CN111848814B - Recombinant porcine IL-29 fusion protein and preparation method and application thereof - Google Patents

Recombinant porcine IL-29 fusion protein and preparation method and application thereof Download PDF

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CN111848814B
CN111848814B CN202010570157.3A CN202010570157A CN111848814B CN 111848814 B CN111848814 B CN 111848814B CN 202010570157 A CN202010570157 A CN 202010570157A CN 111848814 B CN111848814 B CN 111848814B
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glu
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CN111848814A (en
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罗昊澍
师磊
钟小荣
于金库
韩国
熊剑胜
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Beijing Weijiexin Biotechnology Co ltd
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

The invention discloses a recombinant porcine IL-29 fusion protein and a preparation method and application thereof, wherein the fusion protein is formed by connecting porcine IL-29 with an immunoglobulin Fc fragment or Porcine Serum Albumin (PSA) directly or indirectly through a connecting element. The porcine IL-29 fusion protein can be prepared by utilizing a mammalian cell expression system based on a genetic engineering technology. Compared with the natural porcine IL-29, the recombinant porcine IL-29 fusion protein provided by the invention has longer half-life and higher antiviral activity, and can be used for preparing medicines for preventing and treating porcine viral diseases.

Description

Recombinant porcine IL-29 fusion protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of prevention and control of biological medicines and animal viral diseases, in particular to a recombinant porcine IL-29 fusion protein and a preparation method and application thereof.
Background
Interleukin 29 (IL-29) is the first cytokine reported in 2003 to have antiviral activity and is highly similar in amino acid sequence to the IL-10 cytokine family; due to the antiviral activity of interferons, it also belongs to the type III interferon or IFN- λ family, also known as IFN- λ 1.
IL-29, like IFN- α/β (type I interferon), by binding to the corresponding receptor, activates the JAK-STAT signaling pathway and induces transcription of downstream interferon-stimulated genes (ISGs), thereby stimulating the expression of proteins associated with antiviral responses, including antiviral proteins such as MxA, OASL, and PKR. MxA is a member of the anti-adhesion viral protein family, and is capable of blocking viral nucleic acid entry intracellularly. OASL is a member of the 2'-5' oligoadenylate synthetase family and inhibits viral protein synthesis and viral infection. PKR is a double-stranded RNA-dependent protein kinase that causes apoptosis and thus blocks viral replication. In vitro studies have shown that human IL-29 can reduce the effects of viral replication and induced cytopathic effects, including Cytomegalovirus (CMV), hepatitis B Virus (HBV), encephalomyocarditis virus (EMCV), vesicular Stomatitis Virus (VSV). The pegylated IFN-lambda 1 (PEG-IL-29) developed by ZymoGenetics Inc. is a new interferon drug for treating hepatitis C, and phase 1 clinical results show that single injection of PEG-IL-29 (1 time in 2 weeks, 1.5 mu g/kg and 3 mu g/kg) or combined use with Ribavirin (1 time per week, 0.5, 0.75 and 2.25 mu g/kg of PEG-IL-29) to HCV (hepatitis C virus) patients shows obvious antiviral effects, and a human body has good safety and tolerance to the drug and no obvious clinical adverse reaction.
Although IL-29 has similar antiviral activity to IFN-. Alpha./β, since IFN-. Alpha./β receptors are widely distributed on various types of cells, IFN-. Alpha./β binding to receptors has immunomodulatory, immunosuppressive and anti-cell-proliferation effects in addition to antiviral effects, and thus, clinical use has very significant side effects including influenza-like syndrome, red and white blood cell depletion, bone marrow cytopenia, and psychiatric disorders (depression, delusions, anxiety, etc.) and the like. IL-28R1, one of the receptors for IL-29, is specifically expressed only in specific tissues, such as keratinocytes, bronchial epithelial cells and hepatocytes, and the specificity of the receptor determines the specificity with which the cells bind IL-29, so that the clinical side effects of IL-29 are significantly lower than those of IFN-. Alpha./β.
In modern large-scale intensive pig farms, outbreaks of viral infectious diseases, such as porcine reproductive and respiratory syndrome, porcine epidemic diarrhea, african swine fever, etc., cause significant economic losses in the pig farm. The porcine IFN-alpha is the existing veterinary interferon, but when used for treating viral infectious diseases, the porcine IFN-alpha is limited in use because of the side effects of fever, weight reduction and the like of the porcine caused by immunosuppression and the influence on energy conversion of the porcine, so the porcine IFN-alpha is commonly used for enhancing the immunity of vaccines. Studies show that the porcine IL-29 can effectively inhibit the replication of viruses such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), vesicular Stomatitis Virus (VSV), pseudorabies virus (PRV), classical Swine Fever Virus (CSFV) and Porcine Epidemic Diarrhea Virus (PEDV). And IL-29 is more effective than IFN-alpha in inducing ISGs expression and inhibiting PEDV infection in ileal organoids. In addition, the porcine IL-29 can be used for treating porcine viral infectious diseases, does not affect the diet, energy conversion and the like of the pigs, and has obviously lower side effect than the porcine IFN-alpha.
