Porcine interferon-like recombinant protein and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to porcine interferon-like recombinant protein and application thereof. The porcine interferon-like recombinant protein Porferon with high activity is obtained by constructing a porcine interferon alpha (PoIFN-alpha) DNA (deoxyribonucleic acid) Shuffling bacterial library and screening the bacterial library, the recombinant protein has a good anti-infection effect on diseases caused by porcine viruses, bacteria, parasites and the like, and the activity of the recombinant protein is remarkably higher than that of porcine natural interferon. Therefore, the porcine interferon-like recombinant protein Porferon can be used for preparing medicines for preventing or treating diseases related to porcine virus, bacterium or parasite infection, and a novel prevention or / and treatment scheme is provided for clinic.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a porcine interferon-like recombinant protein and an application thereof. Background Art

[0002] Interferon (IFN) was first discovered by Isaacs and Lindenmann in 1957. It is a secretory glycoprotein produced by animal cells of the same genus with antiviral, immunomodulatory, and anti-tumor effects. It is currently considered to be an important component of the innate immune response [2-3]. Interferon is a multigene family that is divided into three subtypes based on gene sequence homology, protein structure, evolutionary relationship, cell surface receptors, and functional characteristics. Type I interferons mainly include IFN-α, IFN-β, IFN-ε, IFN-K and IFN-ω (common to all species). In addition, IFN-δ (pigs and horses), IFN-ζ (mice), IFN-τ (cattle), IFN-αω (pigs, horses, cattle) and other interferon subtypes have been found in unique species [3]. Type II interferons mainly include one subtype, IFN-γ. Type III interferons include IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4. Among them, porcine type I interferons include at least 39 functional genes, distributed on chromosomes 1 and 10 of pigs. These functional genes include 17 IFN-α subtypes, 11 IFN-δ subtypes, 7 IFN-ω subtypes, and single IFN-β, IFN-ε, IFN-К, and IFN-αω subtypes [3]. After porcine type I interferons were first discovered for their antiviral activity [1], they were gradually proven to have anti-tumor and immunomodulatory activities. Among them, porcine IFN-α and IFN-β subtypes have the strongest antiviral activity [4]. Currently, type I interferon has been widely used in the treatment of viral diseases in humans and pigs, such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papilloma virus (HPV), porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), etc. However, due to problems such as low systemic toxicity and short half-life, the clinical application of interferon preparations is limited. Therefore, the development of highly effective and low-toxic interferon antiviral preparations is of great significance for the prevention and treatment of current clinical epidemics.

[0003] After viral infection, the body can be induced to produce interferon. Interferon binds to the interferon receptors of cells and exerts effective antiviral function by regulating the cell's metabolic process and inducing the expression of downstream interferon genes (IFN-stimulated genes, ISGs).

[0004] The receptors for type I interferon (IFN) in animals are composed of two subunits, IFNAR-1 and IFNAR-2, which are widely distributed on the surface of various cell types. After type I interferon binds to the receptor, the intracellular regions of IFNAR-1 and IFNAR-2 couple with tyrosine kinases JAK-1 and TYK2 and induce their rapid phosphorylation. The phosphorylated receptor regions recruit signal transducers and activators of transcription (STAT) proteins, such as STAT1 and STAT2. Subsequently, STAT proteins form homo- or heterodimers and bind to interferon response factor 9 (IRF-9) to form a complex called IFN-stimulated gene factor-3 (ISGF-3). The ISGF-3 complex migrates into the cell nucleus and binds to specific DNA elements (IFN-stimulated response elements, ISRE) on the promoters of IFN-stimulated genes (ISGs), activating the transcription of downstream genes. This signaling pathway is the classic JAK-STAT signaling mechanism.

[0005] In recent years, studies have found that type I interferons not only play a role through the classical JAK-STAT pathway, but also regulate a wider range of biological functions by activating other signaling pathways. For example, type I interferons can participate in cell survival and anti-apoptosis regulation through the PI3K / A)KT pathway; in neutrophils, IFN-β exerts anti-apoptotic effects by activating nuclear factor κ-B (NF-κB) and PKC-δ. These non-classical signaling pathways provide new research directions for the diverse functions of type I interferons.

[0006] Interferon can not only directly induce the antiviral state of cells, but also is an important immune factor, playing a key role in immune regulation and enhancing immune response. It can activate the innate immune system, enhance the killing ability of natural killer cells (NK cells), and accelerate the clearance of infected cells; enhance the function of dendritic cells (DCs), promote antigen presentation and IL-12 secretion, induce T cells to differentiate into Th1 type, and improve the clearance ability of viruses and intracellular pathogens. In terms of specific immunity, interferon activates T cells and B cells, enhances the production of antibodies (such as IgG and IgA) and cell-mediated immune responses. Interferon adjuvants can accelerate antigen recognition and processing, increase T cell and B cell activity, and optimize the direction and intensity of immune response by activating the innate immune system. For example, interferon combined with classical swine fever virus (CSFV) vaccine can significantly increase antibody titer and prolong protection time; combined with pseudorabies virus (PRV) vaccine, interferon can significantly enhance CTL activity and virus clearance ability. Microecological preparations integrating interferon genes can regulate intestinal immune status and exert anti-inflammatory effects for a long time. Interferon mRNA also has great potential in adjuvant applications, which can make up for the shortcomings of mRNA vaccines and significantly enhance their immunogenicity. For example, the combination of α interferon mRNA with COVID-19 or influenza mRNA vaccines can increase the level of neutralizing antibodies and enhance the protective efficacy against mutant strains. At the same time, the combination of interferon mRNA with traditional vaccines, such as CSFV inactivated vaccines, can significantly improve the immunogenicity and cellular immunity of vaccines.

[0007] At present, there is still no systematic experimental data on the antiviral activity of different subclasses and subtypes of porcine interferon, which leads to great blindness in the modification and mutation of porcine interferon proteins. At present, the research and development of interferon antiviral preparations is still not very ideal. Studies have shown that the porcine IFN-α17 interferon subtype has the strongest antiviral activity, and the ability of different porcine IFN-α subtypes to induce antiviral responses in the body varies greatly. Although current interferon products have certain antiviral effects, they cannot meet the requirements for the prevention and treatment of various viral diseases in clinical practice. Summary of the invention

[0008] In view of the problems and shortcomings of the prior art, the present invention aims to provide a porcine interferon-like recombinant protein and its application.

[0009] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a porcine interferon-like recombinant protein, which is the following protein A1) or A2) or A3) or A4):

[0011] A1), a protein whose amino acid sequence is shown in Sequence 1 in the sequence listing;

[0012] A2), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 1 in the sequence list;

[0013] A3), a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1 in the sequence list;

[0014] A4), a protein having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the sequence list and having the same function;

[0015] A5), a mutant protein having 1 to 7 amino acid residue mutations compared to the amino acid sequence of the 17 subtypes of porcine natural α interferon protein, wherein the mutation site of the mutant protein is at least one of P(V)4A, A42V, H58Y, E71K, G72D, A74S, and A148V;

[0016] A6) A modified protein obtained by subjecting the protein shown in Sequence 1 in the sequence listing to pharmaceutically acceptable protein modifications.

