CN108822219A - A kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent - Google Patents

A kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent Download PDF

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CN108822219A
CN108822219A CN201810734986.3A CN201810734986A CN108822219A CN 108822219 A CN108822219 A CN 108822219A CN 201810734986 A CN201810734986 A CN 201810734986A CN 108822219 A CN108822219 A CN 108822219A
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protein
pig
leu
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albumen
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CN108822219B (en
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李晶
刘文军
范文辉
刘丽蓉
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Institute of Microbiology of CAS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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]
    • C07K14/55—IL-2
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/555—Interferons [IFN]
    • 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

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Abstract

The invention discloses a kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent.Present invention firstly provides a kind of protein, including following two sections：1 albumen of pig PoIFN λ and pig IL2 albumen.The protein further includes link peptide, and link peptide is located between 1 albumen of pig PoIFN λ and pig IL2 albumen.The present invention also protects the protein preparing the application in pig virus infection disease vaccine.The present invention also protects a kind of pig virus to infect disease vaccine, including the protein.The present invention has major application promotional value for the prevention and control of pig virus infectious disease.

Description

A kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent
Technical field
The present invention relates to a kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent.
Background technique
In pig breeding industry, a variety of viral infectious such as porcine reproductive and respiratory syndrome, porcine pseudorabies, swine flu are multiple. Although swine disease Vaccines classes are various and immune frequent, it still cannot effectively prevent breaking out and popular, above-mentioned viral infection for disease Disease causes biggish economic loss to pig breeding industry every year.Vaccine immunity effect is improved, the swine disease prevention and treatment of research and development efficiently, safe is used Medicine becomes the urgent need in pig breeding industry.
Due to the needs of production of intensive culture, more and more scientific research personnel recognize mucosa-immune antiviral The importance of aspect not only generates immune response in mucous membrane tissue by mucosal vaccination, can also pass through common mucosa-immune Network causes systemic immune response, and studying mucosal vaccine and mating adjuvant also has become new hot spot.
Interferon (Interferon, IFN) is initially in nineteen fifty-seven by British scientist Isaacs research avian influenza virus It is found when interference phenomenon, is a kind of glycoprotein with broad anti-viral activity, by the interferon inducer of virus and other types It leads agent to come produced by stimulating endothelial cell, macrophage, lymphocyte and body cell, there is antiviral and antitumor, immunological regulation And induction differentiation isoreactivity.The performance of interferon effect is not to directly act on virus, but it is a variety of wide to stimulate cell to generate Antiviral protein is composed, antivirus action can be played by direct or indirect approach.
According to structure and the difference of receptor, can be divided to mammal IFN is two classes：I type IFN and Type II IFN.Mammal I type IFN mainly includes IFN-α, IFN-β, IFN- ω and IFN- τ, to acid and thermostabilization, with efficient disease-resistance cytotoxic activity, wherein IFN-α is mainly generated by leucocyte, and IFN-β is mainly generated by fibroblast.Mammal Type II IFN includes IFN-γ, by T Cell and NK cell generate, and are that the main macrophage of mammal is living to acid and thermally labile, mainly immunoregulation effect Change the factor.III type interferon (IFN- λ), is a kind of new forms of interferon, is interferon family newcomer, has both I type (antiviral) With the function of II type (immunological regulation), in conjunction with its specific receptor after, play antiviral and antitumor and immunoregulatory activity. III type interferon family includes IFN- λ 1, IFN- λ 2 and IFN- λ 3.The functional receptor compound of IFN- λ be by IFN- λ R1 and The heterodimer of IL-10R β chain composition, IFN- λ are integrated to inducing receptor heterodimerisation on receptor, lead to Jak-STAT signal The activation of transduction pathway, to play biological effect similar with I type IFN.The many biological activities of IFN- λ with clinically answer It is quite similar with extensive IFN α/β, but its expression of receptor limits to, and toxic side effect is relatively small, therefore antiviral and antitumor Aspect has broad application prospects.
Summary of the invention
The object of the present invention is to provide a kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent.
Present invention firstly provides a kind of protein (protein first), including following two sections：1 albumen of pig PoIFN λ and Pig IL2 albumen.
The protein further includes link peptide, and link peptide is located between 1 albumen of pig PoIFN λ and pig IL2 albumen.
The 1 1-172 amino acids residue of sequence of 1 albumen such as sequence table of pig PoIFN λ or 3 1-172 of sequence of sequence table Shown in amino acids residue.