The natural interferon cytokine of the pig has short half-life period in vivo, generally 2 to 4 hours, and needs to be injected for 1 to 2 times per day for antiviral control of the pig. In addition, the commercial porcine IFN-alpha is produced by inducing porcine leukocyte by chicken Newcastle Disease Virus (NDV), and has insufficient yield and uneven quality. The production of interferon protein by using an escherichia coli system has been successful, but bacteria belong to a prokaryotic system, cannot correctly process and fold heterologous protein, and often appear in the form of inclusion bodies, so that the difficulty of operation is increased.
In view of the lack of long-acting and safe antiviral interferon products in the current pig breeding, the patent focuses on the use of gene recombination technology and mammalian protein expression technology to develop long-acting pig IL-29 fusion protein for the prevention and treatment of pig viral diseases.
Disclosure of Invention
The invention aims to provide a novel recombinant porcine IL-29 fusion protein, and a preparation method and application thereof.
The concept of the invention is as follows: the porcine IL-29 is fused with Fc or porcine albumin (PSA) by utilizing a genetic engineering technology to generate novel recombinant proteins pIL-29-Fc and pIL-29-PSA, so that the high biological activity of the pIL-29 is retained, and a longer half-life period and higher antiviral activity can be obtained; meanwhile, the recombinant protein with high purity and large-scale production can be obtained. The closest natural protein molecule in terms of molecular structure, physicochemical properties and biological function can be obtained by expressing recombinant proteins using mammalian expression systems, in particular chinese hamster ovary Cells (CHO).
In order to realize the purpose, the invention provides the recombinant porcine IL-29 fusion protein which is pIL-29Fc and pIL-29PSA, and the fusion protein is formed by connecting the porcine IL-29 with a fusion partner directly or indirectly through a connecting element.
The fusion partner comprises immunoglobulin, albumin and the like. Wherein the Fc fragment of an immunoglobulin comprises an immunoglobulin hinge region, CH2 and CH3 regions; preferably mammalian immunoglobulins, including human or canine, feline, porcine, murine, equine, bovine mammals, more preferably porcine IgG2a immunoglobulins. The serum albumin is preferably mammalian serum albumin, including human or mammals such as dog, cat, pig, mouse, horse, and cow, and more preferably Porcine Serum Albumin (PSA).
The fusion protein is formed by connecting the porcine IL-29 and a fusion partner directly or indirectly through a Linker. Wherein the Linker is a flexible polypeptide consisting of 2-20 flexible amino acids, and the flexible amino acids are selected from at least one of Gly, ser, ala and Thr; preferably, the Linker is (Gly-Gly-Gly-Gly-Ser) n, wherein n is an integer between 2 and 5, and more preferably n is 3.
The novel recombinant pig pIL-29 fusion protein pIL-29-Fc is shown as follows;
(a) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2; or
(b) And (b) a protein derived from (a) and having 90% or more homology with the amino acid sequence shown in SEQ ID NO. 2 and the same function.
The novel recombinant porcine pIL-29 fusion protein pIL-29-PSA is as follows;
(c) A protein consisting of an amino acid sequence shown as SEQ ID NO. 4; or
(d) And (c) a protein having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO. 4 and having the same function.
The invention also provides biological materials such as expression cassettes, expression vectors, cloning vectors, engineering bacteria or transgenic cell lines and the like, which comprise the nucleotide encoding the fusion protein.
The invention provides a preparation method of a recombinant porcine IL-29 fusion protein, which comprises the following steps: respectively and artificially synthesizing encoding genes of pIL-29-Fc and pIL-29-PSA fusion proteins, carrying out codon optimization, respectively constructing the optimized genes into eukaryotic expression vectors, transferring the eukaryotic expression vectors into eukaryotic cells, expressing the eukaryotic expression vectors in the eukaryotic cells, and separating and purifying target proteins.
The eukaryotic expression vector includes but is not limited to pcDNA3.1, and the eukaryotic cell includes but is not limited to 293 and CHO cells.
