CN110357971B

CN110357971B - Pig compound interferon for emergency prevention of African swine fever

Info

Publication number: CN110357971B
Application number: CN201910772921.2A
Authority: CN
Inventors: 李晶; 范文辉; 张鹤; 刘文军
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-08-21
Filing date: 2019-08-21
Publication date: 2021-01-15
Anticipated expiration: 2039-08-21
Also published as: CN110357971A

Abstract

The invention discloses a pig compound interferon for emergently preventing African swine fever. The invention provides a protein (porcine gamma interferon-connecting peptide-porcine antibacterial peptide fragment fusion protein) as shown in a sequence 7 of a sequence table. The gene encoding the protein also belongs to the protection scope of the invention. The invention also protects a complex comprising the protein and lentinan; in the compound, each 1.0 × 10^‑9The mol protein proportion is 15 mu g lentinan. The invention also protects the application of the protein or the compound in preparing the African swine fever vaccine. The invention also protects an African swine fever vaccine, and the active ingredient of the African swine fever vaccine is the protein or the compound. The protein and the compound provided by the invention have good immune protection effect and high stability (can be stored at room temperature and has long storage period). The invention has great application and popularization value for prevention and control of African swine fever.

Description

Pig compound interferon for emergency prevention of African swine fever

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a pig compound interferon for emergency prevention of African swine fever.

Background

China is a big country for pig production and marketing, the consumption of pork accounts for 50% of the consumption of pork in the world, and pig breeding areas are located throughout the country. According to the latest revised data issued by 'Chinese statistical yearbook 2018', the quantity of domestic pigs in stock reaches 44158 thousands of pigs, and the number of the pigs in the first place in the world is the top. However, the pig breeding level and the biological safety control degree in China are different at present, and various production main bodies such as scattered farmers, medium and small farms, large farms and the like exist. Although the proportion of large-scale breeding of 2600 ten thousand pig farms in China is increased year by year, more than 500 pigs are grown every year and account for less than 1%, and scattered farmers still exist for a long time and have higher biological safety risk.

African Swine Fever (ASF) is an acute, hemorrhagic and virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs, European wild pigs, etc.). The world animal health Organization (OIE) classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a type of animal epidemic disease which is mainly prevented in China. The clinical symptoms of African swine fever are similar to those of swine fever, and diagnosis can be confirmed only by means of laboratory monitoring. The first African swine fever epidemic situation is diagnosed in China in 8 months and 3 days in 2018.

After the highly pathogenic ASFV strain is infected, the clinical symptoms are mainly manifested as high fever, severe depression, anorexia, cyanosis of skin, hemorrhagic lesion and the like, and the ASF is an important problem to be solved urgently by the world pig industry. Interferon is a protein with broad-spectrum antiviral action, which itself has no direct killing effect on viruses, but can inhibit virus replication. Cellular mechanisms of interferon antiviral: the coding gene of the antiviral protein exists in the body cell, the gene is in a suppression state under normal conditions, when the interferon is combined with a cell membrane receptor and enters a human cell, the suppression state of the gene is released, the body cell synthesizes the antiviral protein, so that the virus nucleic acid cannot be combined with the ribonucleoprotein body of the host cell, and the virus cannot be replicated.

Disclosure of Invention

The invention aims to provide a porcine consensus interferon for emergency prevention of African swine fever.

The invention provides a protein (porcine gamma interferon-connecting peptide-porcine antibacterial peptide fragment fusion protein) as shown in a sequence 7 of a sequence table.

The gene encoding the protein also belongs to the protection scope of the invention.

The coding region of the gene is shown as a sequence 8 in a sequence table.

The recombinant expression vector containing the gene also belongs to the protection scope of the invention. The recombinant expression vector can be specifically a recombinant plasmid IV. Inserting a double-stranded DNA molecule shown by 4 th-582 th nucleotides of a sequence 8 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid IV.

Recombinant bacteria containing the gene also belong to the protection scope of the invention. The recombinant bacterium can be specifically a recombinant bacterium IV. The recombinant plasmid IV is introduced into escherichia coli BL21(DE3) to obtain a recombinant bacterium IV.

The invention also protects a complex comprising the protein and lentinan; in the compound, each 1.0 × 10^-9The mol protein proportion is 15 mu g lentinan.

The compound consists of the protein, the lentinan and a buffer solution; 1.0X 10 of the compound per ml^-9mol of the protein and 15 mu g of the lentinan. The buffer may specifically be a PBS buffer, more specifically a PBS buffer of pH 7.4.

The preparation method of the lentinan comprises the following steps:

(1) pulverizing dried Lentinus Edodes, and sieving to obtain powder;

(2) adding boiling water into the powder obtained in the step (1), standing and extracting at room temperature for 30min, then centrifuging at 3000rpm for 10min, and collecting supernatant; 1L of boiling water is mixed with each 100g of powder;

(3) and (3) adding absolute ethyl alcohol into the supernatant obtained in the step (2) to enable the volume percentage of the ethyl alcohol in the system to be 15-30%, standing for 1 hour at room temperature, then centrifuging for 10min at 3000rpm, collecting the precipitate, and freeze-drying to obtain the product, namely the lentinan.

The volume percentage of the ethanol in the system can be specifically 15%.

The volume percentage of the ethanol in the system can be 30 percent.

The screening can be specifically 80-mesh screening.

The invention also protects the application of the protein or the compound in the preparation of the African swine fever vaccine.

The invention also provides an African swine fever vaccine, the active ingredient of which is the protein or the compound.

The vaccine can be used for emergency prevention of African swine fever.

The protein and the compound provided by the invention have good immune protection effect and high stability (can be stored at room temperature and has long storage period).

The invention has great application and popularization value for prevention and control of African swine fever.

Drawings

FIG. 1 shows the electrophoresis patterns of protein I solution, protein II solution, protein III solution and protein IV solution.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, the cell culture conditions were: 37 ℃ and 5% CO₂。

pET30a (+) vector: the product number of the Beijing Ding Guosheng biotechnology Limited liability company is MCV 031. Vesicular Stomatitis Virus (VSV): china institute for veterinary medicine.