The 1 183-316 amino acids residue of sequence of pig IL2 albumen such as sequence table or 3 183-316 of sequence of sequence table Shown in amino acids residue.
The protein is as shown in the sequence 1 of sequence table or the sequence 3 of sequence table.
The present invention also protects a kind of protein (protein second), for following (a1), (a2) or (a3)：
(a1) contain the fusion protein of above-mentioned protein first；
(a2) fusion protein that small peptide of the connection containing label obtains in the end of above-mentioned protein first；
(a3) fusion protein that connection label obtains in the end of above-mentioned protein first.
The DNA molecular of coding any description above protein also belongs to protection scope of the present invention.
The DNA molecular specifically can be as shown in the sequence 2 of sequence table or the sequence 4 of sequence table.
The present invention also protects any description above protein preparing the application in pig virus infection disease vaccine.
The present invention also protects a kind of pig virus to infect disease vaccine, including any description above protein.
The present invention also protects the application of any description above protein, for following (b1) or (b2)：
(b1) it is used as pig immunopotentiator；
(b2) pig immunopotentiator is prepared.
The present invention also protects a kind of product, including any description above protein；The function of the product is following (b1) Or (b2)：
(b1) it is used as pig immunopotentiator；
(b2) pig immunopotentiator is prepared.
Any description above pig virus infectious disease is swine flu caused by swine influenza virus.The swine influenza virus is H1N1 swine influenza virus.The H1N1 swine influenza virus is A/California/04/2009A (H1N1) strain.
Any description above pig is Landrace.
The present invention has major application promotional value for the prevention and control of pig virus infectious disease.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.VSV virus (vesicular stomatitis virus)：China Veterinery Drug Inspection Office.Following reality The quantitative test in example is applied, three repeated experiments are respectively provided with, results are averaged.
Embodiment 1, the building of recombinant bacterium and the preparation of destination protein
One, the building of recombinant plasmid
NdeI the and BamHI digestion position of insertion pET28a (+) carrier of double chain DNA molecule shown in the sequence 2 by sequence table Between point, recombinant plasmid pET28a-PoIFN λ 1-IL2 is obtained.According to sequencing result, to recombinant plasmid pET28a-PoIFN λ 1- IL2 carries out structure and is described as follows：The sequence of sequence table is inserted between NdeI the and BamHI restriction enzyme site of pET28a (+) carrier DNA molecular shown in column 2.Protein shown in the sequence 1 of DNA molecular polynucleotide shown in the sequence 2 of sequence table.By sequence Protein shown in the sequence 1 of list is named as PoIFN λ 1-IL2 fusion protein after optimization.In the sequence 1 of sequence table, 1- 1 albumen of pig PoIFN λ after 172 amino acids residue compositional optimizations, the pig after 183-316 amino acids residue compositional optimization IL2 albumen.
NdeI the and BamHI digestion position of insertion pET28a (+) carrier of double chain DNA molecule shown in the sequence 4 by sequence table Between point, recombinant plasmid first is obtained.According to sequencing result, structure is carried out to recombinant plasmid first and is described as follows：It is carried in pET28a (+) DNA molecular shown in the sequence 4 of sequence table is inserted between NdeI the and BamHI restriction enzyme site of body.Shown in the sequence 4 of sequence table DNA molecular polynucleotide sequence 3 shown in protein.Protein shown in sequence 3 by sequence table is named as optimization Preceding PoIFN λ 1-IL2 fusion protein.Pig PoIFN λ in the sequence 3 of sequence table, before 1-172 amino acids residue compositional optimization 1 albumen, the pig IL2 albumen before 183-316 amino acids residue compositional optimization.
NdeI the and BamHI digestion position of insertion pET28a (+) carrier of double chain DNA molecule shown in the sequence 5 by sequence table Between point, recombinant plasmid second is obtained.According to sequencing result, structure is carried out to recombinant plasmid second and is described as follows：It is carried in pET28a (+) DNA molecular shown in the sequence 5 of sequence table is inserted between NdeI the and BamHI restriction enzyme site of body.Shown in the sequence 5 of sequence table DNA molecular code optimization before 1 albumen of pig PoIFN λ.
Two, it the building of recombinant bacterium and prepares the ability of destination protein and compares
1, the acquisition of recombinant bacterium
Recombinant plasmid pET28a-PoIFN λ 1-IL2 is imported into Escherichia coli BL-21 (DE3), obtains 68 plants of recombinant bacteriums.