The invention also provides application of the two recombinant porcine IL-29 fusion proteins in preparation of medicaments or combined medicaments for preventing and treating livestock virus infection diseases. Wherein, the animals include but are not limited to pigs, cows, sheep, etc.; preferably a pig.
The invention provides any one of the following applications of the recombinant porcine IL-29 fusion protein:
1) The composition is used for preventing and treating the antiviral infection of the pigs, and comprises the steps of preventing the viral infection or eliminating the virus after the infection;
2) Can be used for preparing pig antiviral drugs or compositions.
The invention provides a pig antiviral drug or a composition, and the active ingredient is any one of the recombinant pig IL-29 fusion proteins.
Such viruses include, but are not limited to, porcine reproductive and respiratory syndrome virus, pseudorabies virus, vesicular stomatitis virus, porcine epidemic diarrhea virus, porcine circovirus, classical swine fever virus, transmissible gastroenteritis virus, and the like.
The modified protein, including fusion protein pIL-29Fc, pIL-29-PSA or porcine IL-29, is acetylated, pegylated, glycosylated or combined with BSA, etc., and belongs to the protection scope of the invention.
The modified protein, including fusion protein pIL-29Fc, fusion protein pIL-29-PSA, or fusion protein formed by fusing pig IL-29 with pig Fc, pig PSA or other proteins and not changing the protein activity, belongs to the protection scope of the invention.
Based on the technical scheme, the invention at least has the following advantages and beneficial effects:
the recombinant porcine IL-29 fusion protein provided by the invention is used for preventing and treating porcine viral infectious diseases, has obviously reduced side effects compared with the prior I-type interferon (IFN-alpha/beta) for livestock, and does not influence the diet and energy conversion of sick pigs; the half life period is obviously longer than that of natural interferon (containing porcine IL-29), and the administration frequency is reduced.
1) In vitro inhibition cytopathic test results show that pIL-29Fc and pIL-29-PSA can inhibit PEDV-induced Vero cytopathic effect, and the pIL-29Fc and pIL-29-PSA have anti-PEDV activity.
2) In vitro experiments show that pIL-29Fc and pIL-29-PSA can induce mRNA transcription of porcine primary pulmonary macrophage antiviral proteins OASL and MxA, and the two fusion proteins can be used for preventing and treating Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
3) The half-lives of pIL-29Fc and pIL-29-PSA in rats were 53h and 74h, respectively, which were significantly higher than the half-life of native pIL-29.
4) The fusion proteins pIL-29-Fc and pIL-29-PSA have higher antiviral activity relative to pIL-29.
Drawings
FIG. 1 is a non-reducing SDS-PAGE electrophoresis of pIL-29-Fc in example 2 of the present invention, wherein MK is a protein Marker; lane 1 is the pool.
FIG. 2 is an SDS-PAGE non-reduced electrophoretogram of pIL-29-PSA in example 2 of the present invention, wherein MK is a protein Marker; lane 1 is the pool.
FIG. 3 is a graph showing that pIL-29-Fc and pIL-29-PSA in example 4 of the present invention inhibit PEDV-induced Vero cell pathology.
FIG. 4 is a graph of pIL-29-Fc induced transcriptional expression of OASL and MxA in porcine primary lung macrophages in vitro as in example 5 of the invention, where a significant difference (p < 0.05) was observed compared to the CK group and a very significant difference (p < 0.01) was observed compared to the CK group.
FIG. 5 is a graph of the expression of OASL and MxA transcripts in porcine primary lung macrophages induced by pIL-29-PSA in vitro as shown in example 5 of the present invention, where the difference is significant compared to the CK group (p < 0.05) and the difference is very significant compared to the CK group (p < 0.01).
Detailed Description
The technical scheme of the invention is as follows: the amino acid sequence of the porcine IL-29 is fused with Fc fragment and PSA protein directly or indirectly through a connecting element to synthesize recombinant porcine IL-29 fusion protein pIL-29-Fc and pIL-29-PSA.
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction of pIL-29Fc and pIL-29-PSA fusion protein recombinant cell lines
Search of UniProt and GenBank databases for nucleotides of porcine IL-29, porcine Fc, and porcine PSAOrAn amino acid sequence. According to hamster codon preference, pIL-29-Fc and pIL-29-PSA are subjected to codon optimization, and related nucleotides are artificially synthesized, wherein the codon-optimized pIL-29-Fc nucleotide sequence is shown as SEQ ID NO:1, and the codon-optimized pIL-29-PSA nucleotide sequence is shown as SEQ ID NO: 3. The synthesized pIL-29-Fc and pIL-29-PSA nucleotides are respectively constructed in a pcDNA3.1 vector to obtain recombinant expression vectors pcDNA3.1-pIL-29-Fc and pcDNA3.1-pIL-29-PSA. After linearization of the pcDNA3.1-pIL-29-Fc recombinant vector, electrotransformation into CHO cells, and pressure screening to obtain a stable transgenic cell line pIL-29-Fc/CHO of pIL-29-Fc; after linearization, pcDNA3.1-pIL-29-PSA recombinant vector is electrically transferred into CHO cells, and a stable transfer cell line pIL-29-PSA/CHO of pIL-29-PSA is obtained through pressure screening.