2 XYT culture solution: 16g/L of peptone, 10g/L of yeast powder, 5g/L of sodium chloride and the balance of water.

Fermentation ofCulture medium (ph 7.2): peptone 5g/L, yeast powder 5g/L, KH₂PO₄ 2g/L、K₂HPO₄ 4g/L、Na₂HPO₄·12H₂O 7g/L、(NH₄)₂SO₄ 1.2g/L、NH₄Cl 0.2g/L、MnSO₄·5H₂O 0.001g/L、CoCl₂·6H₂O 0.004g/L、Na₂MoO₄·2H₂O 0.002g/L、ZnCl₂ 0.002g/L、CuSO₄·5H₂O 0.001g/L、H₃BO₄0.005g/L、FeSO₄·7H₂O 0.02g/L、CaCl·2H₂O 0.02g/L、MgSO₄·7H₂0.3g/L of O, 0.2g/L of antifoaming agent, 0.1mg/ml of ampicillin and the balance of water. Defoaming agent: shanghai elisa Biotechnology Inc., model number OED60K, product number Q04M10X 81687; the main component is polyalkylene oxide ether.

Feed medium (ph 7.2): 150ml/L of glycerol, 30g/L of peptone and 30g/L, MgSO of yeast powder₄·7H₂O5.5 mg/L, and the balance of water.

The PBS buffers used in the examples were all PBS buffers (pH7.2) unless otherwise specified. PBS buffer (ph 7.2): NaCl 137mmol/L, KCl 2.7mmol/L, Na₂HPO₄ 4.3mmol/L、KH₂PO₄1.4mmol/L, and the balance of water.

Denaturation buffer (ph 8.0): 6mol/L guanidine hydrochloride, 2mmol/L EDTA, 50mmol/L Tris-HCl, 10mmol/L DTT and the balance of water.

Renaturation buffer (ph 8.0): 0.5mol/L L-Arg, 2mmol/L EDTA, 20% (volume ratio) glycerol, 0.9mmol/L GSSG, 0.1mol/L Tris & HCl, and the balance of water.

Example 1 preparation of protein solution

Preparation of recombinant plasmid

Inserting a double-stranded DNA molecule shown by 4 th-435 th nucleotides in a sequence 2 of a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid I. The recombinant plasmid I has an open reading frame shown in a sequence 2 of a sequence table, and expresses protein shown in a sequence 1 of the sequence table.

Inserting a double-stranded DNA molecule shown by 4 th-948 th nucleotides of a sequence 4 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid II. The recombinant plasmid II has an open reading frame shown in a sequence 4 of a sequence table, and expresses protein shown in a sequence 3 of the sequence table.

Inserting a double-stranded DNA molecule shown by 4 th to 552 th nucleotides of a sequence 6 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid III. The recombinant plasmid III has an open reading frame shown in a sequence 6 of a sequence table, and expresses protein shown in a sequence 5 of the sequence table.

Inserting a double-stranded DNA molecule shown by 4 th-582 th nucleotides of a sequence 8 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid IV. The recombinant plasmid IV has an open reading frame shown in a sequence 8 of a sequence table, and expresses protein shown in a sequence 7 of the sequence table.

The double-stranded DNA molecule shown by the 4 th-498 th nucleotides of the sequence 10 in the sequence table is inserted between NdeI and XhoI enzyme cutting sites of pET30a (+) vector to obtain recombinant plasmid V. The recombinant plasmid V has an open reading frame shown in a sequence 10 of a sequence table, and expresses protein shown in a sequence 9 of the sequence table.

The double-stranded DNA molecule shown by the 4 th-1011 th nucleotide of the sequence 12 in the sequence table is inserted between NdeI and XhoI enzyme cutting sites of pET30a (+) vector to obtain a recombinant plasmid VI. The recombinant plasmid VI has an open reading frame shown in a sequence 12 of a sequence table and expresses protein shown in a sequence 11 of the sequence table.

Inserting a double-stranded DNA molecule shown by 4 th-615 th nucleotides of a sequence 14 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid VII. The recombinant plasmid VII has an open reading frame shown in a sequence 14 of a sequence table and expresses protein shown in a sequence 13 of the sequence table.

Inserting a double-stranded DNA molecule shown by nucleotides 4-645 of a sequence 16 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET30a (+) vector to obtain a recombinant plasmid VIII. The recombinant plasmid VIII has an open reading frame shown in a sequence 16 of a sequence table and expresses protein shown in a sequence 15 of the sequence table.

The above eight recombinant plasmids were sequence verified.

Second, preparation of recombinant bacterium

The recombinant plasmid I was introduced into E.coli BL21(DE3) to obtain recombinant strain I.

The recombinant plasmid II was introduced into E.coli BL21(DE3) to obtain recombinant strain II.

The recombinant plasmid III was introduced into E.coli BL21(DE3) to obtain recombinant strain III.

The recombinant plasmid IV is introduced into escherichia coli BL21(DE3) to obtain a recombinant bacterium IV.

The recombinant plasmid V was introduced into E.coli BL21(DE3) to obtain recombinant strain V.

The recombinant plasmid VI is introduced into Escherichia coli BL21(DE3) to obtain a recombinant strain VI.

The recombinant plasmid VII is introduced into Escherichia coli BL21(DE3) to obtain a recombinant bacterium VII.

The recombinant plasmid VIII is introduced into escherichia coli BL21(DE3) to obtain a recombinant strain VIII.

Preparation of protein

1. Preparation of seed liquid

Inoculating the recombinant strain to 250mL of 2 XYT culture solution, and performing shake culture at 30 deg.C and 220rpm for 12h to obtain seed solution (OD of the seed solution)_450nmThe value is 1).

2. Fermentation of

250mL of the seed solution was inoculated into 5L of the fermentation medium and cultured. OD of the system was continuously monitored during the culture_450nmValue when OD of the system_450nmAt a value of 15 (usually about 12 hours of culture), IPTG (at a concentration of 50. mu.M in the system) was added, and the culture was continued for 8 hours.

The temperature was 37 ℃ throughout the cultivation. Aeration and stirring at 600rpm were continued throughout the culture. In the whole culture process, the pH value of the system is adjusted by using 25% ammonia water solution and 20% phosphoric acid water solution, and the pH value is kept to be 7.0-7.4. In the culture process, the carbon source in the fermentation medium is gradually consumed along with the growth of the thalli, the thalli DO not grow after the carbon source is consumed, and the Dissolved Oxygen (DO) rises again. And continuously monitoring DO, feeding the feed culture medium when the DO is monitored to rise back to 30%, and then controlling whether the feed culture medium is fed or not by monitoring the DO value (the DO is more than 30% of the feed culture medium, and the DO is less than 30% of the feed culture medium, and the feed culture medium is stopped feeding), wherein the feeding speed of the feed culture medium is 26-35 ml/h.

3. Extraction and purification of proteins

(1) And (3) taking the fermentation system which finishes the step 2, centrifuging at 5000rpm for 10 minutes, and collecting the thallus precipitate.