2, the ability that each strain recombinant bacterium prepares destination protein compares
The 68 plants of recombinant bacteriums respectively prepared by step 1 carry out following steps：
(1) recombinant bacterium single colonie is seeded to the LB liquid medium of 3ml kanamycins containing 0.1mg/ml, 37 DEG C, 200rpm shaken cultivation 12 hours, obtain seed liquor.
(2) the liquid LB that the seed liquor that 20ml step (1) obtains is seeded to 1980ml kanamycins containing 0.1mg/ml is trained Support base, 37 DEG C, 200rpm shaken cultivation to system OD600nmValue is 0.6, and IPTG, which is added, simultaneously makes its concentration 1mmol/L, then 37 DEG C, 200rpm shaken cultivation 4h.
(3) after completing step (2), by entire cultivating system, thalline were collected by centrifugation, is suspended with the PBS buffer solution of pH8.0, so It carries out afterwards ultrasonication (300W, ultrasonic 6s stop 12s, 99 times), then 5000g is centrifuged 10 minutes, collects precipitating (inclusion body).
(4) precipitating for taking step (3) to obtain successively is washed with Washing buffer and Resuspension buffer.
Washing buffer(pH8.0)：Containing 0.5%Triton-100,50mM Tris, 300mM NaCl, 10mM EDTA, 10mM DTT, surplus are water.
Resuspension buffer(pH8.0)：Tris containing 50mM, 100mM NaCl, 10mM EDTA, 10mM DTT, Surplus is water.
(5) complete step (4) after, be completely dissolved inclusion body with Dissolution buffer, then 4 DEG C, 10000g from The heart 20 minutes, collect supernatant.
Dissolution buffer(pH8.0)：Gua-HCl containing 6M, 10% glycerol, 50mM Tris, 100mM NaCl, 10mM EDTA, 10mM DTT, surplus are water.
(6) supernatant for taking step (5) to obtain, is slowly dropped in Refolding buffer, 4 DEG C, 200rpm stirring 24h。
Refolding buffer(pH8.0)：Tris containing 100mM, 400mM L-Arg HCl, 2mM EDTA, 5mM GSH, 0.5mM GSSG, surplus are water.
(7) whole system for completing step (6) is collected, is concentrated under the conditions of 4 DEG C, bonus point when being concentrated into 20ml or so Son sieve buffer then proceedes to be concentrated under the conditions of 4 DEG C, molecular sieve buffer is added when being concentrated into 20ml or so to 100ml To 100ml, then proceedes to be concentrated under the conditions of 4 DEG C, be concentrated into 4ml, as concentrate.
Molecular sieve buffer (pH8.0)：Tris containing 20mM, 150mM NaCl, surplus are water.
(8) concentrate for taking 1ml step (7) to obtain, 4 DEG C, 12000rpm centrifugation 10min, with Superdex 7510/ 300GL sieve chromatography column purification.
Column volume is 24ml；Filler is 15 100-200 of sephadex G.
Eluent (pH8.0)：Tris containing 20mM, 150mM NaCl, surplus are water.
Eluent flow rate：0.3ml/min.
Collect solution, as destination protein solution after crossing column that retention volume is 12.8ml-15.1ml.
68 plants of recombinant bacteriums are subjected to the destination protein solution that above-mentioned steps obtain and carry out polyacrylamide gel electrophoresis respectively, Show single band to get the electrophoretically pure destination protein arrived.PoIFN λ 1-IL2 fusion protein after destination protein optimizes.
The protein concentration in destination protein solution that detection obtains each recombinant bacterium progress above-mentioned steps, 67 plants of recombinant bacteriums The obtained protein concentration in destination protein solution is between 3.7-4.0mg/ml, and the destination protein that 1 plant of recombinant bacterium obtains is molten Protein concentration in liquid is that this plant of recombinant bacterium (is named as escherichia coli BL21/pET28a-PolFN λ 1- by 24.1mg/ml IL2)。
3, the preservation of recombinant bacterium
Escherichia coli (Escherichia coli) BL21/pET28a-PolFN λ 1-IL2, in 06 month 2018 Being preserved within 08th China Committee for Culture Collection of Microorganisms's common micro-organisms center, (abbreviation CGMCC, address are：Beijing The institute 3 of Chaoyang District North Star West Road 1), deposit number is CGMCC No.15920.