[0017] According to the above-mentioned porcine interferon-like recombinant protein, preferably, in order to facilitate the purification of the protein in A1), a tag can be connected to the amino terminus or carboxyl terminus of the protein shown in sequence 1 in the sequence table, and the tag can be selected from Poly-Arg (usually 5 residues, the sequence is shown in RRRRR), Poly-His (usually 6-8 residues, the sequence is shown in HHHHHH, HHHHHHHH), FLAG (usually 8 residues, the sequence is shown in DYKDDDDK), Strep-tagII (usually 8 residues, the sequence is shown in WSHPQFEK), c-myc (usually 10 residues, the sequence is shown in EQKLISEEDL), etc.

[0018] According to the above-mentioned porcine interferon-like recombinant protein, preferably, in A5), the mutation site P(V)4A means that the amino acid residue at position 4 in the amino acid sequence of the protein of the 17 subtypes of porcine natural α interferon is mutated from P or V to A; the mutation site A42V means that the amino acid residue at position 42 in the amino acid sequence of the protein of the 17 subtypes of porcine natural α interferon is mutated from A to V; the mutation site H58Y means that the amino acid residue at position 58 in the amino acid sequence of the protein of the 17 subtypes of porcine natural α interferon is mutated from H to Y; the mutation site E71K means that the amino acid residue at position 58 in the amino acid sequence of the protein of the 17 subtypes of porcine natural α interferon is mutated from H to Y; The amino acid residue at position 71 in the amino acid sequence of the protein of 17 subtypes of natural α-interferon mutates from E to K; the mutation site G72D means that the amino acid residue at position 72 in the amino acid sequence of the protein of 17 subtypes of porcine natural α-interferon mutates from G to D; the mutation site A74S means that the amino acid residue at position 74 in the amino acid sequence of the protein of 17 subtypes of porcine natural α-interferon mutates from A to S; the mutation site A148V means that the amino acid residue at position 148 in the amino acid sequence of the protein of 17 subtypes of porcine natural α-interferon mutates from A to V.

[0019] According to the above-mentioned porcine interferon-like recombinant protein, preferably, in A6), protein modification includes but is not limited to PEGylation, forming a fusion protein with a long half-life protein or polypeptide such as human serum albumin (HSA), immunoglobulin Fc fragment or other long-acting sequences, forming a long-acting fusion protein with a specific polypeptide tag such as XTEN polypeptide, forming a glycosylation modification through engineering design or subsequent chemical modification, etc.

[0020] The second aspect of the present invention provides a biological material related to the protein described in the first aspect, which is any of the following:

[0021] B1), a nucleic acid molecule encoding the protein according to claim 1;

[0022] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0025] B5), mRNA containing the nucleic acid molecule described in B1);

[0026] B6), an adjuvant containing the nucleic acid molecule described in B1);

[0027] B7) A vaccine containing the nucleic acid molecule described in B1).

[0028] According to the above biological material, preferably, the coding sequence of the nucleic acid molecule in B1) is as shown in Sequence 2 in the sequence listing.

[0029] The third aspect of the present invention provides any of the following uses of the protein provided in the first aspect or the biomaterial provided in the second aspect:

[0030] C1) Application in the preparation of drugs for preventing or treating porcine viral infection-related diseases;

[0031] C2) Application in the preparation of products for inhibiting porcine viral infection;

[0032] C3) Application in the preparation of anti-swine virus products;

[0033] C4) Use in the preparation of drugs for preventing or treating diseases related to bacterial infection;

[0034] C5) Application in the preparation of products for inhibiting bacterial infection;

[0035] C6) Use in the preparation of drugs for preventing or treating parasitic diseases;

[0036] C7) Use in the preparation of immune adjuvants, wherein the vaccine is used to prevent diseases related to pig viral, bacterial and / or parasitic infections;

[0037] C8) Application in feed additives or feed;

[0038] C9) Application in microecological preparations;

[0039] C10) Application in mRNA products;

[0040] C11), application in the preparation of products for regulating and / or improving pig immunity.

[0041] According to the above application, preferably, the porcine virus includes but is not limited to vesicular stomatitis virus, pseudorabies virus, classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), swine influenza virus (SIV), porcine circovirus disease (PCV), porcine adenovirus (Porcine Adenovirus), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV), porcine acute diarrhea syndrome coronavirus (Porcine Acute Diarrhea Syndrome Coronavirus, SADS-CoV), swine pox virus (Swine Pox Virus), Porcine Parvovirus (PPV), Japanese Encephalitis Virus (JVE), Porcine Encephalomyocarditis Virus, Porcine Enterovirus, Porcine Alphavirus Encephalitis Virus, Porcine Orthoreovirus, Seneca Valley Virus Infection (SVA), (Swine Dysentery caused by BredaVirus), Porcine Rubulavirus In, and Porcine Cytomegalovirus (PCMV).

[0042] According to the above application, preferably, the porcine bacteria include but are not limited to at least one of Streptococcus suis, Escherichia coli and Salmonella suis.

[0043] According to the above application, preferably, the pig parasites include but are not limited to at least one of Toxoplasma gondii, Scabies mites, and Trichinella spiralis.

[0044] According to the above application, preferably, the product includes but is not limited to at least one of a drug, a vaccine additive, a feed additive, a probiotic preparation, a reagent, and a kit.

[0045] According to the above application, preferably, in C7), the porcine interferon-like recombinant protein can be used as an immune adjuvant of the vaccine to enhance the vaccine-induced immune response, increase the antibody titer and the level of cellular immune response, and shorten the immune onset time.

[0046] According to the above application, preferably, in C11), the product that regulates and / or improves pig immunity is an immunomodulator, and the immunomodulator contains the protein provided by the first aspect or the biological material provided by the second aspect; the immunomodulator can fight against viral, bacterial and parasitic infections by directly activating immune cells and regulating immune responses, while enhancing the effect of vaccines.

[0047] According to the above application, preferably, the nucleic acid molecule or expression cassette described in the second aspect can be integrated into the probiotic genome by genetic engineering methods, so that the probiotic can express biologically active interferon; wherein, the probiotic strain includes but is not limited to the following strains: lactic acid bacteria (LacticAcidBacteria), Bifidobacterium (Bifidobacterium spp.), Bacillus subtilis (Bacillus subtilis), Bacillus (Bacillus spp.), Enterococcus (Enterococcus spp.), Propionibacterium (Propionibacteriumspp.), yeast (Yeast), actinomycetes (Streptomyces spp.) and the like. After providing the probiotic to pigs through oral administration, the intestinal immune barrier function can be significantly enhanced and pathogen infection can be inhibited. More preferably, the nucleic acid molecule described in the second aspect can be integrated into the host bacterial genome by the CRISPR / Cas9 system and stably expressed.

