CN106674354B

CN106674354B - Fusion protein of chicken interferon IFN-lambda and IFN-alpha

Info

Publication number: CN106674354B
Application number: CN201710078269.5A
Authority: CN
Inventors: 任涛; 丁诗月; 谢鹏
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-02-14
Filing date: 2017-02-14
Publication date: 2020-10-16
Anticipated expiration: 2037-02-14
Also published as: CN106674354A

Abstract

The invention belongs to the technical field of biological engineering, and relates to gene fusion expression of interferon fusion preparations of chicken lambda and alpha, a production method and clinical application thereof. The bioengineering interferon fusion preparation is characterized in that a CEF cell total RNA is extracted, a specific primer is designed, chicken lambda and alpha interferon genes are cloned and fused through a complementary hydrophobic flexible amino acid joint to obtain a complete chicken lambda and alpha interferon fusion gene, and the complete chicken lambda and alpha interferon fusion gene is cloned to a 19-T vector to ensure that a large amount of cloning expression of the chicken lambda and alpha interferon fusion gene is successful and then is connected with a pET-32a vector to express a large amount of chicken lambda and alpha interferon genes; identifying the product, and performing denaturation, purification and renaturation after determining that the product is expressed in an inclusion body; and (3) detecting the antiviral activity of the compound and evaluating the clinical application effect. The invention successfully constructs the recombinant fusion chicken lambda and alpha interferon prokaryotic expression plasmid pET32 a-chIFN-lambda + alpha and realizes the 1:1 fusion expression of the chicken lambda and alpha interferon on an escherichia coli prokaryotic expression system.

Description

Fusion protein of chicken interferon IFN-lambda and IFN-alpha

Technical Field

The invention belongs to gene fusion expression in the technical field of bioengineering, and particularly relates to a fusion chicken lambda and alpha interferon gene and a preparation method and application thereof.

Background

Interferon (IFN) is a cytokine with broad-spectrum antiviral, antitumor and immune-enhancing functions produced by host cells under the action of a specific inducer, and is essentially a secreted multifunctional glycoprotein. The interactions between viruses were first discovered in 1957 by lsaacs, a scientist in the united kingdom, and are proteins in nature, which are induced by the virus, interfere with viral replication, and limit viral infection. According to the source, amino acid sequence and biological activity of interferon, the interferon is divided into three categories, i type interferon, including IFN-alpha, IFN-beta, IFN-kappa, IFN-omega and IFN-tau, wherein the research of IFN-alpha and IFN-beta is more mature; the type II interferon is IFN-gamma, but the function of the type II interferon is mainly to regulate the immune function, and the antiviral effect is not strong; type III interferons include IFN-. lambda.1 (IL-29), IFN-. lambda.2 (IL-28A), IFN-. lambda.3 (IL-28B) and IFN-. lambda.4 newly discovered in 2013. IFN-alpha belongs to type I interferon and is composed of 582 nucleic acids, and after 93 signal peptides are removed, 162 amino acids are encoded. IFN-alpha of different breeders is relatively conserved, and the nucleotide homology is more than 99.3 percent. Although not directly acting on virions, type i interferons have been one of the research hotspots because they rapidly induce host cells to produce antiviral proteins. Research shows that the chicken IFN-alpha can inhibit the propagation of the H5N1 virus and has certain influence on the immunity of the Newcastle disease vaccine. IFN- λ (interferon λ), also known as type III interferons, is a novel class of interferons found after interferons I, II. Through combination with a specific heterodimer complex receptor (IFN-lambda R1/IL-10R2), JAK-STAT signal pathway is activated, thereby exerting antiviral effect. Because of the receptor specificity, IFN-lambda has other advantages different from type I interferon, human IFN-lambda is increasingly researched, but chicken IFN-lambda is still shallow, and a small amount of research shows that the chicken IFN-lambda can inhibit the proliferation of influenza virus in a tracheal ring experiment. There is no example of tandem expression of these two chicken interferons and testing for antiviral activity.