(2) And (2) taking the thallus precipitate obtained in the step (1), washing and re-suspending the thallus precipitate by using a PBS buffer solution, then carrying out ultrasonic disruption (the ultrasonic disruption parameter is phi 10 probe, stopping 5 seconds every 7 seconds of ultrasound, and the total time is 90 minutes), then centrifuging the thallus precipitate for 10 minutes at 4 ℃ and 10000rpm, and collecting the precipitate.

(3) And (3) taking the precipitate obtained in the step (2), washing the precipitate for 3 times by using PBS (phosphate buffer solution), then washing the precipitate for 3 times by using PBST (PBST), then dissolving the precipitate by using a denatured buffer solution, then centrifuging the precipitate for 10 minutes at 4 ℃ and 10000rpm, and collecting the supernatant.

(4) And (4) slowly adding the supernatant obtained in the step (3) into a renaturation buffer solution, standing for 48 hours at 4 ℃, centrifuging for 20 minutes at 4 ℃ and 10000g, and collecting the supernatant.

(5) And (4) taking the supernatant obtained in the step (4), adjusting the pH to 7.4 by using NaOH, and then carrying out anion exchange chromatography.

The anion exchange chromatography parameters were as follows:

a chromatographic column: HiTrap Q FF (GE Healthcare); the specification is 5X 5 mL;

sample is added at the speed of 0.5 mL/min, and the sample loading amount is 500 mL;

and (3) an elution process: the elution time is 60 min; the mobile phase at the initial moment is PBS buffer solution with pH7.4, the mobile phase at the termination moment is 1M NaCl aqueous solution, the mobile phase between the initial moment and the termination moment is composed of the PBS buffer solution with pH7.4 and the 1M NaCl aqueous solution, and the volume fraction of the PBS buffer solution with pH7.4 in the mobile phase from the initial moment to the termination moment is linearly reduced to 0 percent from 100 percent; the flow rate of the mobile phase is 1 ml/min; when the volume fraction of PBS buffer solution with pH7.4 in the mobile phase is 70%, the target protein is effectively eluted, an ultraviolet detector is used for detecting at the wavelength of 280nm in the elution process, and a post-column solution corresponding to a target elution peak is collected (the collection amount is 250 mL).

(6) Passing the solution obtained in step (5) through a Desalting column (GE Healthcare) to replace the solvent with PBS buffer solution (pH7.4), thereby obtaining a protein solution.

And (3) carrying out the third step on the recombinant bacterium I, and naming the obtained protein solution as a protein I solution (the protein I is the porcine gamma interferon). The protein concentration of the protein I solution was 11.8. mu.M.

And (3) carrying out the third step on the recombinant bacterium II, and naming the protein II solution (namely the protein II is the porcine gamma interferon-porcine antibacterial peptide fusion protein) by the obtained protein solution. The protein concentration of the protein II solution was 5.5. mu.M.

And (3) carrying out a third step on the recombinant bacterium III, and naming the obtained protein solution as a protein III solution (protein III is porcine gamma interferon-porcine antibacterial peptide fragment fusion protein). The protein III solution had a protein concentration of 9.3. mu.M.

And (3) carrying out a third step on the recombinant bacterium IV, and naming the obtained protein solution as a protein IV solution (the protein IV is porcine gamma interferon-connecting peptide-porcine antibacterial peptide fragment fusion protein). The protein concentration of the protein IV solution is 9.0 mu M.

And (3) carrying out the third step on the recombinant bacterium V, and naming the obtained protein solution as a protein V solution (namely the porcine beta interferon). Protein V solution protein concentration is 10.2. mu.M.

And (3) carrying out the third step on the recombinant bacterium VI, and naming the protein VI solution (namely the pig beta interferon-pig antibacterial peptide fusion protein) as the obtained protein solution. Protein VI solution had a protein concentration of 5.1. mu.M.

And (3) carrying out a third step on the recombinant bacteria VII, and naming the obtained protein solution as a protein VII solution (the protein VII is the swine beta interferon-swine antibacterial peptide fragment fusion protein). The protein concentration of the protein VII solution was 8.2. mu.M.

And (3) carrying out a third step on the recombinant bacterium VIII to obtain a protein solution named as a protein VIII solution (the protein VIII is the fusion protein of the porcine beta interferon-connecting peptide-the porcine antibacterial peptide fragment). The protein concentration of the protein VIII solution was 8.0. mu.M.

The electrophoretograms of the protein I solution, the protein II solution, the protein III solution and the protein IV solution are shown in figure 1. The target band was recovered and sequenced with the N-terminal 10 amino acid residues, all consistent with the N-terminal 10 amino acid residues of the target protein.

Example 2 detection of Interferon Activity

The test solutions were: the protein I solution, the protein II solution, the protein III solution or the protein IV solution prepared in example 1 were all prepared freshly.

1. Cell plating

Taking well-grown PK-15 cells, carrying out pancreatin digestion, and then suspending with DMEM medium containing 100U/mL penicillin, 100U/mL streptomycin and 10% (volume ratio) FBS to obtain the cell concentration of 1 × 10⁶one/mL of cell suspension, the cell suspension was added to a 96-well cell culture plate (100. mu.l/well), and then placed at 37 ℃ with 5% CO₂The cells were cultured for 8 hours (to form a monolayer of cells).

2. The test solution was diluted to 25-fold, 250-fold, 2500-fold or 25000-fold volume with DMEM medium containing 10% (volume ratio) FBS to obtain each dilution.

3. Taking the cell culture plate which is finished with the step 1, and sucking and removing the supernatant; test wells, adding the diluent (100 μ l/well) obtained in step 2, and setting 3 multiple wells for each dilution; setting 3 positive control wells and 3 negative control wells, adding DMEM medium (100. mu.l/well) containing 10% (volume ratio) FBS; the cell culture plate was then placed at 37 ℃ with 5% CO₂The culture box is kept still for 24 hours.

4. Diluting vesicular stomatitis virus with DMEM medium containing 100U/mL penicillin and 100U/mL streptomycin to obtain 100TCID₅₀Viral fluid (TCID)/mL₅₀Against PK-15 cells).

5. Taking the cell culture plate after the step 3, discarding the supernatant, and washing the cell culture plate once by using PBS buffer; adding the virus solution (100 mu l/well) obtained in the step 4 into the test well and the positive control well; negative control wells, DMEM medium containing 100U/mL penicillin and 100U/mL streptomycin (100. mu.l/well) was added; then placing at 37 ℃ and containing 5% CO₂The culture box is kept still for 24 hours.

6. Taking the cell culture plate which completes the step 5, abandoning the supernatant, and washing with PBS buffer solutionAfter crystal violet staining (100. mu.l of crystal violet staining solution was added to each well and allowed to stand at room temperature for 30min) and destaining (100. mu.l of destaining solution was added and allowed to stand at room temperature for 10min) in this order, OD was measured with a microplate reader_570nmAnd (6) value and recording.