Example 2 purification of pIL-29Fc and pIL-29-PSA fusion proteins
Respectively carrying out fermentation culture on the recombinant cells pIL-29-Fc/CHO and pIL-29-PSA/CHO in a fermentation tank, removing cells and cell debris from fermentation liquor by a two-stage deep filtration membrane package, and then filtering by a 0.22 mu m filter membrane to obtain clear fermentation liquor. pIL-29Fc and pIL-29-PSA were purified using the following methods, respectively.
purification of pIL-29 Fc: the fermentation broth was first purified by affinity chromatography Protein A (MabSelect SureTM, GE Healthcare): the column was equilibrated to baseline with an equilibration solution (50 mM glycine, 0.15M NaCl, pH 7.2), and then eluted with an eluent (50 mM glycine, pH 3.0) and collected. The Protein A pool was purified by anion exchange Capto Q (GE Healthcare) column chromatography: the collected solution was adjusted to pH 8.0 with 1M NaOH, equilibrated to baseline with an equilibration solution (10 mM Tris-HCl pH 8.0), and then eluted with an eluent (10 mM Tris-HCl,0.2M NaCl, pH 8.0) to collect the purified protein. Concentration by ultrafiltration and subsequent buffer exchange. The purified target protein was subjected to SDS-PAGE non-reducing gel electrophoresis (FIG. 1).
purification of pIL-29-PSA: the fermentation broth was first purified with the affinity chromatography packing Blue Sepharose TM 6Fast Flow (GE Healthcare): first, use the equilibrium solution (25 mM KH) 2 PO4, pH 6.8-7.0) to baseline, and then eluting with an eluent (25 mM Tris, 1.5M NaCl pH 9.0-9.1) to collect the eluent. Concentration by ultrafiltration and subsequent buffer exchange. The purified target protein was subjected to SDS-PAGE non-reducing gel electrophoresis (FIG. 2).
Example 3 determination of the biological Activity of pIL-29Fc and pIL-29-PSA fusion proteins
The potency of pIL-29-Fc and pIL-29-PSA was determined using the cytopathic inhibition method. Well-grown MDBK (bovine kidney cell) cell suspension is expressed by 2X 10 5 cells/mL were plated at a density of 100. Mu.L/well in 96-well cell culture plates overnight in a 5% CO2 cell incubator at 37 ℃. pIL-29, pIL-29-Fc and pIL-29-PSA were diluted separately in 2% FBS-DMEM in 4-fold gradients, with 8 replicates per gradient, 100. Mu.L protein/well added and incubated for a further 24h at 37 ℃ in a 5% CO2 cell incubator. Cell culture was discarded and cells were infected with VSV at 100 TCID50 per well for 24h post infection. Discarding the culture solution, adding 50 mu L of crystal violet staining solution into each hole, staining for 30 min at room temperature, washing off the staining solution with running water, adding 100 mu L of destaining solution into each hole, and standing for 3 to 5 min at room temperature. After mixing, absorbance was measured at a wavelength of 570 nm with a microplate reader using 630 nm as a reference wavelength. A cell control group and a virus control group are simultaneously arranged. The antiviral activity of pIL-29 was found to be 5.34X 10 by calculation 4 The antiviral activity of IU/mL and pIL-29-Fc is 3.55X 10 5 IU/mL, pIL-29-PSA has an antiviral activity of 8.29X 10 5 IU/mL, the fusion proteins pIL-29-Fc and pIL-29-PSA have higher antiviral activity than pIL-29.
Example 4 pIL-29Fc and pIL-29-PSA inhibition of PEDV-induced Vero cytopathic assay
Vero cell (African green monkey kidney cell line) suspension was expressed at 2X 10 5 cells/mL were plated at a density of 100. Mu.L/well in 96-well cell culture plates and incubated overnight at 37 ℃ in a 5% CO2 cell incubator. After 24h, cell supernatant is discarded, 50 mug/mL of pIL-29-Fc fusion protein and 50 mug/mL of pIL-29-PSA fusion protein are respectively added into sample wells, cultivation is continued for 24h, supernatant is discarded, and the cells are infected by PEDV (attenuated strain) of 3000 TCID50. And simultaneously setting a negative control hole and a positive control hole, wherein the negative control hole is a hole without the fusion protein and the PEDV, and the positive control hole is a hole without the fusion protein of only the PEDV. Each experimental group was provided with 4 replicate wells. Culturing at 37 deg.C in 5% CO2 cell culture box for 48-72 hr, staining with 0.05% crystal violet staining solution for 10 min, washing off the staining solution with running water, and observing cytopathic condition under microscope.