Newcastle Disease (ND) is an acute, highly contagious Disease of chickens and various birds caused by Newcastle Disease virus. The main pathogenesis of the disease is characterized by dyspnea, nervous disorder, diarrhea, mucosal and serosal hemorrhage. Because of the different strains of newcastle disease, the severity of newcastle disease animals varies greatly. The disease is spread rapidly, the death rate is very high, the poultry industry is seriously damaged, and huge loss is caused to the world economy.

Disclosure of Invention

In order to make up for the defects of the two interferons, the complementary advantages of the two interferons are realized. The fusion protein of the chicken interferon IFN-lambda and IFN-alpha is simple and convenient to produce, low in cost, high in activity and good in treatment effect on newcastle disease and influenza.

The first purpose of the invention is to provide the chicken IFN-lambda and IFN-alpha fusion protein.

Another purpose of the invention is to provide a production method of the fusion protein and a preparation method of prokaryotic expression plasmid pET32a-IFN lambda-linker-IFN alpha.

The invention also aims to provide the application and the effect of the fusion protein in inhibiting virus propagation on cells and clinically treating animal viral epidemic diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the purpose of the invention is realized by the following technical scheme: a fusion gene of chicken IFN-lambda and IFN-alpha (IFN lambda-linker-IFN alpha) has the following nucleotide sequence:

CAGGTCACCCCGAAGAAGAGCTGCAGCCTCTCCAAGTACCAGTTCCCTGCACCTTTGGAGTTGAAGGCAGTGTGGAGGATGAAGGAGCAGTTTGAAGACATCATGCTGTTAACAAACAGAAAATGCAACACCAGACTCTTCCATCGGAAGTGGGACATAGCTGAGCTGTCGGTACCTGACCGAATCACCCTGGTGGAGGCTGAGCTGGACCTCACCATCACCGTGCTCACAAACCCCACAACCCAGAGACTGGCAGAGACGTGCCAACAGCCCCTGGCCTTCCTTACCCAAGTCCAGGAGGACCTGCGAGACTGCTTGGCCCTCGAGGCACCTTCACATCAGCCCTCTGGGAAACTGAGGCACTGGCTGCAGAAGCTGGAGACAGCCAAGAAGAAGGAGACCGCCGGCTGCCTGGAGGCCTCAGCCATCCTCCACATCTTCCAAGTACTGAACGACCTGCGGTGCGCAGCCCAGCGCGAGGATTGCACTTCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTCGCCCCCAGGATGCCACCTTCTCTCACGACAGCCTCCAGCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTGCTCCTTCAACGACACCATCCTGGACACCAGCAACACCCGGCAAGCCGACAAAACCACCCACGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAACGACAGCCAACGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCAATGCTTGGACAGCAGCGACACGCGCTCCCGGACGCGATGGCCTCGCAACCTTCACCTCACCATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAACGATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAACACGCGCACTTAG

the chicken IFN-lambda and IFN-alpha fusion gene is formed by fusing two interferon genes through an overlap extension PCR (sequence assisted polymerase chain reaction, SOE-PCR), removing a signal peptide after the two interferon genes are amplified respectively, connecting the chicken IFN-lambda and the IFN-alpha through a hydrophobic flexible amino acid linker (linker) (G4S)3, cloning to a prokaryotic expression vector pET32a (+), and detecting the antiviral activity in vivo and in vitro respectively after the chicken IFN-lambda and the IFN-alpha are expressed and purified through a BL21 system.