7. And (3) data processing, wherein the content of the interferon capable of inhibiting 50% of cytopathic effect is defined as an activity unit, and the titer of the interferon is calculated by using a Reed-Muench method, namely the dilution factor capable of inhibiting 50% of cytopathic effect.

The interferon titer of the protein I solution is 1.7 multiplied by 10⁶U/ml, i.e. the interferon titer of protein I is 1.44X 10¹⁴U/mol。

The interferon titer of the protein II solution is 7.6 multiplied by 10⁵U/ml, i.e. the interferon titer of protein II is 1.38X 10¹⁴U/mol。

The interferon titer of the protein III solution was 2.5X 10⁷U/ml, i.e. the interferon titer of protein III is 2.69X 10¹⁵U/mol。

The interferon titer of the protein IV solution is 3.7 multiplied by 10⁷U/ml, i.e. the interferon titer of protein IV is 4.11X 10¹⁵U/mol。

Example 3 stability of protein solutions

Freshly prepared protein I solution, protein II solution, protein III solution or protein IV solution prepared in example 1 were allowed to stand at room temperature for 3 months, respectively, and then used as test solutions (each solution was set for 10 parallel treatments).

First, form observation

After standing at room temperature for 3 months, the protein I solution, the protein II solution and the protein III solution all became turbid.

The protein IV solution remained clear after 3 months at room temperature.

Secondly, detecting the activity of interferon

The procedure is as in example 2. The results were averaged.

The interferon titer of the protein I solution after standing at room temperature for 3 months is 8.2 multiplied by 10⁵U/ml, 48% of freshly prepared.

The interferon titer of the protein II solution after being placed at room temperature for 3 months is 3.3 multiplied by 10⁵U/ml, 43% of freshly prepared.

The interferon titer of the protein III solution after standing at room temperature for 3 months is 1.1X 10⁷U/ml, 44% of freshly prepared.

The interferon titer of the protein IV solution after being placed at room temperature for 3 months is 3.4 multiplied by 10⁷U/ml, 92% of freshly prepared.

The results show that the protein IV is most stable and has the best preservation performance.

Example 4 preparation of lentinan

1. Pulverizing commercially available dried Lentinus Edodes, and sieving with 80 mesh sieve to obtain powder.

2. And (3) taking 100g of the powder obtained in the step (1), adding 1L of boiled deionized water, standing and extracting for 30min at room temperature, then centrifuging for 10min at 3000rpm, and collecting supernatant.

3. And (3) adding absolute ethyl alcohol into the supernatant obtained in the step (2) to enable the volume percentage of the ethyl alcohol in the system to be 15%, standing for 1 hour at room temperature, then centrifuging for 10 minutes at 3000rpm, collecting precipitates, and freeze-drying to obtain the lentinan I.

4. And (3) adding absolute ethyl alcohol into the supernatant obtained in the step (2) to ensure that the volume percentage of the ethyl alcohol in the system is 30%, standing for 1 hour at room temperature, then centrifuging for 10min at 3000rpm, collecting the precipitate, and freeze-drying to obtain the product, namely the lentinan II.

Example 5 preparation of composites and comparison of Effect

The freshly prepared protein I solution, protein II solution, protein III solution, protein IV solution, protein V solution, protein VI solution, protein VII solution or protein VIII solution of example 1 was used. Lentinan I or lentinan II prepared in example 4 was used.

Preparation of composite

1. Adding lentinan I into the protein I solution, and then supplementing the volume with PBS buffer solution with pH7.4 to obtain a compound I-I. 1.0X 10 of the compound I-I per ml^-9mol protein I and 15 mu g lentinan I.

2. Adding lentinan into the protein I solutionII, then make up the volume with PBS buffer solution of pH7.4, get complex I-II. 1.0X 10 of the complex I-II per ml^-9mol protein I and 15 mu g lentinan II.

3. Adding lentinan I into the protein II solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound II-I. 1.0X 10 of the compound II-I per ml^-9mol protein II and 15 mu g lentinan I.

4. Adding lentinan II into the protein II solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound II-II. 1.0X 10 of compound II-II per ml^-9mol protein II and 15 mu g lentinan II.

5. Adding lentinan I into the protein III solution, and then complementing the volume by PBS buffer solution with pH7.4 to obtain a compound III-I. 1.0X 10 of compound III-I per ml^-9mol protein III and 15 mu g lentinan I.

6. Adding lentinan II into the protein III solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound III-II. 1.0X 10 of compound III-II per ml^-9mol protein III and 15 mu g lentinan II.

7. Adding lentinan I into the protein IV solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound IV-I. 1.0X 10 of compound IV-I per ml^-9mol protein IV and 15 mu g lentinan I.

8. Adding lentinan II into the protein IV solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound IV-II. Per ml of the complex IV-II, contains 1.0X 10^-9mol protein IV and 15 mu g lentinan II.

9. Adding lentinan I into the protein V solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound V-I. 1.0X 10 of compound V-I per ml^-9mol protein V and 15. mu.g lentinan I.

10. Adding lentinan II into the protein V solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound V-II. Every milli of Chinese characterIn the liter of complex V-II, 1.0X 10^-9mol protein V and 15. mu.g lentinan II.

11. Adding lentinan I into the protein VI solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound VI-I. Per ml of the compound VI-I, 1.0X 10^-9mol protein VI and 15 mu g lentinan I.

12. Adding lentinan II into the protein VI solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound VI-II. Per ml of the compound VI-II, contains 1.0X 10^-9mol protein VI and 15 mu g lentinan II.

13. Adding lentinan I into the protein VII solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound VII-I. 1.0X 10 of complex VII-I/ml^-9mol protein VII and 15 mu g lentinan I.

14. Adding lentinan II into the protein VII solution, and then complementing the volume with PBS buffer solution with pH7.4 to obtain a compound VII-II. 1.0X 10 of compound VII-II per ml^-9mol protein VII and 15 mu g lentinan II.

15. Adding lentinan I into the protein VIII solution, and then complementing the volume by PBS buffer solution with pH7.4 to obtain a compound VIII-I. 1.0X 10 of compound VIII-I/ml^-9mol protein VIII and 15 mu g lentinan I.

16. Adding lentinan II into the protein VIII solution, and then complementing the volume by PBS buffer solution with pH7.4 to obtain a compound VIII-II. 1.0X 10 of compound VIII-II per ml^-9mol protein VIII and 15 mu g lentinan II.