As shown in figure 3, pIL-29-Fc and pIL-29-PSA both can significantly inhibit PEDV-induced Vero cytopathic effect, wherein more than 85% of cells in positive control wells have pathological death, and pIL-29-Fc and pIL-29-PSA well cells are similar to negative controls, and most of the cells maintain normal growth state.
Example 5 pIL-29Fc and pIL-29-PSA induce mRNA transcription of OASL and MxA, primary porcine lung macrophages
OASL and MxA are important interferon-induced antiviral proteins, where MxA is a member of the anti-adhesion viral protein family, and is capable of blocking viral nucleic acid entry into the cell. OASL is a member of the 2'-5' oligoadenylate synthetase family, and inhibits viral protein synthesis and viral infection. The experiment researches whether pIL-29Fc and pIL-29-PSA can induce the transcription of primary pig lung macrophages OASL and MxA.
Taking fresh intact pig lung with intact trachea from slaughterhouse, slowly pouring DPBS (double antibody) from trachea mouth, gently kneading pig lung surface, recovering lavage fluid after 3-5 min, repeating lavage for 2 times, recovering lavage fluid, and sending to laboratory. And (3) filtering with double-layer sterile gauze under an aseptic condition, collecting filtrate, centrifuging at 1000 rpm for 5 min, removing supernatant, resuspending cell sediment with sterile PBS, and cleaning for 2-3 times according to the method to obtain the primary porcine alveolar macrophage. Subjecting primary pig to alveolar macrophagocytosisCells were seeded in 6-well plates, 1X 10 6 cells/well were loaded with pIL-29 at 100 ng/mL and pIL-29-Fc and pIL-29-PSA at different concentrations (10, 100 and 1000 ng/mL). A negative control group (CK group) was also set. Culturing for 16 h at 37 ℃ in a 5% CO2 cell culture box, collecting cells, extracting RNA, and quantitatively detecting the mRNA expression levels of the antiviral proteins OASL and MxA by RT-qPCR, wherein the pig Actin is used as an internal reference gene.
As shown in FIGS. 4 and 5, compared with the negative control, both pIL-29-Fc and pIL-29-PSA can induce the transcription enhancement of the antiviral proteins OASL and MxA in the porcine primary pulmonary macrophages. Wherein, compared with a control group, the 100 ng/mL pIL-29-Fc can induce the transcriptional up-regulation of OASL by about 164 times and the transcriptional up-regulation of MxA by about 49 times; 100 ng/mL pIL-29-PSA induces transcriptional upregulation of OASL by about 350-fold and MxA by about 142-fold.
Example 6 pharmacokinetic testing of pIL-29-Fc and pIL-29-PSA in rats
12 adult SD rats with the body weight of 180-220 g are selected, and divided into two groups, namely a pIL-29-Fc group and a pIL-29-PSA group. Two groups of mice were injected subcutaneously with 0.2 mg/kg of pIL-29-Fc or pIL-29-PSA, and blood was collected before, after, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 168, 214, 262 h of drug injection, and serum was isolated. The pig IL-29 ELISA detection kit is adopted to detect the content of pIL-29-Fc and pIL-29-PSA in serum, and WinNonlin software is used to analyze the parameters of the drug. The results show that the half-life of pIL-29-Fc is about 53h, the half-life of pIL-29-PSA is about 74h, and the pIL-29-Fc fusion protein and the pIL-29-PSA fusion protein are remarkably prolonged in vivo in rats.