The chicken IFN-lambda and IFN-alpha fusion gene is obtained by the following steps:

(1) primer design

The primer design is that according to the gene sequence of chicken IFN-lambda (GenBank No. KF680102.1) and the gene sequence of IFN-alpha (GenBank No. AM049251.1), signal peptide is predicted and removed through SignalP, two pairs of primers are designed, and two enzyme cutting sites of EcoR1 and Hind3 are respectively inserted at the upstream and the downstream, namely IFN-lambda-F, IFN-lambda-R1, IFN-alpha-F1 and IFN-alpha-R. Then 2 primers IFN-lambda R2 and IFN-alpha F2 containing complementary hydrophobic flexible amino acid linkers were designed downstream of IFN-lambda and upstream of IFN-alpha, respectively. The sequence is as follows:

IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA

IFN-λ-R1：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC

IFN-λ-R2：

CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC

IFN-α-F1：CCGGAATTCTGCAACCACCTTC

IFN-α-F2：

TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC

IFN-α-R：CCCAAGCTTCTAAGTGCGCGTGTTGCC

(2) gene cloning and identification

Self-made Chick Embryo Fibroblast (CEF) cells by SPF chick embryos, use a Newcastle disease GM strain to challenge, extract RNA, and reverse to obtain a template cDNA of amplified chick IFN-lambda and IFN-alpha genes, wherein IFN-lambda-F, IFN-lambda-R1, IFN-alpha-F1 and IFN-alpha-R primers are used to amplify gene fragments of the chick IFN-lambda and IFN-alpha with signal peptide removed.

Then, the chicken IFN-lambda and IFN-alpha gene are fused by an SOE-PCR method: firstly, amplifying two gene segments containing a linker by using IFN-lambda-F, IFN-lambda-R2, IFN-alpha-F2 and IFN-alpha-R primers respectively; secondly, taking the gel recovery product of the first step as a template, and performing PCR amplification for 10-15 cycles without adding a primer; and step three, taking the PCR product of the step two as a template, and amplifying PCR by using IFN-lambda-F and IFN-alpha-R to obtain products, namely chicken IFN-lambda and IFN-alpha gene fragments.

Construction of prokaryotic expression vector of chicken IFN-lambda and IFN-alpha fusion protein (chIFN-lambda + alpha)

(1) Cloning of the fusion Gene into the pMD-19T vector

Connecting the fusion gene of chicken IFN-lambda and IFN-alpha to pMD-19T vector, connecting for more than 4h at 16 ℃, transforming to an agar plate containing benzyl amine, selecting monoclonal bacteria, putting the monoclonal bacteria into LB culture medium, shaking bacteria, identifying positive clone by bacteria liquid PCR, performing amplification culture after correct sequencing, and extracting plasmid pMD-19T-chIFN-lambda + alpha.

(2) pET32 a-chIFN-lambda + alpha recombinant plasmid construction

Respectively carrying out double enzyme digestion on pMD-19T-chIFN-lambda + alpha and pET32a empty carriers by using EcoR1 and Hind3 fast cutting enzymes, recycling glue, connecting the glue by using T4 ligase at normal temperature for more than 30min, transforming the glue into an agar plate containing aminobenzyl, selecting monoclonal bacteria, putting the monoclonal bacteria into an LB culture medium for shaking the bacteria, carrying out PCR (polymerase chain reaction) on the bacteria liquid to identify positive clones, carrying out amplification culture after correct sequencing, and extracting a recombinant plasmid pET32 a-chIFN-lambda + alpha.

The invention adopts a genetic engineering method to fuse chicken IFN-lambda and IFN-alpha, and has the following beneficial effects:

(1) the invention utilizes the receptor distribution difference rule of I and III type interferons and fuses the two by using a genetic engineering method, thereby being beneficial to comprehensively exerting the effects of the two interferons at a higher level;

(2) the invention successfully extracts chIFN-lambda + alpha fusion protein from the inclusion body, and can extract protein with higher concentration after denaturation, revivification and purification, thereby laying a good foundation for subsequent experiments. (ii) a

(3) The extracted fusion protein chIFN-lambda + alpha has good antiviral effect in vivo and in vitro experiments.

Drawings

FIG. 1 is a PCR-amplified agarose nucleic acid electrophoresis picture of chicken IFN-lambda gene; wherein, Lane M is DNA markerDL2000, Lane 1 is a negative control, and Lane 2 is the chIFN-lambda gene amplification result.