Three, it the building of recombinant bacterium and prepares the ability of destination protein and compares
1, the acquisition of recombinant bacterium
Recombinant plasmid first is imported into Escherichia coli BL-21 (DE3), obtains 20 plants of recombinant bacteriums.
2, the ability that each strain recombinant bacterium prepares destination protein compares
The 20 plants of recombinant bacteriums respectively prepared by step 1 detect, method with step 22.
20 plants of recombinant bacteriums are subjected to the destination protein solution that above-mentioned steps obtain and carry out polyacrylamide gel electrophoresis respectively, Show single band to get the electrophoretically pure destination protein arrived.PoIFN λ 1-IL2 fusion protein before destination protein optimizes.
The protein concentration in destination protein solution that detection obtains each recombinant bacterium progress above-mentioned steps, 20 plants of recombinant bacteriums The obtained protein concentration in destination protein solution is between 2.5-2.7mg/ml.
The strongest one plant of recombinant bacterium of destination protein ability will be prepared in 20 plants of recombinant bacteriums is named as recombination fungus beetle.
Four, the ability for constructing and its preparing destination protein of recombinant bacterium compares
1, the acquisition of recombinant bacterium
Recombinant plasmid second is imported into Escherichia coli BL-21 (DE3), obtains 20 plants of recombinant bacteriums.
2, the ability that each strain recombinant bacterium prepares destination protein compares
The 20 plants of recombinant bacteriums respectively prepared by step 1 detect, method with step 22.
20 plants of recombinant bacteriums are subjected to the destination protein solution that above-mentioned steps obtain and carry out polyacrylamide gel electrophoresis respectively, Show single band to get the electrophoretically pure destination protein arrived.1 albumen of pig PoIFN λ before destination protein optimizes.
The protein concentration in destination protein solution that detection obtains each recombinant bacterium progress above-mentioned steps, 20 plants of recombinant bacteriums The obtained protein concentration in destination protein solution is between 1.1-1.3mg/ml.
The strongest one plant of recombinant bacterium of destination protein ability will be prepared in 20 plants of recombinant bacteriums is named as recombinant bacterium second.
Step 2: step 3 and step 4 the result shows that：Using identical expression vector and host strain, before optimization The yield of PoIFN λ 1-IL2 fusion protein is significantly higher than the yield for optimizing preceding 1 albumen of pig PoIFN λ, PoIFN λ 1-IL2 after optimization The yield of fusion protein is significantly higher than the yield for optimizing preceding PoIFN λ 1-IL2 fusion protein.
Embodiment 2, the function of destination protein (animal experiment verifying)
Experimental animal is Landrace (40 ages in days, every weight are 10kg).
Using big in the step of PoIFN λ 1-IL2 fusion protein is embodiment 1 after used optimization in the present embodiment two Intestines Escherichia BL21/pET28a-PolFN λ 1-IL2 is prepared.PoIFN λ 1- before used optimization in the present embodiment It is prepared in the step of IL2 fusion protein is embodiment 1 three using recombination fungus beetle.In the present embodiment before used optimization It is prepared in the step of 1 albumen of pig PoIFN λ is embodiment 1 four using recombinant bacterium second.
Composition I is made of PoIFN λ 1-IL2 fusion protein, white oil and dilution after optimizing, and mixes well emulsification.Combination Object II is made of PoIFN λ 1-IL2 fusion protein, white oil and dilution before optimizing, and mixes well emulsification.Composition III is by optimizing Preceding 1 albumen of pig PoIFN λ, white oil and dilution composition, mix well emulsification.The function of white oil be adjuvant, SEPPIC S.A, MONTANIDE ISA 11R VG.Dilution is the PBS buffer solution of pH7.2.