[0048] According to the above applications, preferably, the mRNA products or DNA vaccines in B5) and C10) can be used alone or in combination with mRNA vaccines and traditional vaccines for the prevention and / or treatment of swine infectious diseases. The fourth aspect of the present invention provides a drug, which comprises the porcine interferon-like recombinant protein provided in the first aspect.

[0049] According to the above-mentioned medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier; the medicine is used to prevent or treat diseases related to pig viral, bacterial and / or parasitic infections.

[0050] According to the above-mentioned drugs, preferably, the carrier / excipients include (but are not limited to): diluents; excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc.; fillers such as starch, sucrose, etc.; binders such as monosyrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; humectants such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and soap clay, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0051] According to the above-mentioned medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier package.

[0052] According to the above-mentioned drugs, preferably, the carrier encapsulation refers to encapsulating the drugs into lipid nanoparticles, polymer nanoparticles or other intelligent delivery systems, which can achieve a sustained release effect, prolong its residence time in the body, and improve tissue distribution and targeting performance.

[0053] According to the above-mentioned medicine, preferably, the medicine can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drops, eye drops, oral administration, drinking water, through penetration, absorption, physical or chemical mediation methods; or introduced into the body after being mixed or wrapped with other substances.

[0054] The fifth aspect of the present invention provides a feed additive, which comprises the coding gene of the porcine interferon-like recombinant protein provided in the first aspect and / or the porcine interferon-like recombinant protein provided in the second aspect.

[0055] The sixth aspect of the present invention provides an immune adjuvant, which comprises the coding gene of the porcine interferon-like recombinant protein provided in the first aspect and / or the porcine interferon-like recombinant protein provided in the second aspect.

[0056] The seventh aspect of the present invention provides an mRNA preparation, which comprises the coding gene of the porcine interferon-like recombinant protein provided in the first aspect and / or the porcine interferon-like recombinant protein provided in the second aspect.

[0057] The eighth aspect of the present invention provides a method for preparing the recombinant protein provided in the first aspect, the preparation method comprising: constructing a recombinant expression vector containing a nucleic acid molecule encoding the porcine interferon-like recombinant protein described in the first aspect, introducing the recombinant expression vector into a host cell, obtaining a recombinant cell expressing the recombinant protein, culturing the recombinant cell, and obtaining the recombinant protein by separation and purification.

[0058] In the present invention, the term "interferon-like" refers to having the functional and / or structural characteristics exhibited by interferon or interferon analogs or expected similar functional and / or structural characteristics. For example, "interferon-like biological activity" includes antiviral, antiproliferative and immunomodulatory activities.

[0059] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0060] The present invention constructs a porcine interferon α (PoIFN-α) DNA Shuffling bacterial library and screens the bacterial library to obtain a highly active porcine interferon-like recombinant protein Porferon. The porcine interferon-like recombinant protein Porferon has a good anti-infection effect on porcine viruses (such as porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine circovirus, etc.), bacteria (such as suis streptococci, porcine intestinal bacteria, etc.), parasites (such as toxoplasma, scabies, trichinella, etc.) and other related infectious diseases, and the activity of anti-virus, bacteria and parasite infection is significantly higher than that of natural porcine PoIFN-α17. Therefore, the porcine interferon-like recombinant protein Porferon of the present invention can be used to prepare drugs for preventing or treating porcine viral, bacterial or parasitic infection-related diseases, and provides a new prevention and treatment scheme for porcine anti-virus, bacterial and parasitic infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The amino acid sequence comparison results of Porferon protein and 17 kinds of porcine interferon-α;

[0062] Figure 2 The phylogenetic tree diagram of the amino acid sequence alignment of Porferon protein and 17 pig interferon-α;

[0063] Figure 3 The comparison result of the nucleotide sequence encoding Porferon protein and PoIFN-α17; because the three bases ATG at the 5' end of the nucleotide sequence encoding Porferon protein are artificially added start codons, Figure 3 The nucleotide sequence is numbered starting from the fourth base at the 5' end, and the number of bases counted is 474;

[0064] Figure 4 The amino acid sequence alignment result of Porferon protein and PoIFN-α17; Figure 4 When the amino acids of Porferon protein are numbered, the methionine (Met) translated by the start codon added in the Porferon protein for gene expression is regarded as the 0th amino acid, and the first amino acid after the methionine (Met) translated by the start codon, i.e., cystine (the first amino acid of the mature protein of porcine natural interferon is also cystine), is numbered as the first amino acid. Therefore, the number of amino acid residues in the statistical amino acid sequence is 158;

[0065] Figure 5 The figure shows the test result of Porferon protein inhibiting PRRSV replication in MARC-145 cells;

[0066] Figure 6 The figure shows the test results of natural PoIFN-α17 and Porferon protein inhibiting PRRSV replication in MARC-145 cells;

[0067] Figure 7 The statistical results of piglet survival and clinical symptoms in the clinical experiment; A is the statistical result of piglet body temperature; B is the statistical result of piglet survival rate; C is the clinical scoring result of piglets;

[0068] Figure 8 The results of the PRRSV genome cDNA copy number detection;

[0069] Fig. 9 These are photos of lung lesions in piglets in the virus control group and the Porferon treatment group; the left photo is a photo of lung lesions in piglets that died in the virus control group, and the right photo is a photo of lung lesions in piglets in the Porferon treatment group. DETAILED DESCRIPTION

[0070] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0071] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, components and / or combinations thereof.

[0072] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0073] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0074] Example 1: Obtaining the porcine interferon-like recombinant protein Porferon

[0075] 1. Construction of the initial bacterial library of porcine interferon α (PoIFN-α) DNA Shuffling

[0076] There are 17 interferon α subtypes in pigs, namely: PoIFN-α1 (Gene bank record number GQ415055), PoIFN-α2 (Gene bank record number GQ415056), PoIFN-α3 (Gene bank record number GQ415057), PoIFN-α4 (Gene bank record number GQ415058), PoIFN-α5 (Gene bank record number GQ415059), PoIFN-α6 (Gene bank record number GQ415060), PoIFN-α7 (Gene bank record number GQ415061), PoIFN-α8 (Gene bank record number GQ415062), PoIFN-α9 (Gene bank record number GQ415063), PoIFN-α10 (Gene bank record number GQ415064), PoIFN-α11 (Gene bank record number GQ415065), PoIFN-α12 (Gene bank record number GQ415066), PoIFN-α13 (Gene bank record number GQ415067), PoIFN-α14 (Gene bank record number GQ415068), PoIFN-α15 (Gene bank record number GQ415069), PoIFN-α16 (Gene bank record number GQ415070), PoIFN-α17 (Gene bank record number GQ415071), PoIFN-α18 (Gene bank record number GQ415072), PoIFN-α19 (Gene bank record number GQ415083), PoIFN-α10 (Gene bank record number GQ415084), PoIFN-α11 (Gene bank record number GQ415085), PoIFN-α12 (Gene bank record number GQ415086), PoIFN-α13 =PoIFN-α17 (Gene bank record number GQ415070); based on the nucleotide sequences of the above 17 interferon α subtype mature protein genes, the following 15 pairs of degenerate mutated oligodeoxynucleotide molecular fragments that can cover all DNA sequences of porcine natural interferon subtype α were designed and synthesized as primers. The specific nucleotide sequences are as follows (only the forward fragment nucleotide sequences of the 15 pairs of oligodeoxynucleotide molecular fragments are listed here, and the reverse complementary sequences are not listed):