Two, group immunization

17 pig houses with the same conditions were prepared, 20 square meters each. Long white pigs (SPF pig breeding management center, Beijing, about 40 days old, each weighing 20kg) were divided into 17 groups of 10 pigs each. Each group was bred in the same pigsty.

The groups of test animals were treated separately as follows:

group 1 did not receive any inoculation treatment;

group 2 complexes I-I (fresh) were inoculated in 1ml doses each time;

group 3 complexes I-II (fresh) were inoculated in 1ml doses each time;

group 4 inoculation of complexes II-I (fresh) at 1ml dose per inoculation;

group 5 inoculation of complexes II-II (fresh), 1ml of each inoculum;

group 6 inoculation of complexes III-I (fresh), 1ml of each inoculum;

group 7 inoculation of complexes III-II (fresh), 1ml of each inoculum;

group 8 complexes IV-I (fresh) were inoculated in 1ml doses each time;

group 9 complexes IV-II (fresh) were inoculated in 1ml doses each time;

group 10 inoculation of complex V-I (fresh), 1ml per inoculation dose;

group 11 inoculation complexes V-II (fresh), each inoculation dose was 1 ml;

group 12 complexes VI-I (fresh) were inoculated in 1ml doses per inoculation;

group 13 inoculation of complexes VI-II (fresh), 1ml of each inoculum;

group 14 complexes VII-I (fresh) were inoculated in 1ml doses per batch;

group 15 complexes VII-II (fresh) were inoculated at 1ml dose per inoculation;

group 16 inoculation complexes VIII-I (fresh) at 1ml dose per inoculation;

group 17 inoculation complexes VIII-II (fresh) at 1ml dose per inoculation;

groups 2 to 17 were inoculated for 3 consecutive days (as test day 1, test day 2 and test day 3), once daily, all orally.

Thirdly, imitating the natural infection African swine fever virus

Taking 17 diseased long white pigs, wherein the 17 diseased long white pigs are from the same population, have the disease character of African swine fever, and have African swine fever virus in tissues after tissue separation and identification.

On day 4 of the experiment, one sick pig was placed in each pig house, simulating natural infection.

After 14 days of the pigs, the mortality rate of each group was counted.

The mortality rate in group 1 was 100%.

The mortality rate in group 2 was 30%.

The mortality rate in group 3 was 40%.

Mortality in group 4 was 40%.

Mortality in group 5 was 40%.

Mortality in group 6 was 10%.

Mortality in group 7 was 20%.

The mortality rate in group 8 was 0%.

Mortality in group 9 was 0%.

The mortality rate in group 10 was 70%.

Mortality in group 11 was 60%.

The mortality rate in group 12 was 80%.

Mortality in group 13 was 60%.

The mortality rate in group 14 was 70%.

Mortality in group 15 was 50%.

Mortality in group 16 was 60%.

Mortality in group 17 was 50%.

The compound IV-I and the compound IV-II have the best protection effect.

Example 6 storage Properties of composites

The freshly prepared complexes I-I, complexes I-II, complexes II-I, complexes II-II, complexes III-I, complexes III-II, complexes IV-I and complexes IV-II of step one of example 5 were each allowed to stand at room temperature for 3 months.

One, group immunization

9 pig houses with the same conditions were prepared, each 20 square meters. Long white pigs (SPF pig breeding management center, Beijing, about 40 days old, each weighing 20kg) were divided into 9 groups of 10 pigs each. Each group was bred in the same pigsty.

The groups of test animals were treated separately as follows:

group 1 did not receive any inoculation treatment;

group 2 complexes I-I (room temperature for 3 months) were inoculated at 1ml per dose;

group 3 complexes I-II (incubated at room temperature for 3 months) were inoculated at 1ml per dose;

group 4 inoculation of complexes II-I (3 months at room temperature), 1ml of each inoculation dose;

group 5 inoculation of complexes II-II (3 months at room temperature), 1ml of each inoculation dose;

group 6 inoculation of Complex III-I (3 months at room temperature), 1ml per inoculation dose;

group 7 inoculation of complexes III-II (3 months at room temperature), 1ml of each inoculation dose;

group 8 complexes IV-I (room temperature for 3 months), each inoculation dose is 1 ml;

group 9 complexes IV-II (room temperature for 3 months), each inoculation dose is 1 ml;

groups 2 to 9 were inoculated for 3 consecutive days (as test day 1, test day 2 and test day 3), once daily, all orally.

Second, imitate the African swine fever virus of natural infection

And taking 9 diseased long white pigs, wherein the 9 diseased long white pigs are from the same population, have the disease character of the African swine fever, and have the African swine fever virus in the tissues after tissue separation and identification.

After 14 days of the pigs, the mortality rate of each group was counted.

The mortality rate in group 1 was 100%.

The mortality rate in group 2 was 60%.

The mortality rate in group 3 was 60%.

Mortality in group 4 was 70%.

The mortality rate in group 5 was 70%.

Mortality in group 6 was 40%.

Mortality in group 7 was 50%.

The mortality rate in group 8 was 0%.

Mortality in group 9 was 20%.

The complexes IV-I are the most stable and have the best preservation properties.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> a porcine consensus interferon for emergency prevention of African swine fever