FIG. 2 is a PCR-amplified agarose nucleic acid electrophoresis picture of chicken IFN-alpha gene; wherein, Lane M is DNA markerDL5000,

Lanes

1 and 2 are chIFN-alpha gene amplification results, and Lane 3 is a negative control.

FIG. 3 is a diagram of the agarose nucleic acid electrophoresis of the CHIFN-. lambda. + alpha gene amplified by SOE-PCR; wherein, Lane M is DNAmarker DL2000, Lane 1 is the chIFN-lambda + alpha gene amplification result, and Lane 2 is the negative control.

FIG. 4 is a graph showing the results of SDS-PAGE identifying chIFN-. lambda. + alpha protein expression; wherein, Lane M is a low molecular weight protein Marker, Lane 1 is pET-32a empty vector control, Lane 2 is chIFN-lambda + alpha supernatant, Lane 3 is chIFN-lambda + alpha inclusion body precipitate.

FIG. 5 is a diagram showing the result of identifying chIFN-lambda + alpha protein expression by Western blot; wherein, Lane M is a low molecular weight protein Marker, Lane 1 is pET-32a empty vector control, Lane 2 is chIFN-lambda + alpha supernatant, Lane 3 is chIFN-lambda + alpha inclusion body precipitate.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The invention is not so limited.

Example 1 construction of prokaryotic expression vector of fusion gene of chicken IFN-lambda and IFN-alpha (chIFN-lambda + alpha)

(1) Design and synthesis of related primers

IFN-λ-F：CCGGAATTCCAGGTCACCCCGAAGAA

IFN-λ-R1：CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC

IFN-λ-R2：

CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC

IFN-α-F1：CCGGAATTCTGCAACCACCTTC

IFN-α-F2：

TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC

IFN-α-R：CCCAAGCTTCTAAGTGCGCGTGTTGCC

(2) gene cloning and identification

Taking 10-day-old SPF chick embryos, removing heads, limbs and internal organs by using tweezers, cutting chicken, digesting the chicken by using pancreatin for 4min, filtering to prepare CEF cells, after passage once, using a Newcastle disease GM strain to detoxify, after 12h, extracting RNA, reversing to obtain a template cDNA of amplified chicken IFN-lambda and IFN-alpha genes, and amplifying by using IFN-lambda-F, IFN-lambda-R1, IFN-alpha-F1 and IFN-alpha-R primers, wherein the reaction system is as follows: 25 μ l Premix ExTaq enzyme, 2.5 μ l template DNA (cDNA), 0.5 μ l upstream primer, 0.5 μ l downstream primer, and distilled water to make up to 50 μ l. The reaction procedure was as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 40s, extension at 72 ℃ for 7min, with 35 cycles of steps 2 to 4. The sizes of the nucleic acid electrophoresis bands are about 561bp and 582bp respectively.

Then, the chicken IFN-lambda and IFN-alpha gene are fused by an SOE-PCR method: firstly, amplifying an IFN-lambda-linker fragment containing a linker by using IFN-lambda-F and IFN-lambda-R2 primers respectively, amplifying an IFN-lambda-linker fragment containing a linker by using IFN-alpha-F2 and IFN-alpha-R primers respectively, and carrying out gel recovery on the two fragments respectively; and step two, taking the recovered product of the first step of gel as a template, mixing the template in an equimolar way, not using a primer, carrying out PCR amplification, and carrying out a reaction system as follows: mu.l of Premix ExTaq enzyme, 8.4. mu.l of distilled water, and the first step gel recovery product were mixed equimolar for a total of 1.6. mu.l, and the reaction procedure was as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 1min, annealing at 60 ℃ for 30s, extension at 72 ℃ for 40s, and extension at 72 ℃ for 7min, wherein 15 cycles of the steps 2 to 4 are performed; and thirdly, taking the PCR product of the second step as a template, and amplifying PCR by using IFN-lambda-F and IFN-alpha-R to obtain a product, namely the chIFN-lambda + alpha gene fragment.