Experimental animal is divided into 7 groups, every group 24, is handled respectively following (being single-dose)：
First group：By way of collunarium, every experimental animal gives 1mL composition I, and PoIFN λ 1-IL2 melts after optimization The dosage of hop protein is 0.1mg/kg experimental animal, and the dosage of white oil is 0.1mg/kg experimental animal；
Second group：By way of intramuscular injection, every experimental animal gives 1mL composition I, PoIFN λ 1- after optimization The dosage of IL2 fusion protein is 0.1mg/kg experimental animal, and the dosage of white oil is 0.1mg/kg experimental animal；
Third group：By way of collunarium, every experimental animal gives 1mL compositions II, and PoIFN λ 1-IL2 melts before optimizing The dosage of hop protein is 0.1mg/kg experimental animal, and the dosage of white oil is 0.1mg/kg experimental animal；
4th group：By way of intramuscular injection, every experimental animal gives 1mL compositions II, PoIFN λ 1- before optimizing The dosage of IL2 fusion protein is 0.1mg/kg experimental animal, and the dosage of white oil is 0.1mg/kg experimental animal；
5th group：By way of collunarium, every experimental animal gives 1mL composition III, 1 albumen of pig PoIFN λ before optimizing Dosage be 0.1mg/kg experimental animal, the dosage of white oil is 0.1mg/kg experimental animal；
6th group：By way of intramuscular injection, every experimental animal gives 1mL composition III, pig PoIFN λ 1 before optimizing The dosage of albumen is 0.1mg/kg experimental animal, and the dosage of white oil is 0.1mg/kg experimental animal；
7th group：By way of collunarium, every experimental animal gives 1mL physiological saline.
The administration same day carries out attacking poison on the 21st day as test the 1st day, test.It attacks poison and uses intranasally inoculated mode, every head Experimental animal is inoculated with 1ml virus liquid.Virus liquid is the virus liquid of swine influenza virus A/California/04/2009A (H1N1), The content of swine influenza virus A/California/04/2009A (H1N1) is 1000PFU in 1ml virus liquid.
It tests the 24-26 days, counts the flu episode rate of each group.The period occur in following three symptom two with The pig of upper symptom is judged as morbid pig：1. not searching for food；2. having difficulty in breathing；3. body temperature is 40.5 DEG C or more.
First group of flu episode rate is 8.33%.Second group of flu episode rate is 12.5%.The influenza of third group is sent out Sick rate is 20.83%.4th group of flu episode rate is 25%.5th group of flu episode rate is 41.67%.6th group of stream Feeling disease incidence is 50%.7th group of flu episode rate is 100%.
Embodiment 3, interferon activity detection
Cell culture fluid：DMEM culture solution containing 10%FBS.
One, interferon activity detection method
1, prepared by cell
Well-grown PK-15 cell is taken, is suspended after digestion with cell culture fluid, obtaining cell concentration is 5 × 105A/ The cell suspension of mL；Cell suspension is added into 96 porocyte culture plates (100 hole μ l/), in 37 DEG C, 5%CO2Under the conditions of cultivate 8- 10h becomes cell monolayer.
2, it uses cell culture fluid for solvent, first test substance is dissolved or diluted, makes protein concentration 0.001mg/ml, 4 times of gradient dilutions are carried out as mother liquor, then by mother liquor, 6 gradients is diluted, obtains various dilutions.
3, the tissue culture plate of step 1 is taken into, inhales and abandons supernatant；Cell culture fluid (100 μ l/ are added in 3 Positive control wells Hole), cell culture fluid (100 hole μ l/) is added in 3 negative control holes, and dilution (the 100 μ l/ that step 2 obtains are added in other holes Hole), 3 multiple holes are arranged in every kind of dilution；Then in 37 DEG C, 5%CO2Under the conditions of cultivate 12-15h.
4, it takes VSV viral, is diluted with serum-free DMEM culture solution, obtaining virus concentration is 1000TCID50The virus of/ml is dilute Release liquid.
5, the tissue culture plate of step 3 is taken into, inhales and abandons supernatant；It is dilute that virus prepared by step 4 is added in 3 Positive control wells It releases in liquid (100 hole μ l/), serum-free DMEM culture solution (100 hole μ l/) is added in 3 negative control holes, and step 4 system is added in other holes Standby viral dilution (100 hole μ l/)；Then in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
6, the tissue culture plate of step 5 is taken into, inhales and abandons supernatant, is added in violet staining liquid (100 hole μ l/), then room Temperature places 30min.
7, the tissue culture plate of step 6 is taken into, inhales and abandons supernatant, rinsed with distilled water, destainer (100 μ l/ are then added Hole), then it is placed at room temperature for 10min.
8, OD is measured using microplate reader570nmIt is worth and records.
The interference cellulose content of 50% cytopathy can be inhibited to be defined as an active unit, with Reed-Muench method Interferon potency is calculated, the extension rate of 50% cytopathy can be inhibited, the results are shown in Table 1.
Table 1 calculates interferon potency with Reed-Muench method
X：The OD of negative control hole570Average value；
Y：The OD of Positive control wells570Average value.