<130> GNCYX191862

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 144

<212> PRT

<213> Artificial Sequence

<400> 1

Met Gln Ala Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe

1 5 10 15

Asn Ala Ser Thr Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu

20 25 30

Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser

35 40 45

Gln Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn

50 55 60

Gln Ala Ile Gln Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln

65 70 75 80

Arg Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu

85 90 95

Ile Lys Ile Pro Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser

100 105 110

Glu Leu Ile Lys Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg

115 120 125

Lys Arg Lys Arg Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

130 135 140

<210> 2

<211> 435

<212> DNA

<213> Artificial Sequence

<400> 2

atgcaggcgc cgttctttaa ggagatcacc attctgaaag actacttcaa cgcgagcacc 60

agcgatgtgc cgaacggtgg cccgctgttt ctggaaattc tgaagaactg gaaagaggaa 120

agcgacaaga aaatcattca gagccaaatc gtgagcttct acttcaagtt ctttgagatc 180

ttcaaggata accaggcgat tcaacgtagc atggacgtta tcaagcagga tatgttccaa 240

cgttttctga acggtagcag cggcaaactg aacgactttg agaagctgat caaaattccg 300

gtggataacc tgcagattca gcgtaaggcg atcagcgaac tgattaaagt tatgaacgac 360

ctgagcccgc gtagcaacct gcgtaagcgt aaacgtagcc aaaccatgtt ccagggtcaa 420

cgtgcgagca agtaa 435

<210> 3

<211> 315

<212> PRT

<213> Artificial Sequence

<400> 3

Met Gln Ala Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe

1 5 10 15

Asn Ala Ser Thr Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu

20 25 30

Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser

35 40 45

Gln Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn

50 55 60

Gln Ala Ile Gln Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln

65 70 75 80

Arg Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu

85 90 95

Ile Lys Ile Pro Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser

100 105 110

Glu Leu Ile Lys Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg

115 120 125

Lys Arg Lys Arg Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

130 135 140

Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly Arg Trp Ser Leu Trp Leu

145 150 155 160

Leu Leu Leu Gly Leu Val Val Pro Ser Ala Ser Ala Gln Ala Leu Ser

165 170 175

Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu Asn Glu Gln Ser

180 185 190

Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp Gln Pro Pro Lys

195 200 205

Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser Phe Thr Val Lys

210 215 220

Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro Pro Glu Leu Cys Asp

225 230 235 240

Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val Gly Thr Val Thr Leu

245 250 255

Asn Pro Ser Ile His Ser Leu Asp Ile Ser Cys Asn Glu Ile Gln Ser

260 265 270

Val Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro

275 280 285

Pro Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro

290 295 300

Pro Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg

305 310 315

<210> 4

<211> 948

<212> DNA

<213> Artificial Sequence

<400> 4

atgcaggcgc cgttctttaa ggagatcacc attctgaaag actacttcaa cgcgagcacc 60

agcgatgtgc cgaacggtgg cccgctgttt ctggaaattc tgaagaactg gaaagaggaa 120

agcgacaaga aaatcattca gagccaaatc gtgagcttct acttcaagtt ctttgagatc 180

ttcaaggata accaggcgat tcaacgtagc atggacgtta tcaagcagga tatgttccaa 240

cgttttctga acggtagcag cggcaaactg aacgactttg agaagctgat caaaattccg 300

gtggataacc tgcagattca gcgtaaggcg atcagcgaac tgattaaagt tatgaacgac 360

ctgagcccgc gtagcaacct gcgtaagcgt aaacgtagcc aaaccatgtt ccagggtcaa 420

cgtgcgagca aggagaccca gagggccagc ctgtgcctgg ggcgctggtc actgtggctt 480

ctgctgctgg gactcgtggt gccctcggcc agcgcccagg ccctcagcta cagggaggcc 540

gtgcttcgtg ctgtggatcg cctcaacgag cagtcctcgg aagctaatct ctaccgcctc 600

ctggagctgg accagccgcc caaggccgac gaggacccgg gcaccccgaa acctgtgagc 660

ttcacggtga aggagactgt gtgtcccagg ccgacccggc agcccccgga gctgtgtgac 720

ttcaaggaga acgggcgagt gaagcagtgt gtggggacag tcaccttgaa cccatccatt 780

cactcactgg acatctcctg taatgagatt cagagtgtca ggagacgtcc ccgaccccca 840

tatttgccaa ggccaaggcc acctccgttt ttcccaccaa ggctcccacc aaggatccca 900

ccagggttcc caccaaggtt cccaccacgg ttccccggaa aacggtaa 948

<210> 5

<211> 183

<212> PRT

<213> Artificial Sequence

<400> 5

Met Gln Ala Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe

1 5 10 15

Asn Ala Ser Thr Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu

20 25 30

Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser

35 40 45

Gln Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn

50 55 60

Gln Ala Ile Gln Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln

65 70 75 80

Arg Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu

85 90 95

Ile Lys Ile Pro Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser

100 105 110

Glu Leu Ile Lys Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg

115 120 125

Lys Arg Lys Arg Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

130 135 140

Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro

145 150 155 160

Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro

165 170 175

Arg Phe Pro Pro Arg Phe Pro

180

<210> 6

<211> 552

<212> DNA

<213> Artificial Sequence

<400> 6

atgcaggcgc cgttctttaa ggagatcacc attctgaaag actacttcaa cgcgagcacc 60

agcgatgtgc cgaacggtgg cccgctgttt ctggaaattc tgaagaactg gaaagaggaa 120

agcgacaaga aaatcattca gagccaaatc gtgagcttct acttcaagtt ctttgagatc 180

ttcaaggata accaggcgat tcaacgtagc atggacgtta tcaagcagga tatgttccaa 240

cgttttctga acggtagcag cggcaaactg aacgactttg agaagctgat caaaattccg 300

gtggataacc tgcagattca gcgtaaggcg atcagcgaac tgattaaagt tatgaacgac 360

ctgagcccgc gtagcaacct gcgtaagcgt aaacgtagcc aaaccatgtt ccagggtcaa 420

cgtgcgagca agaggagacg tccccgaccc ccatatttgc caaggccaag gccacctccg 480

tttttcccac caaggctccc accaaggatc ccaccagggt tcccaccaag gttcccacca 540

cggttcccct aa 552

<210> 7

<211> 193

<212> PRT

<213> Artificial Sequence

<400> 7

Met Gln Ala Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe

1 5 10 15

Asn Ala Ser Thr Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu

20 25 30

Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser

35 40 45

Gln Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn

50 55 60

Gln Ala Ile Gln Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln

65 70 75 80

Arg Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu

85 90 95

Ile Lys Ile Pro Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser

100 105 110

Glu Leu Ile Lys Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg

115 120 125

Lys Arg Lys Arg Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg Arg Pro Arg Pro