(3) Cloning of the fusion Gene into the pMD-19T vector

Connecting the chIFN-lambda + α fusion gene to pMD-19T vector, wherein the reaction system comprises 5.0 mul of LigationSolution I, 0.5 mul of pMD 19-T vector, 4.5 mul of chIFN-lambda + α PCR recovery product, connecting for more than 4h at 16 ℃, carrying out competent transformation to an agar plate containing benzyl ammonia by DH5 α, standing overnight at 37 ℃, picking up a monoclonal bacterium, placing the monoclonal bacterium in an ampicillin-containing LB culture medium, shaking for 6-8h, identifying a positive clone by bacterium liquid PCR, and the reaction system comprises 7.5 mul of Premix rTaq enzyme and 6.1 mul of ddH₂O, 0.2 mul upstream primer IFN-lambda-F, 0.2 mul downstream primer IFN- α -R and 1.0 mul bacterial liquid, the reaction program is that pre-denaturation at 94 ℃ is carried out for 4min, denaturation at 94 ℃ is carried out for 1min, annealing at 55 ℃ is carried out for 30s, extension at 72 ℃ is carried out for 40s, extension at 72 ℃ is carried out for 7min, wherein 30 cycles from the 2 nd step to the 4 th step are carried out, positive samples are selected and identified, the sequencing is carried out, amplification culture is carried out after the sequencing is correct, plasmids are extracted

pMD-19T-chIFN-λ+α。

(4) pET32 a-chIFN-lambda + alpha recombinant plasmid construction

pMD-19T-chIFN-. lambda. + α and pET32a were digested separately with EcoR1 and Hind3 fast-cutting enzymes in a double digestion reaction system of 1.5. mu.l EcoR1 enzyme, 1.5. mu.l Hind3 enzyme, 5. mu.l 10 × Buffer, 2-5. mu.g plasmid, ddH₂Supplementing O to 50 mu l, carrying out water bath at 37 ℃ for 30min, recycling glue, adding T4 ligase, adding 8 mu l of 1 mu l T4 ligase and 1 mu l T4 ligase into enzyme digestion products according to the proportion of pMD-19T-chIFN-lambda + α: pET32a empty vector which is 3:1, connecting Buffer at 1 mu l T4, connecting at normal temperature for more than 30min, transforming to an agar plate containing benzyl, selecting monoclonal bacteria, putting the monoclonal bacteria into an ampicillin-containing LB culture medium for shaking, identifying positive clones by bacteria liquid PCR, sending sequencing, carrying out amplification culture after correct sequencing, and extracting recombinant plasmid pET32 a-chIFN-lambda + α.

Example 2 protein expression and characterization

The recombinant plasmid pET32 a-chIFN-lambda + alpha is subjected to competence transformation by BL21, after the amplification culture of monoclonal bacteria, ampicillin LB culture medium is inoculated according to the proportion of 1:100, shaking culture is carried out at 37 ℃ until the OD600nm value is about 0.6, IPTG is added for induction expression, bacteria liquid is centrifuged, supernatant is discarded, PBS is resuspended and centrifugally cleaned for 2 times, an ultrasonication instrument 200W is used for cracking for 15min, precipitates are centrifugally separated at 4 ℃, precooled PBS is used for cleaning for 2 times, an inclusion body is dissolved by a lysine equivalent disruption Buffer containing 8M urea, the mixture is incubated at room temperature for 60min, insoluble impurities are centrifugally removed at 4 ℃, the supernatant is filtered by a 0.45 mu M filter, the mixture is combined with a Ni column, imidazole eluent with different concentrations is used for Elution until the OD280 value is 0, and finally an Elution Buffer containing 250mmol/L imidazole is used for Elution, and filtrate is collected. The filtrate was placed in dialysis bags treated with EDTA, and subjected to gradient dialysis with 8M,6M,4M,2M,0M and PBS, each at 12h intervals, and finally identified by SDS-PAGE.