[0077] Frag1: 5'-CGAAGCATTGGTTAAAAATTAAGGAGGAAGGATCCATGGCCCCA(A / T)CCTCA(G / A)C(C / T)(T / C)TC(C / T)TCA(C / T)(A / G)G(C / T)C CTGGTGCTAC-3';

[0078] <h2 style=";text-align:left;direction:ltr">Frag2:5'-C(C / T)TCA(C / T)(A / G)G(C / T)CCTGGTGCTAC TCAGCT(G / A)CAA(T / G)GCCATCT(G / A)CT(G / C)TCTGGGCT GC(G / A)ACC(T / C)G(C / G)(C / T)-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] <h2 style=";text-align:left;direction:ltr"> Frag3:5'-T(G / C)TCTGGGCTGC(G / A)ACC(T / C)G(C / G)(C / T)TCAGACC(C / T)ACAGCCTGGCTC ACACCAGGGC CCTGAGGC(T / C)C-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> Frag4:5'-ACACCAGGGCCCTGAGGC(T / C)CCTGGCACAAATGAGGAGAAT CTCCCCCTTCTCCTGCCTGG-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] <h2 style=";text-align:left;direction:ltr"> Frag5: 5'-CTCCCCCTTCTCCTGCCTGGACCACAGAA(G / A)GGACTTTGGA T(T / C)CCC(C / T)CA(A / T)G AGGC(C / T)TT(G / T)GG-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0082] <h2 style=";text-align:left;direction:ltr"> Frag6:5'-T(T / C)CCC(C / T)CA(A / T)GAGGC(C / T)TT(G / T)GG GGGCAACCAGGTCCAGAAGG CTCAAGCCAT GGCTCTGGTG-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0083] <h2 style=";text-align:left;direction:ltr"> Frag7:5'-CTCAAGCCAT GGCTCTGGTG CATGAGATGC TCCAGCAGAC CT(T / C)CCAGCTCTTCAGCACAG-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] <h2 style=";text-align:left;direction:ltr"> Frag8:5'-CT(T / C)CCAGCTCTTCAGCACAGAGGGCTC(G / A)GC TGCTGCCTGG(G / A)ATGA(G / C)AGCC TCCTGCACCA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> Frag9:5'-(G / A)ATGA(G / C)AGCC TCCTGCACCAGTTCTGCACT GGACTGGATCAGCAGCTCAG GGACCTGGAA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] Frag10: 5'-AGCAGCTCAG GGACCTGGAAGCCTGTGTCAT(G / A)CAGGAGG(C / T)(G / C)GGGCTGGAA GGGAC(C / G)CCCC-3';

[0087] Frag11: 5'-(G / C)GGGCTGGAA GGGAC(C / G)CCCC TGCTGGAGGAGG(A / G)CTCCATCCTGGCTGTGA GGAAATACTT-3';

[0088] Frag12: 5'-CTGGCTGTGAGGAAATACTT CCACAGACTCA(C / T)CCTCTATC TGCA(A / G)GAGAA GA(G / A)CTACAGC-3';

[0089] Frag13: 5'-TGCA(A / G)GAGAAGA(G / A)CTACAGC CCTCTGTGCCT GGGAGATC(G / A)TCAGGGCAGAA GTCATGA(G / C)A(G / T)-3';

[0090] Frag14: 5'-CAGGGCAGAAGTCATGA(G / C)A(G / T)CTTCTCTCTTC CTCCA(C / G)A(A / C)ACCTGCAAGAC A GACTCAGGA(A / G)-3';

[0091] Frag15: 5'-CTGCAAGACAGACTCAGGA(A / G)GAAGGAG CTCGAG CAC CAC CAT CAC CATCAC CAT CAC TAA GT GAATTCTGCA GATATCCATC-3'.

[0092] Among them, the Frag1 fragment contains a BamHI restriction enzyme site and the start codon ATG for protein translation, and the Frag5 fragment contains 8 His codons, a TAA termination codon and an EcoRI restriction enzyme site.

[0093] The 15 paired oligodeoxynucleotide fragments were mixed in equal moles and assembled by PCR according to the method described in the literature (Stemmer WP. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 1994; 91(22): 10747-10751). The obtained products were digested with restriction endonucleases (RE) BamHI and EcoRI and cloned into the Escherichia coli expression vector ppCMH (the expression vector ppCMH is a derivative expression vector of pBV220, and its multiple cloning region contains BamHI site and EcoRI site, for details, see the literature Zhang ZQ. et al. Construction and application of a high level expression vector containing PRPL promoter. Chinese J of Virol. 1990: 2: 18-23). The constructed expression plasmid library was verified by DNA sequence analysis (PE Applied Biosystems, US) for sequence accuracy and recombination efficiency, and then transformed into Escherichia coli DH5α competent cells to form the initial library of porcine interferon α (PoIFN-α) DNA Shuffling. In the process of assembling the gene by PCR with the above primers, conventional DNA polymerase (New England Biolab, MA, US) was used instead of high-fidelity DNA polymerase.

[0094] The initial library of porcine interferon α (PoIFN-α) DNA Shuffling was transfected into Escherichia coli DH5α according to conventional methods to obtain the initial bacterial library of porcine interferon α DNA Shuffling.

[0095] 2. Construction of secondary bacterial library of porcine interferon α (PoIFN-α) DNA Shuffling:

[0096] The bacterial DNA of the bacterial clones selected by the primary library screening or the secondary library (see step 3) was amplified with the first pair of PCR primers (CMHF3: 5'-ACCATGAAGGTGACGCTC-3' and CMHR3: 5'-AATCTTCTCTCATCCGC-3'), which were the flanking sequences upstream and downstream of the multiple cloning site of the pCMH plasmid vector, respectively. The PCR products obtained after amplification were digested with DNase I and assembled by primer-free PCR, and then amplified with the second pair of primers (CMHF1: 5'-GAAGGCTTTGGGGTGTGTGATA-3') and CMHR1: 5'-TGGCGGATGAGAGAAGAT-3'). The PCR products after further amplification were digested with BamHI and ECoRI, and cloned into the Escherichia coli expression vector pCMH to construct a plasmid library, which was transformed into Escherichia coli DH5α competent cells and analyzed by DNA sequence analysis (PE After the sequence accuracy and recombination efficiency were verified by Applied Biosystems, US, the resulting library was the DNA Shuffling secondary bacterial library of porcine interferon α.

