, BlucBacII (pETL), p2Bac, p2Blue, p89B310, pAc360, pAc373, pAcAB3, pAcAB 4, pAcAS3, pAcC129, pAcC4, DZI, pAcGP67, pAcIEl, pAcJPl, cpALF 2, pAcMLF7, pAcMULF 8, pAPlcM, cpAP 2, pAcRP23, pAcRP25, pAcRW4, pAMAG, pAcUWl, pAcUW21, pAcUW2A, pAcUW2B, pAcUW3, pAcUW31, pAcUpYNcVNW 874, pAcMAG, pAcUpYNV 42, pApYNpYNV 3611, pApYNpYNpYVC 369872, pApYNpYVC 42, pApYVC 3611, pAcVEcVpVEpVIV, pApVEpVEpVEpVIV, pApVEpVIV, pApVEpVEpVEpVIV or pApPSpSIVC 36pVEpVIV 3611, pAcVpVEpVIV 36pVEpVIV 36pVIV 8, pAcJV 36pVEpVEpVEpVEpVEpVEpVIV 8, pAcJV # 4, pAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJpAcJV # 4, pAcJpAcJpAcJpAcJ;

the baculovirus is selected from bombyx mori baculovirus parent strain BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV; the insect host is selected from the group consisting of Bombyx mori (Bombyx mori), Bombyx mori (Bombyx mandarina), Ricinus communis (Philosamia cynthia ricim), Bombyx mori (Dictyoloca japonica), Ailanthus altissima (Philosamia cynthia pryeri), Antheraea pernyi (Antheraea pernyi), Antheraea japonica (Antheraea yamamai), Bombyx mori (Antheraea polyphylla), Autographa californica (Atogaria californica), Ectropicalis gigas (Ectropis obliqua), Trichoplusia (Mamestra brassicae), Spodoptera littoralis (Spodoptera littoralis), Spodoptera frugiperda (Spodoptera frugiperda), Trichoplusia ni (Spodoptera armyworm, Heliothis armyworm (Heliothis virens), Heliothis virtus, Helicosa (tobacco), Heliothis virens (tobacco), and Helicoverpa armigera (tobacco);

the infection refers to the infection of 1-5-year-old insect larvae or pupae bodies by the recombinant baculovirus through ingestion or penetration of the epidermis.

6. The method of claim 5, wherein the baculovirus transfer vector is pVL 1393; the baculovirus is a parent strain BmBacmid of silkworm baculovirus; the insect host is silkworm (Bombyx mori).

7. Use of a mutant cat omega 2 interferon of claim 1 in the manufacture of a medicament or agent for the prophylaxis or treatment of a viral disease in a cat; the feline viral disease is feline distemper, feline infectious rhinotracheitis, feline calicivirus infection, feline leukemia virus infection, feline immunodeficiency virus infection, or vesicular stomatitis virus infection.

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