CN108822219B

CN108822219B - Fusion interferon and application thereof in preparation of mucosal immunopotentiator

Info

Publication number: CN108822219B
Application number: CN201810734986.3A
Authority: CN
Inventors: 李晶; 刘文军; 范文辉; 刘丽蓉
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-07-06
Filing date: 2018-07-06
Publication date: 2020-08-18
Anticipated expiration: 2038-07-06
Also published as: CN108822219A

Abstract

The invention discloses a fusion interferon and application thereof in preparation of a mucosal immunopotentiator. The invention firstly provides a protein, which comprises the following two sections: porcine PoIFN λ 1 protein and porcine IL2 protein. The protein also includes a linker peptide between the porcine PoIFN λ 1 protein and the porcine IL2 protein. The invention also protects the application of the protein in preparing the vaccine for the porcine viral infectious diseases. The invention also protects a vaccine for porcine viral infectious diseases, which comprises the protein. The invention has great application and popularization value for preventing and controlling the porcine viral infectious diseases.

Description

Fusion interferon and application thereof in preparation of mucosal immunopotentiator

Technical Field

The invention relates to a fusion interferon and application thereof in preparation of a mucosal immunopotentiator.

Background

In the pig industry, various viral infectious diseases such as porcine reproductive and respiratory syndrome, porcine pseudorabies, swine influenza and the like are frequent. Although the pig disease vaccine is various and frequent in immunity, outbreak and epidemic of the disease cannot be effectively prevented, and the viral infectious diseases cause great economic loss to the pig industry every year. The improvement of vaccine immunity efficacy and the research and development of high-efficiency and safe swine disease prevention and treatment drugs are urgent needs in the swine industry.

Due to the actual production needs of intensive culture, more and more scientific researchers recognize the importance of mucosal immunity in the aspect of antivirus, and mucosal vaccination vaccines not only generate immune response in mucosal tissues, but also can cause systemic immune response through a common mucosal immune network, so that research on mucosal vaccines and matched adjuvants is increasingly a new hotspot.

Interferon (IFN), originally discovered in 1957 by Isaacs, UK scientists, in the study of the interference phenomenon of avian influenza virus, is a glycoprotein with broad-spectrum antiviral activity, produced by viruses and other Interferon inducers to stimulate endothelial cells, macrophages, lymphocytes and somatic cells, and has antiviral, antitumor, immunomodulatory and differentiation inducing activities. The interferon plays a role not directly acting on viruses but stimulating cells to produce various broad-spectrum antiviral proteins, and can play an antiviral role through a direct or indirect way.

Depending on the structure and receptor, mammalian IFNs can be divided into two classes: type I IFN and type II IFN. Mammalian type I IFNs are primarily acid and heat stable, highly effective antiviral activities including IFN-alpha, IFN-beta, IFN-omega, and IFN-tau, where IFN-alpha is primarily produced by leukocytes and IFN-beta is primarily produced by fibroblasts. Mammalian type II IFNs, including IFN-gamma, are produced by T cells and NK cells, are acid and heat labile, are primarily immunomodulatory, and are the major macrophage activating factor in mammals. Type III interferon (IFN-lambda) is a novel interferon, is a new member of interferon family, has the functions of type I (antivirus) and type II (immunoregulation), and plays antiviral, antitumor and immunoregulation activities after being combined with a specific receptor thereof. The type III interferon family includes IFN-lambda 1, IFN-lambda 2 and IFN-lambda 3. The functional receptor complex of IFN- λ is a heterodimer consisting of IFN- λ R1 and the IL-10R β chain, and binding of IFN- λ to the receptor induces heterodimerization of the receptor, resulting in activation of the Jak-STAT signal transduction pathway, thereby exerting biological effects similar to those of type I IFN. Many biological activities of IFN-lambda are similar to that of IFN alpha/beta which is widely applied clinically, but the receptor expression is limited, and the toxic and side effects are relatively small, so that the IFN-lambda has wide application prospects in the aspects of antivirus and antitumor.