[0097] 3. Screening of DNA Shuffling Bacterial Library

[0098] Newly transfected E. coli DH5α cells were grown on LB plates at 37°C overnight. Single colonies were picked and inoculated into 100 μl of LB medium containing 50 μg / ml ampicillin in a 96-well plate. The colonies were shaken at 250 rpm at 30°C. After overnight culture, 10 μl of the bacterial culture was inoculated in duplicate into 100 μl of LB medium containing 50 μg / ml ampicillin in a 96-well plate. The initial plate (the so-called stock plate) was temporarily stored at 4°C. The cells in the duplicate plates were cultured at 30°C until OD600 became 0.4 and then induced at 42°C. After 4 hours of heat induction, the bacterial culture was directly transferred to a -80°C freezer to start the freeze-thaw cycle. After 2 cycles of freeze-thaw, the bacterial suspension / lysate was diluted to the desired concentration and exposed to the PK15-VSV system for antiviral activity test to screen positive clones. The specific antiviral activity test method is referred to Cheng et al. (Cheng G, Chen W, Li Z, Yan W, Zhao X, Xie J, Liu M, Zhang H, Zhong Y, Zheng Z. Characterization of the porcine alpha interferon multigene family. Gene 382: 28–38, 2006.) The method described in the literature was carried out. That is, on the first day, PK-15 cells (purchased from China Food and Drug Inspection Institute) were seeded in a 96-well plate at a density of 14,000 cells / well and incubated at 37°C; on the second day, 2-fold serial dilutions of recombinant bacterial lysate, PoIFN-α17 or blank culture medium were added to each well and incubated at 37°C for another 24 hours; on the third day, the culture medium was removed and replaced with a culture medium containing 1,000 PFU of vesicular stomatitis virus (VSV, ATCC, catalog number VR-1421); the cells were incubated at 37°C for 24 hours again, and the cytopathic effect (CPE) was observed under a microscope. The recombinant bacterial clone corresponding to the well that could inhibit cytopathic effect by more than 50% was the positive clone.

[0099] First, select 100 recombinant bacterial clones and make serial dilutions. The dilution at which only 1%-2% of the bacterial clones can inhibit more than 50% of the cells from developing pathological changes is set as the working dilution for this round. The bacterial frozen solution is directly diluted to the working dilution to measure its antiviral effect. If the working dilution is found to be too high or too low later, it can be adjusted separately.

[0100] More than 50,000 colonies were initially screened, and approximately 100 colonies with the highest antiproliferative or antiviral activity were selected for further preliminary validation. The specific operation steps of the preliminary verification are as follows: the selected bacterial culture on the stock plate is streaked on an LB plate containing 50 μ(G / ml ampicillin. A single colony is cultured overnight at 37°C, picked, and inoculated into 1 ml of LB medium containing 50 μ(G / ml ampicillin in a test tube. The bacteria in the test tube are cultured overnight at 30°C with shaking at 250 rpm. Then, 40 μl of the cultured bacteria are inoculated into one of another set of test tubes, which contains 1 ml of LB with ampicillin (50 μ(G / ml), cultured at 30°C until OD600 becomes 0.4, and then induced at 42°C. After 4 hours of heat induction, the bacterial culture is directly transferred to a -80°C freezer to start a freeze-thaw cycle. After 2 cycles of freeze-thaw, the bacterial suspension / lysate is diluted to the desired concentration and tested for antiviral activity in the PK15-VSV system to screen out positive clones.

[0101] After preliminary verification, about 30 colonies with the highest antiviral activity were selected, and the plasmids and their inserts were automatically sequenced. The clones of the insert pCMH with a unique DNA sequence were selected and amplified to obtain PCR amplification products, which were used to construct the next round (secondary) DNA shuffling library.

[0102] pCMH repeated the above steps of "Construction of DNA Shuffling Secondary Library and Screening of DNA Shuffling Bacterial Library". With the increase of screening rounds, the increase of antiviral activity of the screened mutants gradually decreased, and the homology of the screened mutants gradually increased. Among them, the mutants screened in the fourth round were initially verified to have similar antiviral activities, and further DNA sequencing also found that the homology of these mutants was also very high.

[0103] Finally, from the fourth round of screening, 8 colonies with high antiviral activity were selected, and the recombinant proteins expressed were tested for antiviral activity using the PK15-VSV system, and the recombinant proteins with the highest antiviral activity were selected. The specific operation steps are as follows: Each expression plasmid pBVBNF was transformed into DH5α cells, and a single colony was picked and inoculated into 4 ml of LB medium containing 50 μg / ml ampicillin, and cultured at 37°C for 16-18h. The bacteria cultured overnight were inoculated into a large volume of LB medium containing 50 μg / ml ampicillin at a ratio of 1:100, and cultured at 30°C with shaking at 220rpm. When the culture reached the mid-logarithmic growth phase (OD550=0.5-0.6), the culture temperature was quickly raised to 42°C, and the culture was shaken at 220rpm for 4 hours to induce the expression of Porferon. After 4 hours of heat induction, the bacteria were centrifuged at 5000 rpm for 10 min and washed 3 times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, PH = 7.4). The bacteria were suspended with 20 mM tris-HCl PH = 8.0 and then ultrasonically disrupted. The power was set to 300 W, ultrasonic for 5 s and 5 s intervals, and ultrasonic for 25 min until the bacterial solution was clear. After centrifugation, the precipitate was discarded and the supernatant was purified on an AKATA purifier using a cytiva HiTrapCaqto Q ion exchange column (Cat. No.: 29400462) and a Cytiva (GE Life) HiLoad 16 / 600Superdex 75pg gel filtration column (Cat. No.: 28989333). First, the supernatant was loaded onto an ion exchange column, and then (20mM tris-HCL, 100mM-1M NaCL, PH = 8.0) was used for step gradient elution. After collecting the target protein, it was further purified using a gel filtration system. After the target protein purified by the ion exchange column was loaded onto a HiLoad 16 / 600Superdex 75pg gel filtration column, it was eluted using PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, PH = 7.4), and the target protein was collected according to the real-time monitored UV value (the collected target protein was used for antiviral activity using the PK15-VSV system).

[0104] Through the antiviral activity detection of PK15-VSV system, one mutant with the highest antiviral activity was selected from the eight mutant proteins and named Porferon. The amino acid sequence of Porferon protein is shown in Sequence 1, and the corresponding encoding nucleotide sequence of Porferon protein is shown in Sequence 2.

[0105] Sequence 1 is specifically:

[0106] MCNLAQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGFPQEVLGGNQVQKAQAMALVYEMLQQTFQLFSTKDSSAAWDESLLHQFCTGLDQQLRDLEACVMQEVGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRVEVMRAFSSSRNL (as shown in Sequence 1 in the Sequence Listing).