Sequence listing
<110> Beijing Weijixin Biotechnology Ltd
<120> recombinant porcine IL-29 fusion protein, and preparation method and application thereof
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ggaggcggtg ggtcaatttg tccagcttgc gaaagcccag gtccttctgt gttcatcttt 600
cctcctaaac caaaggatac cctcatgatt tcccggacac cacaagtcac ctgtgtcgtt 660
gtggatgttt cacaggagaa tcctgaggtg caattctctt ggtacgtgga cggcgttgaa 720
gtgcacacag cacagacaag acccaaggag gaacagttca actctacata cagggtggtt 780
agtgtcctgc ccattcagca ccaggattgg ctcaacggga aggagttcaa gtgcaaagtg 840
aacaacaagg acctgccagc accaattaca cgcatcatct ccaaggctaa aggacagaca 900
cgggagcctc aggtctacac tctgcctcct catgcagagg agctgtctcg cagcaaggtg 960
tctatcactt gcctggtcat tggtttctac ccacccgaca tcgacgtgga gtggcagcgc 1020
aatgggcagc ccgaaccaga agggaactac cgcacaactc ctcctcaaca ggatgtggat 1080
ggcacctatt tcctgtactc caagttctct gtggacaaag caagttggca aggtggcggt 1140
atcttccagt gcgctgtcat gcacgaggcc ctgcataacc attacaccca gaagtccatt 1200
tcaaagactc ctggcaag 1218
<210> 2
<211> 406
<212> PRT
<213> pig (Sus scrofa)
<400> 2
Val Pro Thr Phe Lys Pro Thr Thr Thr Arg Lys Gly Cys His Met Gly
1 5 10 15
Gln Phe Gln Ser Leu Ser Pro Gln Glu Leu Lys Gly Phe Lys Lys Ala
20 25 30
Lys Asp Ala Leu Glu Glu Ser Leu Ser Leu Lys Asn Trp Ser Cys Ser
35 40 45
Ser Pro Leu Phe Pro Arg Thr Arg Asp Leu Arg Gln Leu Gln Val Trp
50 55 60
Glu Arg Leu Val Ala Leu Glu Ala Glu Leu Asp Leu Thr Leu Lys Val
65 70 75 80
Leu Arg Ala Ala Ala Asp Ser Ser Leu Gly Val Thr Leu Asp Gln Pro
85 90 95
Leu Arg Thr Leu His His Ile His Val Glu Leu Gln Ala Cys Ile Arg
100 105 110
Ala Gln Pro Thr Ala Gly Ser Arg Leu Gln Gly Arg Leu Asn His Trp
115 120 125
Leu His Arg Leu Gln Glu Ala Thr Lys Lys Glu Ser Gln Gly Cys Leu
130 135 140
Glu Ala Ser Val Thr Phe Asn Leu Phe His Leu Leu Val Arg Asp Leu
145 150 155 160
Arg Ser Val Thr Ser Gly Asp Leu His Ile Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Cys Pro Ala Cys Glu Ser
180 185 190
Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
195 200 205
Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val Val Asp Val Ser
210 215 220
Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu
225 230 235 240
Val His Thr Ala Gln Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr
245 250 255
Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Asn
260 265 270
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro
275 280 285
Ile Thr Arg Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg Glu Pro Gln
290 295 300
Val Tyr Thr Leu Pro Pro His Ala Glu Glu Leu Ser Arg Ser Lys Val
305 310 315 320
Ser Ile Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile Asp Val
325 330 335
Glu Trp Gln Arg Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr
340 345 350
Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys
355 360 365
Phe Ser Val Asp Lys Ala Ser Trp Gln Gly Gly Gly Ile Phe Gln Cys
370 375 380
Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile
385 390 395 400
Ser Lys Thr Pro Gly Lys
405
<210> 3
<211> 2304
<212> DNA
<213> pig (Sus scrofa)
<400> 3
gtgcctacat tcaaacctac tacaactcgg aagggctgcc acatgggaca attccagtct 60
ctgtctccac aggaactgaa aggtttcaag aaagcaaagg acgcactgga ggagtccctg 120
tccctcaaga attggtcatg tagcagccca ctctttccca gaactaggga tctcaggcag 180
ctgcaagtgt gggaaaggct cgtggctctg gaagccgaac tcgatctcac cctgaaagtg 240
ctgagagcag cagcagattc atccctcgga gtgacactcg accagcctct gagaacactg 300
catcacatcc acgtggagct gcaagcctgt atcagggctc aaccaacagc tgggagtagg 360
ctgcaaggga gactgaacca ctggctgcat agactccagg aagccacaaa gaaagaaagc 420
cagggttgcc tggaggcttc tgttaccttc aatctcttcc acctgctggt gagggacctg 480
cggtctgtca ccagtggcga cctgcacata ggaggcggag gaagtggagg aggcggtagt 540
ggcggtggtg ggtccgacac atacaagagc gaaattgccc acaggtttaa ggatctgggc 600
gagcagtatt tcaaaggcct cgtcctcatc gcattctccc agcatctcca gcaatgtcct 660