Example 3 in vitro antiviral Activity identification

DF-1 cells were plated into 96-well plates to 80% -90% length, and chIFN-. lambda. + α was diluted in 4-fold gradient 100. mu.l added to each well from 4 separately^-1-4^-10And 24 repeats of each gradient, incubating at 37 ℃ for 12h, adding 100TCID50VSV, NDV and AIV viruses respectively, incubating at 37 ℃ for 1h in 100 mul per well, discarding the virus solution, washing with PBS 2 times, changing with 2% serum DMEM, placing at 37 ℃ for 48h, and detecting the virus content, wherein the results show that chIFN-lambda + α has good protective capacity on DF-1 cells and good VSV, NDV and AIV antiviral activities on DF-1 cells in vitro, 3.2 x 10⁴IU/mg，4.6*10⁴IU/mg，1.5*10⁵IU/mg。

Example 4 in vivo antiviral Activity identification

chIFN-. lambda. + alpha antiviral activity was tested on 2 week old SPF chickens. After the SPF chickens of 2 weeks old are intravenously injected with 100 mu gchIFN-lambda + alpha for 4h, the SPF chickens are intravenously injected again with the same amount, and then are injected with Newcastle disease virus after 2 h. Collecting throat swabs and cloaca swabs in 1, 3, 5, 7, 9 and 11 days later, and detecting and detoxifying by using common chick embryo; observing the feeding and mental conditions of the chickens every day; the death was recorded. The results show that: the protection rate of the chIFN-lambda + alpha group on the SPF chicken can reach 25 percent, while the survival rate of the challenge control group is only 8 percent; in addition, the results of the throat swab and the cloaca swab show that the detoxification of the chIFN-lambda + alpha group can be delayed for 3-7d compared with a detoxification control group, and the detoxification amount is obviously reduced; SPF chickens in the chIFN-lambda + alpha group normally feed and have good spirits.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