Disclosure of Invention

The invention aims to provide a fusion interferon and application thereof in preparing a mucosal immunopotentiator.

The invention firstly provides a protein (protein A), which comprises the following two sections: porcine PoIFN λ 1 protein and porcine IL2 protein.

The protein also includes a linker peptide between the porcine PoIFN λ 1 protein and the porcine IL2 protein.

The porcine PoIFN lambda 1 protein is shown as amino acid residues at 1 st to 172 th positions of a sequence 1 in a sequence table or amino acid residues at 1 st to 172 th positions of a sequence 3 in the sequence table.

The porcine IL2 protein is shown as the amino acid residue at the 183-position 316 of the sequence 1 of the sequence table or the amino acid residue at the 183-position 316 of the sequence 3 of the sequence table.

The protein is shown as a sequence 1 in a sequence table or a sequence 3 in the sequence table.

The invention also protects a protein (protein B) which is (a1), (a2) or (a3) as follows:

(a1) a fusion protein comprising the protein A;

(a2) a fusion protein obtained by connecting a tag-containing short peptide to the end of the protein A;

(a3) the fusion protein is obtained by connecting a tag to the end of the protein A.

DNA molecules encoding any of the above proteins are also within the scope of the invention.

The DNA molecule can be specifically shown as a sequence 2 of a sequence table or a sequence 4 of the sequence table.

The invention also protects the application of any protein in the preparation of the vaccine for the porcine viral infectious diseases.

The invention also provides a vaccine for porcine viral infectious diseases, which comprises any one of the proteins.

The invention also protects the application of any one of the proteins, which is (b1) or (b 2):

(b1) as a porcine immunopotentiator;

(b2) preparing the pig immunopotentiator.

The invention also provides a product comprising any one of the proteins described above; the function of the product is as follows (b1) or (b 2):

(b1) as a porcine immunopotentiator;

(b2) preparing the pig immunopotentiator.

Any one of the swine viral infectious diseases is swine influenza 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 one of the pigs is a Changbai pig.

The invention has great application and popularization value for preventing and controlling the porcine viral infectious diseases.

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. VSV virus (vesicular stomatitis virus): china institute for veterinary medicine. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 construction of recombinant bacteria and production of target protein

Construction of recombinant plasmid

The double-stranded DNA molecule shown in the sequence 2 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector to obtain recombinant plasmid pET28a-PoIFN lambda 1-IL 2. According to the sequencing results, the recombinant plasmid pET28a-PoIFN lambda 1-IL2 was structurally described as follows: the DNA molecule shown in the sequence 2 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector. The DNA molecule shown in the sequence 2 of the sequence table codes the protein shown in the sequence 1 of the sequence table. The protein shown in the sequence 1 of the sequence table is named as the optimized PoIFN lambda 1-IL2 fusion protein. In the sequence 1 of the sequence table, the 1 st-172 th amino acid residues form the optimized porcine PoIFN lambda 1 protein, and the 183 nd-316 th amino acid residues form the optimized porcine IL2 protein.

The double-stranded DNA molecule shown in the sequence 4 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector to obtain recombinant plasmid A. According to the sequencing result, the structure of the recombinant plasmid A is described as follows: the DNA molecule shown in sequence 4 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector. The DNA molecule shown in the sequence 4 of the sequence table codes the protein shown in the sequence 3 of the sequence table. The protein shown in the sequence 3 of the sequence table is named as PoIFN lambda 1-IL2 fusion protein before optimization. In the sequence 3 of the sequence table, the 1 st to 172 th amino acid residues form the porcine PoIFN lambda 1 protein before optimization, and the 183 nd and 316 nd amino acid residues form the porcine IL2 protein before optimization.

The double-stranded DNA molecule shown in the sequence 5 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector to obtain recombinant plasmid B. According to the sequencing result, the structure of the recombinant plasmid B is described as follows: the DNA molecule shown in sequence 5 of the sequence table is inserted between NdeI and BamHI enzyme cutting sites of pET28a (+) vector. The DNA molecule shown in sequence 5 of the sequence table encodes the pig PoIFN lambda 1 protein before optimization.