tatgaggagc atgtgaaact ggtgcgcgaa gtgaccgagt tcgcaaagac ttgtgttgcc 720
gatgaaagtg ctgagaactg cgataagagt attcacacac tgttcggaga caagctgtgt 780
gcaatcccat ccctgcggga gcattatggc gacctggcag actgctgtga gaaagaggag 840
ccagagagaa atgagtgctt cctccagcac aagaatgata atccagatat tcccaaactc 900
aagccagatc ccgtcgccct gtgtgccgac ttccaagagg acgagcagaa gttctggggc 960
aagtatctct acgagatcgc taggaggcac ccatacttct atgcacccga gctgctgtac 1020
tacgccatca tctacaaaga cgtgttctca gagtgctgtc aagccgcaga caaagcagcc 1080
tgtctgctgc ctaagatcga gcacctgagg gagaaggtcc tcacatctgc cgctaaacag 1140
cgcctcaaat gtgcttccat tcagaagttt ggagagagag cattcaaggc ctggagcctg 1200
gcaaggctga gtcagcggtt cccaaaggct gatttcactg agataagcaa gatagttacc 1260
gatctggcca aggtgcacaa agaatgctgt cacggcgacc tgctggagtg cgcagacgac 1320
agggcagacc tggcaaagta tatctgcgag aatcaggata ccatttctac aaagctgaaa 1380
gaatgttgcg acaaacctct gctggagaag tcacactgta tcgccgaggc caagagggat 1440
gaactccctg ccgatctgaa ccctctggag cacgacttcg tcgaggacaa agaagtgtgt 1500
aagaactaca aggaggccaa gcacgtgttt ctgggaacct tcctgtatga gtattcccgc 1560
aggcaccctg attactccgt gtccctgctc ctcaggatcg ctaagatata cgaagcaaca 1620
ctggaagatt gttgtgctaa ggaggaccca cccgcatgtt atgcaaccgt gttcgacaag 1680
tttcaaccac tggtggatga gcccaagaat ctgatcaaac agaactgtga actgtttgag 1740
aagctcggag agtacggttt ccagaacgcc ctcattgtga ggtacactaa gaaagtccct 1800
caggtttcta ctcctaccct cgtggaggtc gcaaggaagc tgggtctggt tggcagtagg 1860
tgctgcaaac ggcctgagga agaaaggctc tcctgtgccg aggattatct gtcactggtg 1920
ctgaaccgcc tctgtgttct gcatgagaag actcctgtgt ctgagaaagt tactaagtgt 1980
tgtacagaga gcctggtgaa cagaaggcca tgcttcagcg cactcactcc tgacgagaca 2040
tacaagccca aggagttcgt ggagggaacc ttcaccttcc acgcagacct gtgtactctg 2100
cccgaagatg agaaacagat aaagaagcaa actgcactgg ttgaactgct gaagcacaag 2160
ccacacgcta ccgaggagca gctccggaca gtgctcggca acttcgctgc cttcgttcag 2220
aagtgttgcg cagctcctga tcacgaggcc tgtttcgccg ttgagggacc caagtttgtg 2280
atcgagatta gaggtattct ggca 2304
<210> 4
<211> 768
<212> PRT
<213> pig (Sus scrofa)
<400> 4
Val Pro Thr Phe Lys Pro Thr Thr Thr Arg Lys Gly Cys His Met Gly
1 5 10 15
Gln Phe Gln Ser Leu Ser Pro Gln Glu Leu Lys Gly Phe Lys Lys Ala
20 25 30
Lys Asp Ala Leu Glu Glu Ser Leu Ser Leu Lys Asn Trp Ser Cys Ser
35 40 45
Ser Pro Leu Phe Pro Arg Thr Arg Asp Leu Arg Gln Leu Gln Val Trp
50 55 60
Glu Arg Leu Val Ala Leu Glu Ala Glu Leu Asp Leu Thr Leu Lys Val
65 70 75 80
Leu Arg Ala Ala Ala Asp Ser Ser Leu Gly Val Thr Leu Asp Gln Pro
85 90 95
Leu Arg Thr Leu His His Ile His Val Glu Leu Gln Ala Cys Ile Arg
100 105 110
Ala Gln Pro Thr Ala Gly Ser Arg Leu Gln Gly Arg Leu Asn His Trp
115 120 125
Leu His Arg Leu Gln Glu Ala Thr Lys Lys Glu Ser Gln Gly Cys Leu
130 135 140
Glu Ala Ser Val Thr Phe Asn Leu Phe His Leu Leu Val Arg Asp Leu
145 150 155 160
Arg Ser Val Thr Ser Gly Asp Leu His Ile Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Tyr Lys Ser Glu Ile
180 185 190
Ala His Arg Phe Lys Asp Leu Gly Glu Gln Tyr Phe Lys Gly Leu Val
195 200 205
Leu Ile Ala Phe Ser Gln His Leu Gln Gln Cys Pro Tyr Glu Glu His
210 215 220
Val Lys Leu Val Arg Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala
225 230 235 240
Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly
245 250 255
Asp Lys Leu Cys Ala Ile Pro Ser Leu Arg Glu His Tyr Gly Asp Leu
260 265 270
Ala Asp Cys Cys Glu Lys Glu Glu Pro Glu Arg Asn Glu Cys Phe Leu
275 280 285
Gln His Lys Asn Asp Asn Pro Asp Ile Pro Lys Leu Lys Pro Asp Pro
290 295 300
Val Ala Leu Cys Ala Asp Phe Gln Glu Asp Glu Gln Lys Phe Trp Gly
305 310 315 320
Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro
325 330 335
Glu Leu Leu Tyr Tyr Ala Ile Ile Tyr Lys Asp Val Phe Ser Glu Cys
340 345 350
Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Ile Glu His
355 360 365
Leu Arg Glu Lys Val Leu Thr Ser Ala Ala Lys Gln Arg Leu Lys Cys
370 375 380
Ala Ser Ile Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Leu