Each entry value is calculated according to upper meter, finally calculates interferon potency (assuming that above-mentioned calculating according to following formula according to G Middle G4 calculated value is greater than 0.5, and G5 calculated values are less than 0.5)：
Interferon potency (U/0.001mg)=beforehand dilution multiple × 4 of test substance(4+(G4-0.5)/(G4-G5))。
Two, the interferon activity of PoIFN λ 1-IL2 fusion protein after optimizing
Large intestine is used in the step of PoIFN λ 1-IL2 fusion protein is embodiment 1 after used optimization in this step two Escherichia BL21/pET28a-PolFN λ 1-IL2 is prepared.
Test substance is respectively as follows：PoIFN λ 1-IL2 fusion protein (three batches) after freshly prepd optimization, will optimization Afterwards -8 DEG C of 4 DEG C of PoIFN λ 1-IL2 fusion protein save 6 months (three batches), will PoIFN λ 1-IL2 fusion protein 4 after optimization DEG C -8 DEG C save 12 months (three batches), will after optimization 4 DEG C -8 DEG C of PoIFN λ 1-IL2 fusion protein save 18 months (three Batch), will after optimization -8 DEG C of 4 DEG C of PoIFN λ 1-IL2 fusion protein save 24 months (three batches), will PoIFN λ 1- after optimization 25 DEG C of IL2 fusion protein save 1 month (three batches), save 25 DEG C of fusion protein of PoIFN λ 1-IL2 after optimization 3 months (three batches), will optimization after 25 DEG C of fusion protein of PoIFN λ 1-IL2 preservations, 6 months (three batches).
Interferon activity is detected according to the method for step 1.
The interferon potency of PoIFN λ 1-IL2 fusion protein is 2 × 10 after freshly prepd optimization5U/mg。
The interferon potency of test substance is shown in Table 2.
Table 2
SEQUENCE LISTING
<110>Institute of Microorganism, Academia Sinica
<120>A kind of fused interferon and its preparing the application in mucosal immunity reinforcing agent
<130> GNCYX181499
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<170> PatentIn version 3.5
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Gly Pro Val Pro Thr Phe Lys Pro Thr Thr Thr Arg Lys Gly Cys His
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Met Gly Gln Cys Gln Ser Leu Ser Pro Gln Glu Leu Lys Gly Phe Lys
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Lys Ala Lys Asp Ala Leu Glu Glu Ser Leu Ser Leu Lys Asn Trp Ser
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Cys Ser Ser Pro Leu Phe Pro Arg Thr Arg Asp Leu Arg Gln Leu Gln
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Val Trp Glu Arg Leu Val Ala Leu Glu Ala Glu Leu Asp Leu Thr Leu
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Lys Val Leu Arg Ala Ala Ala Asp Ser Ser Leu Gly Val Thr Leu Asp
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Gln Pro Leu Arg Thr Leu His His Ile His Val Glu Leu Gln Ala Cys
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Ile Arg Ala Gln Pro Thr Ala Gly Ser Arg Leu Gln Gly Arg Leu Asn
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His Trp Leu His Arg Leu Gln Glu Ala Thr Lys Lys Glu Ser Gln Gly
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Phe Leu Glu Ala Ser Val Thr Phe Asn Leu Phe His Leu Leu Val Arg
145 150 155 160
Asp Leu Arg Ser Val Thr Ser Gly Asp Leu His Ile Gly Gly Gly Gly
165 170 175
Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr
180 185 190
Lys Lys Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Ser Leu Leu Lys
195 200 205
Glu Val Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe
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Lys Phe Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys
225 230 235 240
Leu Val Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln
245 250 255
Ser Lys Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile
260 265 270
Asn Val Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Cys Glu Cys