Second, construction of recombinant bacteria and comparison of capability of preparing target protein

1. Obtaining of recombinant bacteria

The recombinant plasmid pET28a-PoIFN lambda 1-IL2 was introduced into E.coli BL-21(DE3) to obtain 68 recombinant strains.

2. Comparison of the ability of each recombinant bacterium to prepare a target protein

Respectively carrying out the following steps on the 68 recombinant bacteria prepared in the step 1:

(1) the recombinant bacterium single colony is inoculated to 3ml of liquid LB culture medium containing 0.1mg/ml kanamycin and is subjected to shaking culture at 37 ℃ and 200rpm for 12 hours to obtain seed liquid.

(2) Inoculating 20ml of the seed solution obtained in step (1) into 1980ml of liquid LB medium containing 0.1mg/ml kanamycin, and culturing at 37 ℃ and 200rpm with shaking until the OD of the system is reached_600nmThe value was 0.6, IPTG was added to a concentration of 1mmol/L, then 37 deg.CAnd shake-cultured at 200rpm for 4 h.

(3) After completion of step (2), the whole culture system was centrifuged to collect the cells, suspended in PBS buffer solution of pH8.0, sonicated (300W, sonication for 6s for 12s, 99 times), centrifuged at 5000g for 10 minutes, and the pellet (inclusion body) was collected.

(4) And (4) taking the precipitate obtained in the step (3), and Washing the precipitate by using Washing buffer and Resuspension buffer in sequence.

Washing buffer (pH 8.0): contains 0.5% Triton-100, 50mM Tris, 300mM NaCl, 10mM EDTA, 10mM DTT, and the balance water.

Resuspension buffer (pH8.0): 50mM Tris, 100mM NaCl, 10mM EDTA, 10mM DTT, and the balance water.

(5) After completion of step (4), the inclusion bodies were completely solubilized using a solubilization buffer, and then centrifuged at 10000g for 20 minutes at 4 ℃ to collect the supernatant.

Resolution buffer (ph 8.0): containing 6M Gua-HCl, 10% glycerol, 50mM Tris, 100mM NaCl, 10mM EDTA, 10mM DTT, and the balance water.

(6) And (4) taking the supernatant obtained in the step (5), slowly dropwise adding the supernatant into a refining buffer, and stirring at the temperature of 4 ℃ and the rpm of 200 for 24 hours.

Refolding buffer (pH8.0): contains 100mM Tris, 400mM L-Arg HCl, 2mM EDTA, 5mM GSSG, and water in balance.

(7) And (3) collecting the whole system which finishes the step (6), concentrating at 4 ℃, adding the molecular sieve buffer solution to 100ml when the concentration is about 20ml, then continuing to concentrate at 4 ℃, and concentrating to 4ml, thus obtaining the concentrated solution.

Molecular sieve buffer (ph 8.0): containing 20mM Tris, 150mM NaCl, the balance being water.

(8) Taking 1ml of the concentrated solution obtained in step (7), centrifuging at 12000rpm for 10min at 4 ℃, and purifying by using Superdex 7510/300GL molecular sieve chromatographic column.

The column volume is 24 ml; the filler is Sephadex G15100-200.

Eluent (ph 8.0): containing 20mM Tris, 150mM NaCl, the balance being water.

Eluent flow rate: 0.3 ml/min.

Collecting the solution after passing through the column with the retention volume of 12.8ml to 15.1ml, namely the target protein solution.

Respectively carrying out polyacrylamide gel electrophoresis on target protein solutions obtained by carrying out the steps on 68 recombinant bacteria, and displaying a single band, namely obtaining the electrophoretically pure target protein. The target protein is optimized PoIFN lambda 1-IL2 fusion protein.