[0107] Sequence 2 is specifically:

[0108] ATGTGCAACCTGGCTCAGACCCACAGCCTGGCTCACACCCGTGCGCTGCGTCTGCTGGCGCAGATGCGTCGTATCAGCCCGTTCAGCTGCCTGGATCACCGTCGTGATTTCGGCTTCCCGCA GGAAGTGCTGGGCGGTAACCAGGTTCAGAAAGCGCAGGCGATGGCGCTGGTTTACGAAATGCTGCAGCAGACCTTCCAGCTCTTCAGCACAAAGGACTCATCTGCTGCCTGGGATGAGAGCCT CCTGCACCAGTTCTGCACTGGACTGGATCAGCAGCTGCGTGATCTGGAAGCGTGCGTTATGCAGGAAGTTGGCCTGGAAGGTACCCCGCTGCTGGAAGAAGATAGCATCCTGGCGGTTCGTA AATACTTCCACCGTCTGACCCTGTACCTGCAGGAAAAATCTTACAGCCCGTGCGCGTGGGAAATCGTTCGTGTGGAAGTTATGCGTGCGTTCAGCAGCAGCCGTAACCTG (shown as sequence 2 in the sequence listing)

[0109] The amino acid sequences of Porferon protein and 17 naturally occurring porcine PoIFN were compared. Figure 1 As shown. Further, a phylogenetic tree diagram is drawn based on the comparison results, as shown Figure 2 shown.

[0110] Depend on Figure 1 and Figure 2It can be seen that Porferon has the highest homology with PoIFN-α17 at both the nucleotide and amino acid levels. The BLAST comparison results of the coding nucleotide sequence and amino acid sequence of Porferon protein with PoIFN-α17 are shown in Figure 3 and Figure 4 As shown. Figure 3 , Figure 4 It was found that the nucleotide sequence encoding the Porferon protein had about 77.4% (367 / 474) homology with PoIFN-α17, and the amino acid sequence of the Porferon protein had about 95% (150 / 158) homology with PoIFN-α17.

[0111] Example 2: Expression and purification of Porferon protein

[0112] The recombinant clone expressing Porferon protein screened in the fourth round of Example 1 was streaked again on the LB agar plate, and a single colony was picked and inoculated into 4 ml of LB medium containing 50 μg / ml ampicillin, and cultured at 37°C for 16-18 hours. The bacteria cultured overnight were inoculated into a large volume of LB medium containing 50 μg / ml ampicillin at a ratio of 1:100, and cultured at 30°C with shaking at 220 rpm. When the culture reached the mid-logarithmic growth phase (OD550=0.5-0.6), the culture temperature was quickly raised to 42°C and cultured with shaking at 220 rpm for 4 hours to induce the expression of Porferon protein.

[0113] After 4 hours of heat induction, the bacteria were centrifuged at 5000 rpm for 10 min and washed 3 times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, PH = 7.4). The bacteria were suspended with 20 mM tris-HCl PH = 8.0 and then ultrasonically disrupted. The power was set to 300 W, ultrasonic for 5 s and 5 s intervals, and ultrasonic for 25 min until the bacterial solution was clear. After centrifugation, the precipitate was discarded and the supernatant was purified on an AKATA purifier using a cytiva HiTrap Caqto Q ion exchange column (Cat. No.: 29400462) and a Cytiva (GE Life) HiLoad 16 / 600 Superdex 75pg gel filtration column (Cat. No.: 28989333). First, the supernatant was loaded onto a cytiva HiTrap Caqto Q ion exchange column, and then (20mM tris-HCL, 100mM-1M NaCL, PH=8.0) was used for stepwise gradient elution. After the target protein was collected, it was further purified using a gel filtration system. The target protein purified by the cytiva HiTrap Caqto Q ion exchange column was loaded onto a HiLoad 16 / 600 Superdex 75pg gel filtration column, and eluted using a PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, PH=7.4). According to the real-time monitored UV value, the target protein was collected to obtain the Porferon protein. The purity of the Porferon protein was verified by 15% SDS-PAGE analysis, and it was found that the Porferon protein was displayed as a single band with a molecular weight (MW) of 18-19KDa.

[0114] At the same time, in order to facilitate the comparison between Porferon protein and PoIFN-α17, the present invention expresses and purifies PoIFN-α17 in Escherichia coli. The specific operation of expressing and purifying PoIFN-α17 in Escherichia coli is as follows:

[0115] The expression plasmid ppCMH PoIFNa17 of PoIFN-α17 contains the DNA coding sequence of the mature protein of PoIFN-α17 (GeneBank nucleotide accession number GQ415071), and the start signal ATG is artificially added at the 5' end. The expression of PoIFN-α17 is carried out according to the operating procedures described by Joseph S and David WR (see the document "Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli", author: Erik Remaut, Patrick Stanssens, Walter Fiers, journal: Nucleic Acids Research, 1983, Vol. 11, No. 14). The expression plasmid ppCMH PoIFNa17 is transformed into DH5α cells. Single colonies were picked up, inoculated into 4 ml of LB medium containing 50 μg / ml ampicillin, and cultured at 30°C for 8 hours. Then, 2 ml of the cultured bacteria were taken and further cultured at 30°C for 16-18 hours with 50 ml of LB medium containing 50 μg / ml ampicillin. The next morning, the bacterial culture was inoculated into a large volume of LB medium containing 50 μg / ml ampicillin at a ratio of 1:100 and cultured at 30°C with stirring. When the culture reached the mid-logarithmic growth phase (A550=0.5-0.6), the culture temperature was quickly raised to 42°C and maintained for 4 hours to induce the expression of PoIFN-α17.

[0116] After 4 hours of heat induction, the bacteria were centrifuged at 5000 rpm for 10 min and washed 3 times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, PH = 7.4). The bacteria were suspended with 20 mM tris-HCl PH = 8.0 and then ultrasonically disrupted. The power was set to 300 W, ultrasonic for 5 s and 5 s intervals, and ultrasonic for 25 min until the bacterial solution was clear. After centrifugation, the precipitate was discarded and the supernatant was purified on an AKATA purifier using a cytiva HiTrap Caqto Q ion exchange column (Cat. No.: 29400462) and a Cytiva (GE Life) HiLoad 16 / 600Superdex 75pg gel filtration column (Batch No.: 28989333). First, the supernatant was loaded onto an ion exchange column, and then (20mM tris-HCL, 100mM-1M NaCL, PH = 8.0) was used for stepwise gradient elution. After the target protein was collected, it was further purified using a gel filtration system. After the target protein was loaded onto a HiLoad 16 / 600 Superdex 75pg gel filtration column, it was eluted using PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, PH = 7.4), and the target protein was collected according to the real-time monitored UV value. The purity of the purified PoIFN-α17 protein was verified by 15% SDS-PAGE analysis. The pure recombinant PoIFN-α17 protein was displayed as a single band with a molecular weight (MW) of 18-19KDa. Mass spectrometry analysis showed that the purity of the purified PoIFN-α17 molecule was >98% and the molecular weight was 18.16 KDa, which was consistent with the molecular weight of 18158.77 Daltons deduced from its amino acid sequence.