275 280 285
Glu Tyr Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp
290 295 300
Ile Thr Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr
305 310 315
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ggtccggttc cgaccttcaa accgaccacc acccgtaaag gttgccacat gggtcagtgc 60
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cgtcagctgc aggtttggga acgtctggtt gctctggaag ctgaactgga cctgaccctg 240
aaagttctgc gtgctgctgc tgactcttct ctgggtgtta ccctggacca gccgctgcgt 300
accctgcacc acatccacgt tgaactgcag gcttgcatcc gtgctcagcc gaccgctggt 360
tctcgtctgc agggtcgtct gaaccactgg ctgcaccgtc tgcaggaagc taccaaaaaa 420
gaatctcagg gtttcctgga agcttctgtt accttcaacc tgttccacct gctggttcgt 480
gacctgcgtt ctgttacctc tggtgacctg cacatcggcg gtggtggtag cggcggtggt 540
ggtagtgctc cgacctcttc ttctaccaaa aacaccaaaa aacagctgga accgctgctg 600
ctggacctgc agagcctgct gaaagaagtt aaaaactacg aaaacgctga cctgtctcgt 660
atgctgacct tcaaattcta catgccgaaa caggctaccg aactgaaaca cctgcagtgc 720
ctggttgaag aactgaaagc tctggaaggt gttctgaacc tgggtcagtc taaaaactct 780
gactctgcta acatcaaaga atctatgaac aacatcaacg ttaccgttct ggaactgaaa 840
ggttctgaaa cctcttgcga atgcgaatac gacgacgaaa ccgttaccgc tgttgaattc 900
ctgaacaaat ggatcacctt ctgccagtct atctactcta ccctgaccta a 951
<210> 3
<211> 316
<212> PRT
<213> Artificial sequence
<400> 3
Gly Pro Val Pro Thr Phe Lys Pro Thr Thr Thr Arg Lys Gly Cys His
1 5 10 15
Met Gly Gln Phe Gln Ser Leu Ser Pro Gln Glu Leu Lys Gly Phe Lys
20 25 30
Lys Ala Lys Asp Ala Leu Glu Glu Ser Leu Ser Leu Lys Asn Trp Ser
35 40 45
Cys Ser Ser Pro Leu Phe Pro Arg Thr Arg Asp Leu Arg Gln Leu Gln
50 55 60
Val Trp Glu Arg Leu Val Ala Leu Glu Ala Glu Leu Asp Leu Thr Leu
65 70 75 80
Lys Val Leu Arg Ala Ala Ala Asp Ser Ser Leu Gly Val Thr Leu Asp
85 90 95
Gln Pro Leu Arg Thr Leu His His Ile His Val Glu Leu Gln Ala Cys
100 105 110
Ile Arg Ala Gln Pro Thr Ala Gly Ser Arg Leu Gln Gly Arg Leu Asn
115 120 125
His Trp Leu His Arg Leu Gln Glu Ala Thr Lys Lys Glu Ser Gln Gly
130 135 140
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe His Leu Leu Val Arg
145 150 155 160
Asp Leu Arg Ser Val Thr Ser Gly Asp Leu His Ile Gly Gly Gly Gly
165 170 175
Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Asn Thr
180 185 190
Lys Lys Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys
195 200 205
Glu Val Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe
210 215 220
Lys Phe Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys
225 230 235 240
Leu Val Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln
245 250 255
Ser Lys Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile
260 265 270
Asn Val Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Glu Cys
275 280 285
Glu Tyr Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp
290 295 300
Ile Thr Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr
305 310 315
<210> 4
<211> 951
<212> DNA
<213> Artificial sequence
<400> 4
ggtccggttc cgaccttcaa accgaccacc acccgtaaag gttgccacat gggtcagttc 60
cagtctctgt ctccgcagga actgaaaggt ttcaaaaaag ctaaagacgc tctggaagaa 120
tctctgtctc tgaaaaactg gtcttgctct tctccgctgt tcccgcgtac ccgtgacctg 180
cgtcagctgc aggtttggga acgtctggtt gctctggaag ctgaactgga cctgaccctg 240
aaagttctgc gtgctgctgc tgactcttct ctgggtgtta ccctggacca gccgctgcgt 300
accctgcacc acatccacgt tgaactgcag gcttgcatcc gtgctcagcc gaccgctggt 360
tctcgtctgc agggtcgtct gaaccactgg ctgcaccgtc tgcaggaagc taccaaaaaa 420
gaatctcagg gttgcctgga agcttctgtt accttcaacc tgttccacct gctggttcgt 480
gacctgcgtt ctgttacctc tggtgacctg cacatcggcg gtggtggtag cggcggtggt 540
ggtagtgctc cgacctcttc ttctaccaaa aacaccaaaa aacagctgga accgctgctg 600
ctggacctgc agctgctgct gaaagaagtt aaaaactacg aaaacgctga cctgtctcgt 660
atgctgacct tcaaattcta catgccgaaa caggctaccg aactgaaaca cctgcagtgc 720
ctggttgaag aactgaaagc tctggaaggt gttctgaacc tgggtcagtc taaaaactct 780
gactctgcta acatcaaaga atctatgaac aacatcaacg ttaccgttct ggaactgaaa 840