The protein concentration of the target protein solution obtained by subjecting each recombinant bacterium to the above steps was detected, the protein concentration of the target protein solution obtained from 67 recombinant bacteria was 3.7-4.0mg/ml, and the protein concentration of the target protein solution obtained from 1 recombinant bacterium was 24.1mg/ml (the recombinant bacterium was named as Escherichia coli BL21/pET28a-PolFN lambda 1-IL 2).

3. Preservation of recombinant bacteria

Escherichia coli (Escherichia coli) BL21/pET28a-PolFN lambda 1-IL2, which has been deposited in China general microbiological culture Collection center (CGMCC, address: Beijing Shangyang district Beijing Siro No.1 Hospital No. 3) on 08 m 2018, and the deposit number is CGMCC No. 15920.

Thirdly, construction of recombinant bacteria and comparison of capability of preparing target protein

1. Obtaining of recombinant bacteria

The recombinant plasmid A was introduced into E.coli BL-21(DE3) to obtain 20 recombinant strains.

And (3) respectively detecting the 20 recombinant bacteria prepared in the step (1) by the same method as the step (2) in the step (II).

Respectively carrying out polyacrylamide gel electrophoresis on the target protein solutions obtained by carrying out the steps on the 20 recombinant bacteria, and displaying a single band, namely obtaining the electrophoretically pure target protein. The target protein is optimized PoIFN lambda 1-IL2 fusion protein.

And detecting the protein concentration in the target protein solution obtained by carrying out the steps on each recombinant bacterium, wherein the protein concentration in the target protein solution obtained by 20 recombinant bacteria is between 2.5 and 2.7 mg/ml.

And (3) one recombinant bacterium with the strongest capability of preparing the target protein in the 20 recombinant bacteria is named as recombinant bacterium A.

Fourth, construction of recombinant bacteria and comparison of capability of recombinant bacteria to prepare target protein

1. Obtaining of recombinant bacteria

The recombinant plasmid B was introduced into E.coli BL-21(DE3) to obtain 20 recombinant strains.

Respectively carrying out polyacrylamide gel electrophoresis on the target protein solutions obtained by carrying out the steps on the 20 recombinant bacteria, and displaying a single band, namely obtaining the electrophoretically pure target protein. The target protein is optimized pre-pig PoIFN lambda 1 protein.

And detecting the protein concentration in the target protein solution obtained by carrying out the steps on each recombinant bacterium, wherein the protein concentration in the target protein solution obtained by 20 recombinant bacteria is 1.1-1.3 mg/ml.

And (3) one recombinant bacterium with the strongest capability of preparing the target protein in the 20 recombinant bacteria is named as recombinant bacterium B.

The results of the second step, the third step and the fourth step show that: by adopting the same expression vector and host bacteria, the yield of the PoIFN lambda 1-IL2 fusion protein before optimization is obviously higher than that of the PoIFN lambda 1 protein before optimization, and the yield of the PoIFN lambda 1-IL2 fusion protein after optimization is obviously higher than that of the PoIFN lambda 1-IL2 fusion protein before optimization.

Example 2 function of target protein (animal test verification)

The test animals were long white pigs (40 days old, 10kg of each body weight).

The optimized PoIFN λ 1-IL2 fusion protein used in this example was prepared in step two of example 1 using Escherichia coli BL21/pET28a-PolFN λ 1-IL 2. The pre-optimized PoIFN lambda 1-IL2 fusion protein used in this example was prepared using recombinant bacteriA in step three of example 1. The optimized porcine PoIFN λ 1 protein used in this example was prepared by using the recombinant bacterium b in step four of example 1.

The composition I consists of optimized PoIFN lambda 1-IL2 fusion protein, white oil and diluent, and is fully mixed and emulsified. The composition II consists of PoIFN lambda 1-IL2 fusion protein before optimization, white oil and diluent, and is fully mixed and emulsified. The composition III consists of the optimized pig PoIFN lambda 1 protein, white oil and diluent, and is fully mixed and emulsified. The white oil functions as an adjuvant, SEPPIC s.a, montainide ISA 11R VG. The dilution was PBS buffer, pH 7.2.