145 150 155 160

Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro Phe Phe Pro Pro Arg Leu

165 170 175

Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro Arg Phe Pro Pro Arg Phe

180 185 190

Pro

<210> 8

<211> 582

<212> DNA

<213> Artificial Sequence

<400> 8

atgcaggcgc cgttctttaa ggagatcacc attctgaaag actacttcaa cgcgagcacc 60

agcgatgtgc cgaacggtgg cccgctgttt ctggaaattc tgaagaactg gaaagaggaa 120

agcgacaaga aaatcattca gagccaaatc gtgagcttct acttcaagtt ctttgagatc 180

ttcaaggata accaggcgat tcaacgtagc atggacgtta tcaagcagga tatgttccaa 240

cgttttctga acggtagcag cggcaaactg aacgactttg agaagctgat caaaattccg 300

gtggataacc tgcagattca gcgtaaggcg atcagcgaac tgattaaagt tatgaacgac 360

ctgagcccgc gtagcaacct gcgtaagcgt aaacgtagcc aaaccatgtt ccagggtcaa 420

cgtgcgagca agggtggcgg agggagtggt ggcggaggga gtaggagacg tccccgaccc 480

ccatatttgc caaggccaag gccacctccg tttttcccac caaggctccc accaaggatc 540

ccaccagggt tcccaccaag gttcccacca cggttcccct aa 582

<210> 9

<211> 165

<212> PRT

<213> Artificial Sequence

<400> 9

Met Ser Tyr Asp Val Leu Arg Tyr Gln Gln Arg Ser Ser Asn Leu Ala

1 5 10 15

Cys Gln Lys Leu Leu Glu Gln Leu Pro Gly Thr Pro Gln Tyr Cys Leu

20 25 30

Glu Asp Arg Met Asn Phe Glu Val Pro Glu Glu Ile Met Gln Pro Pro

35 40 45

Gln Phe Gln Lys Glu Asp Ala Val Leu Ile Ile His Glu Met Leu Gln

50 55 60

Gln Ile Phe Gly Ile Leu Arg Arg Asn Phe Ser Ser Thr Gly Trp Asn

65 70 75 80

Glu Thr Val Ile Lys Thr Ile Leu Val Glu Leu Asp Gly Gln Met Asp

85 90 95

Asp Leu Glu Thr Ile Leu Glu Glu Ile Met Glu Glu Glu Asn Phe Pro

100 105 110

Arg Gly Asp Met Thr Ile Leu His Leu Lys Lys Tyr Tyr Leu Ser Ile

115 120 125

Leu Gln Tyr Leu Lys Ser Lys Glu Tyr Arg Ser Cys Ala Trp Thr Val

130 135 140

Val Gln Val Glu Ile Leu Arg Asn Phe Ser Phe Leu Asn Arg Leu Thr

145 150 155 160

Asp Tyr Leu Arg Asn

165

<210> 10

<211> 498

<212> DNA

<213> Artificial Sequence

<400> 10

atgagctatg atgtgcttcg ataccaacaa aggagcagca atttggcatg tcagaagctc 60

ctggaacagt tgcctgggac tcctcaatat tgcctcgaag ataggatgaa cttcgaggtc 120

cctgaggaga ttatgcaacc accacaattc cagaaggaag atgcagtatt gattatccac 180

gagatgctcc agcagatctt cggcattctc agaagaaatt tctctagcac tggctggaat 240

gaaaccgtca ttaagactat ccttgtggaa cttgatgggc agatggatga cctggagaca 300

atcctggagg aaatcatgga ggaggaaaat ttccccaggg gagacatgac cattcttcac 360

ctgaagaaat attacttgag cattctgcag tacctgaagt ccaaggagta cagaagctgt 420

gcctggacag tcgtccaagt ggaaatcctc aggaactttt ctttccttaa cagacttaca 480

gattacctcc ggaactga 498

<210> 11

<211> 336

<212> PRT

<213> Artificial Sequence

<400> 11

Met Ser Tyr Asp Val Leu Arg Tyr Gln Gln Arg Ser Ser Asn Leu Ala

1 5 10 15

Cys Gln Lys Leu Leu Glu Gln Leu Pro Gly Thr Pro Gln Tyr Cys Leu

20 25 30

Glu Asp Arg Met Asn Phe Glu Val Pro Glu Glu Ile Met Gln Pro Pro

35 40 45

Gln Phe Gln Lys Glu Asp Ala Val Leu Ile Ile His Glu Met Leu Gln

50 55 60

Gln Ile Phe Gly Ile Leu Arg Arg Asn Phe Ser Ser Thr Gly Trp Asn

65 70 75 80

Glu Thr Val Ile Lys Thr Ile Leu Val Glu Leu Asp Gly Gln Met Asp

85 90 95

Asp Leu Glu Thr Ile Leu Glu Glu Ile Met Glu Glu Glu Asn Phe Pro

100 105 110

Arg Gly Asp Met Thr Ile Leu His Leu Lys Lys Tyr Tyr Leu Ser Ile

115 120 125

Leu Gln Tyr Leu Lys Ser Lys Glu Tyr Arg Ser Cys Ala Trp Thr Val

130 135 140

Val Gln Val Glu Ile Leu Arg Asn Phe Ser Phe Leu Asn Arg Leu Thr

145 150 155 160

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

165 170 175

Trp Ser Leu Trp Leu Leu Leu Leu Gly Leu Val Val Pro Ser Ala Ser

180 185 190

Ala Gln Ala Leu Ser Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Arg

195 200 205

Leu Asn Glu Gln Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu

210 215 220

Asp Gln Pro Pro Lys Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val

225 230 235 240

Ser Phe Thr Val Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln Pro

245 250 255

Pro Glu Leu Cys Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val

260 265 270

Gly Thr Val Thr Leu Asn Pro Ser Ile His Ser Leu Asp Ile Ser Cys

275 280 285

Asn Glu Ile Gln Ser Val Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro

290 295 300

Arg Pro Arg Pro Pro Pro Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile

305 310 315 320

Pro Pro Gly Phe Pro Pro Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg

325 330 335

<210> 12

<211> 1011

<212> DNA

<213> Artificial Sequence

<400> 12

atgagctatg atgtgcttcg ataccaacaa aggagcagca atttggcatg tcagaagctc 60

ctggaacagt tgcctgggac tcctcaatat tgcctcgaag ataggatgaa cttcgaggtc 120

cctgaggaga ttatgcaacc accacaattc cagaaggaag atgcagtatt gattatccac 180

gagatgctcc agcagatctt cggcattctc agaagaaatt tctctagcac tggctggaat 240

gaaaccgtca ttaagactat ccttgtggaa cttgatgggc agatggatga cctggagaca 300

atcctggagg aaatcatgga ggaggaaaat ttccccaggg gagacatgac cattcttcac 360

ctgaagaaat attacttgag cattctgcag tacctgaagt ccaaggagta cagaagctgt 420

gcctggacag tcgtccaagt ggaaatcctc aggaactttt ctttccttaa cagacttaca 480

gattacctcc ggaacgagac ccagagggcc agcctgtgcc tggggcgctg gtcactgtgg 540

cttctgctgc tgggactcgt ggtgccctcg gccagcgccc aggccctcag ctacagggag 600

gccgtgcttc gtgctgtgga tcgcctcaac gagcagtcct cggaagctaa tctctaccgc 660

ctcctggagc tggaccagcc gcccaaggcc gacgaggacc cgggcacccc gaaacctgtg 720

agcttcacgg tgaaggagac tgtgtgtccc aggccgaccc ggcagccccc ggagctgtgt 780