ggttctgaaa cctctttcga atgcgaatac gacgacgaaa ccgttaccgc tgttgaattc 900
ctgaacaaat ggatcacctt ctgccagtct atctactcta ccctgaccta a 951
<210> 5
<211> 519
<212> DNA
<213> Artificial sequence
<400> 5
ggtccggttc cgaccttcaa accgaccacc acccgtaaag gttgccacat gggtcagttc 60
cagtctctgt ctccgcagga actgaaaggt ttcaaaaaag ctaaagacgc tctggaagaa 120
tctctgtctc tgaaaaactg gtcttgctct tctccgctgt tcccgcgtac ccgtgacctg 180
cgtcagctgc aggtttggga acgtctggtt gctctggaag ctgaactgga cctgaccctg 240
aaagttctgc gtgctgctgc tgactcttct ctgggtgtta ccctggacca gccgctgcgt 300
accctgcacc acatccacgt tgaactgcag gcttgcatcc gtgctcagcc gaccgctggt 360
tctcgtctgc agggtcgtct gaaccactgg ctgcaccgtc tgcaggaagc taccaaaaaa 420
gaatctcagg gttgcctgga agcttctgtt accttcaacc tgttccacct gctggttcgt 480
gacctgcgtt ctgttacctc tggtgacctg cacatctaa 519

Claims (10)

1. a kind of protein, including following two sections：1 albumen of pig PoIFN λ and pig IL2 albumen.
2. protein as described in claim 1, it is characterised in that：The protein further includes link peptide, and link peptide is located at pig Between 1 albumen of PoIFN λ and pig IL2 albumen.
3. protein as claimed in claim 1 or 2, it is characterised in that：
The 1 1-172 amino acids residue of sequence of 1 albumen such as sequence table of pig PoIFN λ or 1-172 ammonia of sequence 3 of sequence table Shown in base acid residue；
The 1 183-316 amino acids residue of sequence of pig IL2 albumen such as sequence table or 183-316 ammonia of sequence 3 of sequence table Shown in base acid residue.
4. protein as described in claim 1, it is characterised in that：The sequence 1 of the protein such as sequence table or sequence table Shown in sequence 3.
5. a kind of protein, for following (a1), (a2) or (a3)：
(a1) fusion protein containing the protein any in Claims 1-4；
(a2) fusion protein that end small peptide of the connection containing label of any protein obtains in Claims 1-4；
(a3) fusion protein that the end connection label of any protein obtains in Claims 1-4.
6. encoding the DNA molecular of any protein in claim 1 to 5.
7. any protein is preparing the application in pig virus infection disease vaccine in claim 1 to 5.
8. a kind of pig virus infects any protein in disease vaccine, including claim 1 to 5.
9. the application of any protein in claim 1 to 5, for following (b1) or (b2)：
(b1) it is used as pig immunopotentiator；
(b2) pig immunopotentiator is prepared.
10. a kind of product, including any protein in claim 1 to 5；The function of the product be following (b1) or (b2)：
(b1) it is used as pig immunopotentiator；
(b2) pig immunopotentiator is prepared.
CN201810734986.3A 2018-07-06 2018-07-06 Fusion interferon and application thereof in preparation of mucosal immunopotentiator Active CN108822219B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2007351813A1 (en) * 2006-10-31 2008-10-30 East Carolina University Fusion proteins comprising an anti-inflammatory cytokine and an antigen for treatment of immune disorders
CN106632682A (en) * 2015-08-04 2017-05-10 清华大学 Fusion protein IFN-ELP and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2007351813A1 (en) * 2006-10-31 2008-10-30 East Carolina University Fusion proteins comprising an anti-inflammatory cytokine and an antigen for treatment of immune disorders
CN106632682A (en) * 2015-08-04 2017-05-10 清华大学 Fusion protein IFN-ELP and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DANG WANG ET AL.: "Molecular cloning, expression and antiviral activity of porcine interleukin-29 (poIL-29)", 《DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY》 *
HE,L. ET AL.: "NCBI Reference Sequence: ACT78884.1，IL-2 [Sus scrofa]", 《GENBANK DATABASE》 *
闫若潜 等: "猪α干扰素/白细胞介素2基因的融合表达及活性研究", 《畜牧兽医学报》 *
陆源 等: "白细胞介素分子改造的研究进展", 《中国生物制品学杂志》 *

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