The test animals were divided into 7 groups of 24 animals each and treated separately as follows (all given in a single dose):

a first group: through nasal drip, each test animal is given 1mL of composition I, the optimized administration dose of PoIFN lambda 1-IL2 fusion protein is 0.1mg/kg of test animal, and the administration dose of white oil is 0.1mg/kg of test animal;

second group: administering 1mL of composition I to each test animal by intramuscular injection, wherein the optimized administration dose of PoIFN lambda 1-IL2 fusion protein is 0.1mg/kg of test animal, and the administration dose of white oil is 0.1mg/kg of test animal;

third group: by nasal drip, each test animal is given 1mL of composition II, the dosage of PoIFN lambda 1-IL2 fusion protein before optimization is 0.1mg/kg test animal, and the dosage of white oil is 0.1mg/kg test animal;

and a fourth group: administering 1mL of composition II to each test animal by intramuscular injection, wherein the dosage of PoIFN lambda 1-IL2 fusion protein before optimization is 0.1mg/kg of test animal, and the dosage of white oil is 0.1mg/kg of test animal;

and a fifth group: by nasal drip, each test animal is given 1mL of composition III, the administration dose of the PoIFN lambda 1 protein of the pig before optimization is 0.1mg/kg of test animal, and the administration dose of white oil is 0.1mg/kg of test animal;

a sixth group: administering 1mL of composition III to each test animal by intramuscular injection, wherein the administration dose of PoIFN lambda 1 protein to the pig before optimization is 0.1mg/kg of test animal, and the administration dose of white oil is 0.1mg/kg of test animal;

a seventh group: each test animal was given 1mL of physiological saline by nasal drip.

The day of administration was taken as test day 1 and challenge was performed on test day 21. The challenge was achieved by intranasal inoculation, and each animal was inoculated with 1ml of virus solution. The virus liquid is swine influenza virus A/California/04/2009A (H1N1), and the content of swine influenza virus A/California/04/2009A (H1N1) in 1ml of the virus liquid is 1000 PFU.

On the 24 th to 26 th days of the test, the influenza incidence rate of each group is counted. Pigs showing more than two of the following three symptoms during this time period were judged as sick pigs: firstly, no food is taken; ② difficult breathing; body temperature is above 40.5 ℃.

Influenza incidence in the first group was 8.33%. Influenza incidence in the second group was 12.5%. Influenza incidence in the third group was 20.83%. Influenza incidence in the fourth group was 25%. Influenza incidence in the fifth group was 41.67%. Influenza incidence in the sixth group was 50%. Influenza incidence in group seven was 100%.

Example 3 detection of Interferon Activity

Cell culture solution: DMEM medium containing 10% FBS.

First, interferon activity detection method

1. Cell preparation

Collecting well-grown PK-15 cells, digesting, and suspending with cell culture solution to obtain cell concentration of 5 × 10⁵Cell suspension per mL; the cell suspension was added to a 96-well cell culture plate (100. mu.l/well) at 37 ℃ with 5% CO₂Culturing for 8-10h under the condition to obtain monolayer cells.

2. The cell culture solution is used as a solvent, a substance to be detected is dissolved or diluted to ensure that the protein concentration is 0.001mg/ml, the solution is used as a mother solution, and the mother solution is subjected to 4-time gradient dilution to obtain 6 gradients, so that various dilutions are obtained.

3. Taking the cell culture plate which is finished with the step 1, and sucking and removing the supernatant; cell culture medium (100. mu.l/well) was added to 3 positive control wells, cell culture medium (100. mu.l/well) was added to 3 negative control wells, and the dilution obtained in step 2 was added to the other wellsSolutions (100. mu.l/well) with 3 multiple wells per dilution; then 5% CO at 37 ℃₂Culturing for 12-15h under the condition.

4. Diluting VSV virus with serum-free DMEM culture solution to obtain virus concentration of 1000TCID₅₀Viral dilutions per ml.