[0117] Example 3: Determination of the anti-VSV activity of Porferon protein

[0118] The antiviral activity was determined using the PK15-VSV system according to the method of Cheng et al. (Cheng G, Chen W, Li Z, Yan W, Zhao X, Xie J, Liu M, Zhang H, Zhong Y, Zheng Z. Characterization of the porcine alpha interferon multigene family. Gene 382:28–38, 2006). The specific operation is as follows: On the first day, PK15 cells (purchased from China Food and Drug Inspection Institute) were seeded in a 96-well plate at a density of 14,000 cells / well and incubated at 37°C; On the second day, 2-fold serial dilutions of Porferon or PoIFN-α17 with a stock concentration of 0.5 μg / mL were added to each well, and a medium without Porferon or PoIFN-α17 was used as a blank control, and incubated at 37°C for another 24 hours; On the third day, the medium was removed and replaced with a medium containing 1,000 PFU of vesicular stomatitis virus (VSV, ATCC, catalog number VR-1421). The cells were incubated at 37°C for 24 hours again, and the results were observed under a microscope. First, the "cell control well" and "virus control well" were observed for cytopathic effect (CPE). The potency determination takes the highest dilution that can inhibit 50% of the cytopathic effect as one potency unit, that is, the highest dilution per milliliter of the test sample can still protect half of the cells (50%) from viral attack, and the reciprocal of the dilution is the potency of the sample. Refer to the Reed-Muench formula in the appendix of the "Chinese Veterinary Pharmacopoeia" (Part III of the 2020 edition) for calculation. The logarithm of the highest dilution that inhibits 50% of the cytopathic effect = the logarithm of the dilution with a cytopathic effect inhibition rate greater than or equal to 50% + the logarithm of the distance ratio × the dilution factor. The experiment was repeated 3 times, and the Porferon and PoIFN-α17 samples were measured in the same 96-well plate, and the antiviral activity of purified Porferon and PoIFN-α17 was analyzed in parallel. Among them, the statistical results of the Porferon potency determination are shown in Table 1, and the statistical results of the PoIFN-α17 potency determination are shown in Table 2.

[0119] Table 1 Statistical results of Porferon titer determination

[0120]

[0121] Table 2 Statistical results of PoIFN-α17 titer determination

[0122]

[0123] As shown in Table 1, the logarithm of the dilution of Porferon that can inhibit 50% of the cytopathic effect (Log2X) is 10+0.33, that is, 0.5μ(G / mL Porferon is diluted to 2 10.33 As shown in Table 2, the logarithm of the dilution of PoIFN-α17 that can inhibit 50% of cytopathic effect (Log2X) is 3+0.33, that is, 0.5μ(G / mL sample A is diluted to 2 3.33 The above results show that 0.25n(G / ml purified Porferon protein can protect half of PK-15 from VSV attack, that is, the half protective dose (PD) of this system 50 ) was 0.39n(G / ml, while the PD 50 The data showed that the antiviral activity of Porferon protein was 130 times that of PoIFN-α17.

[0124] Example 4: Determination of the anti-PRRSV proliferation activity of Porferon protein

[0125] MARC-145 cells were seeded into 24-well plates at 5 × 10 cells per well. 5 After the cells grew into a monolayer, 200 μL of Porferon cell culture medium with a concentration of 0.1 ng / mL, 1 ng / mL, and 10 ng / mL was added to the MARC-145 cells, and a cell culture medium control without Porferon was set up. 24 hours later, 1000 TCID50 of porcine reproductive and respiratory syndrome virus (PRRSV) was inoculated, and the cells were collected 48 hours after infection. The indirect immunofluorescence method (IFA) was used to detect the antiviral activity of Porferon on cells against PRRSV. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that the results show that 0.01ng / mLPorferon can effectively inhibit the replication of PRRSV.

[0126] The inhibitory activity of PoIFN-α17 and Povirferon on PRRSV replication in MARC-145 cells was further compared and analyzed. MARC-145 cells were seeded into 24-well plates, with 5×10 5After the cells grew into a monolayer, the culture medium containing 0.1 ng / mL of PoIFN-α17 or Porferon was replaced, and the cell culture medium without PoIFN-α17 or Porferon was set as a control. After another 24 hours, 1000 TCID50 of porcine reproductive and respiratory syndrome virus (PRRSV) was inoculated. The cells were collected 48 hours after infection, and the antiviral activity was detected by indirect immunofluorescence method (IFA). The results are as follows Figure 6 shown.

[0127] Depend on Figure 6 It can be seen that at a concentration of 0.1 ng / mL, the cells in the Porferon incubation well had no specific fluorescence, while under the same conditions, 85% of the cells in the PoIFN-α17 incubation well produced specific fluorescence, indicating that Porferon has a much higher activity in inhibiting the replication of PRSSV than PoIFN-α17.

[0128] Example 5: Determination of the anti-PRV activity of Porferon protein

[0129] PK15 cells were plated in 96-well plates with 14,000 cells per well and cultured in a 37°C 5% CO2 incubator. When the cells grew into a monolayer, the concentrations of 1 μg / ml, 1×10 -1 μg / ml, 1×10 -2 μg / ml, 1×10 -3 μg / ml, 1×10 -4 μg / ml, 1×10 -5 μg / ml, 1×10 -6 μg / ml, 1×10 -7 μg / ml, 1×10 -8 μg / ml, 1×10 -9 μg / ml, 1×10 -10 μg / ml PoIFN-α17 or Porferon culture medium, note that each dilution is repeated 3 times, and blank control and virus control without PoIFN-α17 or Porferon are set up at the same time, and cultured at 37℃5%CO2 for 24h. Then remove the culture medium and inoculate ICID50=100 porcine pseudorabies virus (PRV). Culture for 48h at 37℃5%CO2, and observe the results under a microscope. First observe the "cell control well" and "virus control well". No cell pathological changes (CPE) are indicated by "-", and CPE is indicated by "+". When the virus control well shows "+++" or "++++", that is, the cells in the virus control well show 75% to 100% obvious pathological changes and death and shedding, and the cells in the cell control well all grow well, it indicates that the control system of this experiment is qualified, and the test results can be determined.

[0130] After incubating PK15 cells with porcine PoIFN-α17 and Porferon protein for 24 hours, ICID50=100 PRV was inoculated and cells (CPE) were observed 48 hours after infection. The statistical results are shown in Table 3.

[0131] Table 3 shows the statistical results of IFN-α17 and Porferon protein against PRV

[0132]

[0133] Note: ++++ indicates total cytopathic effect; +++ indicates 75% cytopathic effect; ++ indicates 50% cytopathic effect; + indicates 25% cytopathic effect; - indicates no cytopathic effect

[0134] As shown in Table 3, when the concentration of porcine natural α interferon 17 subtype in the cell culture medium is 0.1μg / ml, 50% of the cells will not produce CPE, while the concentration of Porferon to achieve the same protective effect is 1pg / ml~10pg / ml, indicating that Porferon is 10,000 times more active than PoIFN-α17 in inhibiting PRV proliferation on PK15 cells.