South China Agricultural University

Fusion protein of chicken interferon IFN-lambda and IFN-alpha

(1) Chicken IFN-lambda + alpha gene

CAGGTCACCC CGAAGAAGAG CTGCAGCCTC TCCAAGTACC AGTTCCCTGC ACCTTTGGAG 60

TTGAAGGCAG TGTGGAGGAT GAAGGAGCAG TTTGAAGACA TCATGCTGTT AACAAACAGA 120

AAATGCAACA CCAGACTCTT CCATCGGAAG TGGGACATAG CTGAGCTGTC GGTACCTGAC 180

CGAATCACCC TGGTGGAGGC TGAGCTGGAC CTCACCATCA CCGTGCTCAC AAACCCCACA 240

ACCCAGAGAC TGGCAGAGAC GTGCCAACAG CCCCTGGCCT TCCTTACCCA AGTCCAGGAG 300

GACCTGCGAG ACTGCTTGGC CCTCGAGGCA CCTTCACATC AGCCCTCTGG GAAACTGAGG 360

CACTGGCTGC AGAAGCTGGA GACAGCCAAG AAGAAGGAGA CCGCCGGCTG CCTGGAGGCC 420

TCAGCCATCC TCCACATCTT CCAAGTACTG AACGACCTGC GGTGCGCAGC CCAGCGCGAG 480

GATTGCACTg gtggaggagg cTCTGGTGGA GGCGGTAGCG GAGGCGGAGG GTCGTGCAAC 540

CACCTTCGCC CCCAGGATGC CACCTTCTCT CACGACAGCC TCCAGCTCCT CCGGGACATG 600

GCTCCCACAC TACCCCAGCT GTGCCCACAG CACAACGCGT CTTGCTCCTT CAACGACACC 660

ATCCTGGACA CCAGCAACAC CCGGCAAGCC GACAAAACCA CCCACGACAT CCTTCAGCAC 720

CTCTTCAAAA TCCTCAGCAG CCCCAGCACT CCAGCCCACT GGAACGACAG CCAACGCCAA 780

AGCCTCCTCA ACCGGATCCA CCGCTACACC CAGCACCTCG AGCAATGCTT GGACAGCAGC 840

GACACGCGCT CCCGGACGCG ATGGCCTCGC AACCTTCACC TCACCATCAA AAAACACTTC 900

AGCTGCCTCC ACACCTTCCT CCAAGACAAC GATTACAGCG CCTGCGCCTG GGAACACGTC 960

CGCCTGCAAG CTCGTGCCTG GTTCCTGCAC ATCCACAACC TCACAGGCAA CACGCGCACT1020

TAG 1023

(2) Chicken IFN-lambda gene

ATGGTATGCT ACGGGGTCAC AATTATTTTG GTGGGGACCC TGGGGTCCCT CCTGGTGGGT 60

GCCTTCCCCC AGGTCACCCC GAAGAAGAGC TGCAGCCTCT CCAAGTACCA GTTCCCTGCA 120

CCTTTGGAGT TGAAGGCAGT GTGGAGGATG AAGGAGCAGT TTGAAGACAT CATGCTGTTA 180

ACAAACAGAA AATGCAACAC CAGACTCTTC CATCGGAAGT GGGACATAGC TGAGCTGTCG 240

GTACCTGACC GAATCACCCT GGTGGAGGCT GAGCTGGACC TCACCATCAC CGTGCTCACA 300

AACCCCACAA CCCAGAGACT GGCAGAGACG TGCCAACAGC CCCTGGCCTT CCTTACCCAA 360

GTCCAGGAGG ACCTGCGAGA CTGCTTGGCC CTCGAGGCAC CTTCACATCA GCCCTCTGGG 420

AAACTGAGGC ACTGGCTGCA GAAGCTGGAG ACAGCCAAGA AGAAGGAGAC CGCCGGCTGC 480

CTGGAGGCCT CAGCCATCCT CCACATCTTC CAAGTACTGA ACGACCTGCG GTGCGCAGCC 540

CAGCGCGAGG ATTGCACTTA G 561

(3) Chicken IFN-alpha gene

ATGGCTGTGC CTGCAAGCCC ACAGCACCCA CGGGGGTACG GCATCCTGCT GCTCACGCTC 60

CTTCTGAAAG CTCTCGCCAC CACCGCCTCC GCCTGCAACC ACCTTCGCCC CCAGGATGCC 120

ACCTTCTCTC ACGACAGCCT CCAGCTCCTC CGGGACATGG CTCCCACACT ACCCCAGCTG 180

TGCCCACAGC ACAACGCGTC TTGCTCCTTC AACGACACCA TCCTGGACAC CAGCAACACC 240

CGGCAAGCCG ACAAAACCAC CCACGACATC CTTCAGCACC TCTTCAAAAT CCTCAGCAGC 300

CCCAGCACTC CAGCCCACTG GAACGACAGC CAACGCCAAA GCCTCCTCAA CCGGATCCAC 360

CGCTACACCC AGCACCTCGA GCAATGCTTG GACAGCAGCG ACACGCGCTC CCGGACGCGA 420

TGGCCTCGCA ACCTTCACCT CACCATCAAA AAACACTTCA GCTGCCTCCA CACCTTCCTC 480

CAAGACAACG ATTACAGCGC CTGCGCCTGG GAACACGTCC GCCTGCAAGC TCGTGCCTGG 540

TTCCTGCACA TCCACAACCT CACAGGCAAC ACGCGCACTT AG 582

（4）IFN-λ-F

CCGGAATTCC AGGTCACCCC GAAGAA 26

（5）IFN-λ-R1

CCCAAGCTTC TAAGTGCAAT CCTCGCGCTG GGC 33

（6）IFN-λ-R2

CTCCGCTACC GCCTCCACCA GAGCCTCCTC CACCAGTGCA ATCCTCGCGC TGGGC 55

（7）IFN-α-F1

CCGGAATTCT GCAACCACCT TC 22

（8）IFN-α-F2

TCTGGTGGAG GCGGTAGCGG AGGCGGAGGG TCGTGCAACC ACCTTC 46

（9）IFN-α-R

CCCAAGCTTC TAAGTGCGCG TGTTGCC 27

Claims

1. A chicken interferon lambda and alpha fusion protein is characterized in that: the chicken interferon lambda and alpha gene are fused and expressed in colibacillus through a complementary hydrophobic flexible amino acid joint by the same vector, and the nucleotide sequence of the chicken interferon lambda and alpha gene is shown as SEQ ID NO. 1.