385 390 395 400
Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Thr Glu Ile Ser
405 410 415
Lys Ile Val Thr Asp Leu Ala Lys Val His Lys Glu Cys Cys His Gly
420 425 430
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile
435 440 445
Cys Glu Asn Gln Asp Thr Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp
450 455 460
Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Ala Lys Arg Asp
465 470 475 480
Glu Leu Pro Ala Asp Leu Asn Pro Leu Glu His Asp Phe Val Glu Asp
485 490 495
Lys Glu Val Cys Lys Asn Tyr Lys Glu Ala Lys His Val Phe Leu Gly
500 505 510
Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser
515 520 525
Leu Leu Leu Arg Ile Ala Lys Ile Tyr Glu Ala Thr Leu Glu Asp Cys
530 535 540
Cys Ala Lys Glu Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp Lys
545 550 555 560
Phe Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Ile Lys Gln Asn Cys
565 570 575
Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile
580 585 590
Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val
595 600 605
Glu Val Ala Arg Lys Leu Gly Leu Val Gly Ser Arg Cys Cys Lys Arg
610 615 620
Pro Glu Glu Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Leu Val
625 630 635 640
Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys
645 650 655
Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe
660 665 670
Ser Ala Leu Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu
675 680 685
Gly Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Asp Glu
690 695 700
Lys Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys
705 710 715 720
Pro His Ala Thr Glu Glu Gln Leu Arg Thr Val Leu Gly Asn Phe Ala
725 730 735
Ala Phe Val Gln Lys Cys Cys Ala Ala Pro Asp His Glu Ala Cys Phe
740 745 750
Ala Val Glu Gly Pro Lys Phe Val Ile Glu Ile Arg Gly Ile Leu Ala
755 760 765

Claims (9)

1. The recombinant porcine IL-29 fusion protein is characterized in that the fusion protein is formed by connecting porcine IL-29 with a fusion partner through a Linker, and the sequence of the fusion protein is shown as sequence 2 or sequence 4.
2. Expressing the nucleotide sequence of the fusion protein of claim 1, wherein the sequence is shown as sequence 1 or sequence 3.
3. A biological material comprising a gene encoding the fusion protein of claim 1, said biological material being an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineered bacterium or a transgenic cell line.
4. A method of producing the fusion protein of claim 1, comprising: and (3) artificially synthesizing encoding genes of the fusion proteins shown in the sequence 2 or the sequence 4 respectively, carrying out codon optimization, constructing the optimized genes into a eukaryotic expression vector, transferring the eukaryotic expression vector into eukaryotic cells, expressing the eukaryotic expression vector in the eukaryotic cells, and separating and purifying the target protein.
5. The preparation method according to claim 4, wherein the eukaryotic expression vector is pcDNA3.1 and the eukaryotic cell is a CHO cell.
6. A modified fusion protein of claim 1 comprising acetylation, pegylation, glycosylation or binding to BSA.
7. Use of the fusion protein of claim 1 for the preparation of a medicament or a combination medicament for the prevention and treatment of viral infection diseases in livestock, such as swine.
8. The use according to claim 7, wherein the virus comprises porcine reproductive and respiratory syndrome virus, pseudorabies virus, vesicular stomatitis virus, porcine epidemic diarrhea virus, porcine circovirus, classical swine fever virus, porcine transmissible gastroenteritis virus.
9. A porcine antiviral drug or composition, characterized in that the active ingredient is the fusion protein of claim 1.
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