5. Taking the cell culture plate after the step 3, and sucking and removing the supernatant; adding the virus diluent (100 mu l/hole) prepared in the step 4 into 3 positive control holes, adding serum-free DMEM culture solution (100 mu l/hole) into 3 negative control holes, and adding the virus diluent (100 mu l/hole) prepared in the step 4 into other holes; then 5% CO at 37 ℃₂Culturing for 24h under the condition.

6. The cell culture plate after completion of step 5 was taken, the supernatant was aspirated off, and crystal violet staining solution (100. mu.l/well) was added thereto, followed by standing at room temperature for 30 min.

7. And (3) taking the cell culture plate which finishes the step 6, sucking and removing the supernatant, washing with double distilled water, adding a destaining solution (100 mu l/hole), and standing at room temperature for 10 min.

8. OD determination by means of enzyme-linked immunosorbent assay_570nmAnd (6) value and recording.

The content of interferon capable of inhibiting 50% of cytopathic effect is defined as an activity unit, and the interferon titer, namely the dilution factor capable of inhibiting 50% of cytopathic effect, is calculated by using a Reed-Muench method, and the result is shown in Table 1.

TABLE 1 calculation of interferon potency Using the Reed-Muench method

X: OD of negative control well₅₇₀Average value;

y: OD of Positive control well₅₇₀Average value.

The values are calculated from the above table, and finally the interferon titer is calculated from the G term according to the following formula (assuming that the calculated value of G4 is greater than 0.5 and the calculated value of G5 is less than 0.5 in the above calculation):

the interferon titer (U/0.001mg) of the substance to be tested was × 4 pre-dilution times^{(4+(G4-0.5)/(G4-G5))}。

Secondly, the interferon activity of the optimized PoIFN lambda 1-IL2 fusion protein

The optimized PoIFN lambda 1-IL2 fusion protein used in the step II is prepared by using Escherichia coli BL21/pET28a-PolFN lambda 1-IL2 in the step II of example 1.

The substances to be tested were as follows: freshly prepared optimized PoIFN λ 1-IL2 fusion protein (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 4 ℃ -8 ℃ for 6 months (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 4 ℃ -8 ℃ for 12 months (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 4 ℃ -8 ℃ for 18 months (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 4 ℃ -8 ℃ for 24 months (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 25 ℃ for 1 month (three batches), storing optimized PoIFN λ 1-IL2 fusion protein at 25 ℃ for 3 months (three batches), and storing optimized PoIFN λ 1-IL2 fusion protein at 25 ℃ for 6 months (three batches).

And (5) detecting the interferon activity according to the method of the step one.

The interferon titer of the newly prepared optimized PoIFN lambda 1-IL2 fusion protein is 2 × 10⁵U/mg。

The interferon titres of the substances to be tested are shown in Table 2.

TABLE 2

SEQUENCE LISTING

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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

1. A protein, which consists of the following three segments from N end to C end in sequence: porcine PoIFN λ 1 protein, linker peptide, and porcine IL2 protein;

the porcine PoIFN lambda 1 protein is shown as amino acid residues at 1 st to 172 th positions in a sequence 1 of a sequence table;

the porcine IL2 protein is shown as amino acid residue at the 183-316 position in the sequence 1 of the sequence table.

2. The protein of claim 1, wherein: the protein is shown as a sequence 1 in a sequence table.

3. A protein which is (a1) or (a3) as follows:

(a1) a fusion protein comprising the protein of claim 1 or 2;

(a3) a fusion protein obtained by attaching a tag to the end of the protein of claim 1 or 2.

4. A DNA molecule encoding the protein of any one of claims 1 to 3.

5. Use of a protein according to any one of claims 1 to 3 for the preparation of a vaccine for porcine viral infectious disease.

6. A vaccine for porcine viral infectious disease comprising the protein of any one of claims 1 to 3.

7. Use of a protein according to any one of claims 1 to 3 for the preparation of a porcine immunopotentiator.

8. A product comprising a protein according to any one of claims 1 to 3; the function of the product is as follows (b1) or (b 2):

(b1) as a porcine immunopotentiator;

(b2) preparing the pig immunopotentiator.