gacttcaagg agaacgggcg agtgaagcag tgtgtgggga cagtcacctt gaacccatcc 840

attcactcac tggacatctc ctgtaatgag attcagagtg tcaggagacg tccccgaccc 900

ccatatttgc caaggccaag gccacctccg tttttcccac caaggctccc accaaggatc 960

ccaccagggt tcccaccaag gttcccacca cggttccccg gaaaacggta a 1011

<210> 13

<211> 204

<212> PRT

<213> Artificial Sequence

<400> 13

Met Ser Tyr Asp Val Leu Arg Tyr Gln Gln Arg Ser Ser Asn Leu Ala

1 5 10 15

Cys Gln Lys Leu Leu Glu Gln Leu Pro Gly Thr Pro Gln Tyr Cys Leu

20 25 30

Glu Asp Arg Met Asn Phe Glu Val Pro Glu Glu Ile Met Gln Pro Pro

35 40 45

Gln Phe Gln Lys Glu Asp Ala Val Leu Ile Ile His Glu Met Leu Gln

50 55 60

Gln Ile Phe Gly Ile Leu Arg Arg Asn Phe Ser Ser Thr Gly Trp Asn

65 70 75 80

Glu Thr Val Ile Lys Thr Ile Leu Val Glu Leu Asp Gly Gln Met Asp

85 90 95

Asp Leu Glu Thr Ile Leu Glu Glu Ile Met Glu Glu Glu Asn Phe Pro

100 105 110

Arg Gly Asp Met Thr Ile Leu His Leu Lys Lys Tyr Tyr Leu Ser Ile

115 120 125

Leu Gln Tyr Leu Lys Ser Lys Glu Tyr Arg Ser Cys Ala Trp Thr Val

130 135 140

Val Gln Val Glu Ile Leu Arg Asn Phe Ser Phe Leu Asn Arg Leu Thr

145 150 155 160

Asp Tyr Leu Arg Asn Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg

165 170 175

Pro Arg Pro Pro Pro Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro

180 185 190

Pro Gly Phe Pro Pro Arg Phe Pro Pro Arg Phe Pro

195 200

<210> 14

<211> 615

<212> DNA

<213> Artificial Sequence

<400> 14

atgagctatg atgtgcttcg ataccaacaa aggagcagca atttggcatg tcagaagctc 60

ctggaacagt tgcctgggac tcctcaatat tgcctcgaag ataggatgaa cttcgaggtc 120

cctgaggaga ttatgcaacc accacaattc cagaaggaag atgcagtatt gattatccac 180

gagatgctcc agcagatctt cggcattctc agaagaaatt tctctagcac tggctggaat 240

gaaaccgtca ttaagactat ccttgtggaa cttgatgggc agatggatga cctggagaca 300

atcctggagg aaatcatgga ggaggaaaat ttccccaggg gagacatgac cattcttcac 360

ctgaagaaat attacttgag cattctgcag tacctgaagt ccaaggagta cagaagctgt 420

gcctggacag tcgtccaagt ggaaatcctc aggaactttt ctttccttaa cagacttaca 480

gattacctcc ggaacaggag acgtccccga cccccatatt tgccaaggcc aaggccacct 540

ccgtttttcc caccaaggct cccaccaagg atcccaccag ggttcccacc aaggttccca 600

ccacggttcc cctaa 615

<210> 15

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 15

Met Ser Tyr Asp Val Leu Arg Tyr Gln Gln Arg Ser Ser Asn Leu Ala

1 5 10 15

Cys Gln Lys Leu Leu Glu Gln Leu Pro Gly Thr Pro Gln Tyr Cys Leu

20 25 30

Glu Asp Arg Met Asn Phe Glu Val Pro Glu Glu Ile Met Gln Pro Pro

35 40 45

Gln Phe Gln Lys Glu Asp Ala Val Leu Ile Ile His Glu Met Leu Gln

50 55 60

Gln Ile Phe Gly Ile Leu Arg Arg Asn Phe Ser Ser Thr Gly Trp Asn

65 70 75 80

Glu Thr Val Ile Lys Thr Ile Leu Val Glu Leu Asp Gly Gln Met Asp

85 90 95

Asp Leu Glu Thr Ile Leu Glu Glu Ile Met Glu Glu Glu Asn Phe Pro

100 105 110

Arg Gly Asp Met Thr Ile Leu His Leu Lys Lys Tyr Tyr Leu Ser Ile

115 120 125

Leu Gln Tyr Leu Lys Ser Lys Glu Tyr Arg Ser Cys Ala Trp Thr Val

130 135 140

Val Gln Val Glu Ile Leu Arg Asn Phe Ser Phe Leu Asn Arg Leu Thr

145 150 155 160

Asp Tyr Leu Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg

165 170 175

Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro Phe

180 185 190

Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro Arg

195 200 205

Phe Pro Pro Arg Phe Pro

210

<210> 16

<211> 645

<212> DNA

<213> Artificial Sequence

<400> 16

atgagctatg atgtgcttcg ataccaacaa aggagcagca atttggcatg tcagaagctc 60

ctggaacagt tgcctgggac tcctcaatat tgcctcgaag ataggatgaa cttcgaggtc 120

cctgaggaga ttatgcaacc accacaattc cagaaggaag atgcagtatt gattatccac 180

gagatgctcc agcagatctt cggcattctc agaagaaatt tctctagcac tggctggaat 240

gaaaccgtca ttaagactat ccttgtggaa cttgatgggc agatggatga cctggagaca 300

atcctggagg aaatcatgga ggaggaaaat ttccccaggg gagacatgac cattcttcac 360

ctgaagaaat attacttgag cattctgcag tacctgaagt ccaaggagta cagaagctgt 420

gcctggacag tcgtccaagt ggaaatcctc aggaactttt ctttccttaa cagacttaca 480

gattacctcc ggaacggtgg cggagggagt ggtggcggag ggagtaggag acgtccccga 540

cccccatatt tgccaaggcc aaggccacct ccgtttttcc caccaaggct cccaccaagg 600

atcccaccag ggttcccacc aaggttccca ccacggttcc cctaa 645

Claims

1. A protein is shown as a sequence 7 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein: the coding region of the gene is shown as a sequence 8 in a sequence table.

4. A recombinant expression vector comprising the gene of claim 2 or 3.

5. A recombinant bacterium comprising the gene according to claim 2 or 3.

6. A complex comprising the protein of claim 1 and lentinan; in the compound, each 1.0 × 10^-9The mol protein proportion is 15 mu g lentinan.

7. The composite of claim 6, wherein: the compound consists of the protein, the lentinan and a buffer solution; 1.0X 10 of the compound per ml^-9mol of the eggWhite matter and 15 μ g of the lentinan.

8. The composite of claim 7, wherein:

the preparation method of the lentinan comprises the following steps:

(1) pulverizing dried Lentinus Edodes, and sieving to obtain powder;

9. Use of the protein of claim 1 or the complex of claim 6 or the complex of claim 7 or the complex of claim 8 for the preparation of an african swine fever vaccine.

10. An African swine fever vaccine, the active ingredient of which is the protein of claim 1 or the complex of claim 6 or the complex of claim 7 or the complex of claim 8.