[0135] Example 6: Animal experiment on Porferon protein against PRRSV infection

[0136] Ten healthy piglets aged 4-5 weeks (negative for PRRSV, PCV, PEDV, and ASFV) were numbered and randomly divided into two groups, a virus control group and a Porferon treatment group. 50 1×10 5.5的 All piglets were infected with PRRSV (strain BB0907, kindly donated by the College of Veterinary Medicine of Nanjing Agricultural University) by intramuscular injection of 2 ml and nasal drops of 2 ml.

[0137] Piglets in the virus control group: 1) At the same time as the virus challenge, 2 ml of sterile PBS was injected intramuscularly; 2) 24 hours after the virus challenge, 2 ml of sterile PBS was injected intramuscularly; 3) 96 hours after the virus challenge, 2 ml of sterile PBS was injected intramuscularly.

[0138] Piglets in the Porferon treatment group: 1) At the same time as the challenge, 8ug Porferon (2ml) was injected intramuscularly; 2) 24 hours after the challenge, 8ug Porferon (2ml) was injected intramuscularly; 3) 96 hours after the challenge, 8ug Porferon (2ml) was injected intramuscularly.

[0139] 0-10 days after virus infection, rectal temperature was measured every day, and the survival rate and clinical symptoms of piglets were observed. Figure 7 shown.

[0140] According to statistics, after the virus was inoculated, the virus control group spontaneously developed obvious PRRSV clinical high fever (such as Figure 7 The pigs also showed clinical symptoms such as loss of appetite, lethargy, rough hair, difficulty breathing, periocular edema, and mild diarrhea. 0-10 days after inoculation, 3 of the 5 pigs died (as shown in Figure 1). Figure 7 The Porferon treatment group did not develop clinical fever or obvious clinical symptoms during the trial, and all of them survived in good health. The comprehensive clinical score showed that the Porferon treatment group had significantly reduced symptoms compared with the virus control group (as shown in Figure 2B). Figure 7 The above results fully prove that Porferon protein significantly alleviates the characteristic symptoms caused by PRRSV infection.

[0141] After virus infection, serum was collected on days 1, 4, 7, and 10, and the number of PRRSV genome cDNA copies was determined by qRT-PCR. The results are shown in Figure 8 shown.

[0142] Depend on Figure 8 It can be seen that the virus content in the serum of piglets in the Porferon treatment group was significantly lower than that in the virus control group at 4-10 days, indicating that Porferon protein can significantly reduce the replication of PRRSV in piglets.

[0143] Ten days after the challenge, the animals were euthanized and then autopsied. The macroscopic lesions of the lungs of each group of piglets were observed. Fig. 9 shown.

[0144] Depend on Fig. 9 It can be seen that the lung tissue of the virus control group had obvious lung tissue consolidation and hemorrhage, showing typical gross lesions of porcine reproductive and respiratory syndrome; while the lungs of piglets in the Porferon treatment group also had edema and other lesions, but they were significantly milder than those in the virus control group.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A porcine interferon-like recombinant protein, characterized in that: The protein is A1) or A2) or A3) or A4) or A5) or A6) as follows: A1), a protein whose amino acid sequence is shown in Sequence 1 in the sequence listing; A2), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 1 in the sequence list; A3), a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1 in the sequence list; A4), a protein having 85% or more homology with the amino acid sequence shown in Sequence 1 in the sequence list and having the same function; A5), a mutant protein having 1 to 7 amino acid residue mutations compared to the amino acid sequence of the 17 subtypes of porcine natural α interferon protein, wherein the mutation site of the mutant protein is at least one of P(V)4A, A42V, H58Y, E71K, G72D, A74S, and A148V; A6) A modified protein obtained by subjecting the protein shown in Sequence 1 in the sequence listing to pharmaceutically acceptable protein modification.

2. The biological material related to the protein according to claim 1, which is any one of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), mRNA containing the nucleic acid molecule described in B1); B6), an adjuvant containing the nucleic acid molecule described in B1); B7) A vaccine containing the nucleic acid molecule described in B1).

3. The biomaterial according to claim 2, characterized in that B1) The coding sequence of the nucleic acid molecule is shown in Sequence 2 in the sequence listing.

4. Any of the following uses of the protein according to claim 1 or the biomaterial according to claim 2 or 3: C1) Use in the preparation of drugs for preventing or treating porcine viral infection-related diseases; C2) Application in the preparation of products for inhibiting porcine viral infection; C3) Application in the preparation of products against swine viruses; C4) Use in the preparation of drugs for preventing or treating diseases related to bacterial infection; C5) Application in the preparation of products for inhibiting bacterial infection; C6) Use in the preparation of drugs for preventing or treating parasitic diseases; C7) in the preparation of vaccine immune adjuvants, wherein: The vaccine is used to prevent diseases related to swine viral, bacterial and / or parasitic infections; C8) Application in feed additives or feed; C9) Application in microecological preparations; C10) Application in mRNA products; C11), application in the preparation of products for regulating and / or improving pig immunity.

5. The use according to claim 4, characterized in that: The porcine viruses include but are not limited to at least one of vesicular stomatitis virus, pseudorabies virus, classical swine fever virus, foot-and-mouth disease virus, porcine reproductive and respiratory syndrome virus, African swine fever virus, swine influenza virus, porcine circovirus, porcine adenovirus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine deltacoronavirus, porcine acute diarrhea syndrome coronavirus, porcine poxvirus, porcine parvovirus, porcine Japanese encephalitis virus, porcine encephalomyocarditis virus, porcine enterovirus, porcine forest encephalitis virus, porcine reovirus, porcine Seneca virus infection, porcine measles virus, and porcine cytomegalovirus; the porcine bacteria include but are not limited to suis streptococcus, porcine Escherichia coli, and porcine Salmonella; the porcine parasites include but are not limited to Toxoplasma gondii, scabies, and Trichinella; the products include but are not limited to drugs, vaccine additives, feed additives, probiotic preparations, reagents, and kits.

6. A drug, characterized in that The drug comprises the porcine interferon-like recombinant protein according to claim 1.

7. The drug according to claim 6, characterized in that The medicine also contains a pharmaceutically acceptable carrier; the medicine also contains a pharmaceutically acceptable carrier; the medicine is used for preventing or treating diseases related to pig virus, bacteria and / or parasite infection.

8. The drug according to claim 6, characterized in that The carrier includes but is not limited to diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, wetting agents, lubricants, adsorption carriers, and carrier encapsulation; the drug can be introduced into the body by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or it can be introduced into the body after being mixed or encapsulated with other substances.

9. A feed additive, characterized in that: The feed additive comprises the porcine interferon-like recombinant protein according to claim 1.

10. The method for preparing the recombinant protein according to claim 1, characterized in that: The preparation method comprises: constructing a recombinant expression vector containing a nucleic acid molecule encoding the porcine interferon-like recombinant protein according to claim 1, introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the recombinant protein, culturing the recombinant cell, and obtaining the recombinant protein by separation and purification.

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