2. The chicken interferon lambda and alpha fusion protein of claim 1, which comprises: the preparation method comprises the following steps:

A. primer design

According to the gene sequence of chicken IFN-lambda provided by Genbank, the nucleotide sequence of which is shown as SEQ ID NO. 2 and the gene sequence of IFN-alpha, the nucleotide sequence of which is shown as SEQ ID NO. 3, predicting and removing signal peptide by SignalP, designing two pairs of primers, respectively inserting two enzyme cutting sites of EcoR1 and Hind3 at the upstream and the downstream, wherein the two enzyme cutting sites are IFN-lambda-F, IFN-lambda-R1, IFN-alpha-F1 and IFN-alpha-R; then 2 primers IFN-lambda-R2 and IFN-alpha-F2 containing complementary hydrophobic flexible amino acid linkers are designed at the downstream of IFN-lambda and the upstream of IFN-alpha respectively, and the sequences are as follows:

the sequence of IFN-lambda-F is shown in SEQ ID NO: 4:

CCGGAATTCCAGGTCACCCCGAAGAA；

the sequence of IFN-lambda-R1 is shown in SEQ ID NO: 5:

CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC；

the sequence of IFN-lambda-R2 is shown in SEQ ID NO: 6:

CTCCGCTACCGCCTCCACCAGAGCCTCCTCCACCAGTGCAATCCTCGCGCTGGGC；

the sequence of IFN-alpha-F1 is shown in SEQ ID NO: 7:

CCGGAATTCTGCAACCACCTTC；

the sequence of IFN-alpha-F2 is shown in SEQ ID NO: 8:

TCTGGTGGAGGCGGTAGCGGAGGCGGAGGGTCGTGCAACCACCTTC；

the sequence of IFN-alpha-R is shown in SEQ ID NO: 9:

CCCAAGCTTCTAAGTGCGCGTGTTGCC；

B. gene cloning and identification

Self-making chick embryo fibroblasts by SPF chick embryos, using a Newcastle disease GM strain to challenge, extracting RNA, reversing to obtain a template cDNA for amplifying chicken IFN-lambda and IFN-alpha genes, and amplifying gene fragments of the chicken IFN-lambda and IFN-alpha signal-removed peptides by using IFN-lambda-F, IFN-lambda-R1, IFN-alpha-F1 and IFN-alpha-R primers;

then, the chicken IFN-lambda and IFN-alpha gene are fused by an SOE-PCR method: firstly, amplifying two gene segments containing a linker by using IFN-lambda-F, IFN-lambda-R2, IFN-alpha-F2 and IFN-alpha-R primers respectively; secondly, taking the gel recovery product of the first step as a template, and performing PCR amplification for 10-15 cycles without adding a primer; thirdly, taking the PCR product of the second step as a template, and amplifying PCR by using IFN-lambda-F and IFN-alpha-R to obtain a product, namely chIFN-lambda + alpha gene fragment;

C. construction of prokaryotic expression vector of chicken IFN-lambda and IFN-alpha fusion protein

Connecting the chIFN-lambda + alpha fusion gene to a pMD-19T vector, connecting for more than 4h at 16 ℃, converting to an agar plate containing benzyl amine, selecting monoclonal bacteria, putting the monoclonal bacteria into an LB culture medium, shaking the bacteria, identifying positive clones by bacteria liquid PCR, performing amplification culture after correct sequencing, and extracting a plasmid pMD-19T-chIFN-lambda + alpha;

3. The use of the fusion protein of chicken interferon lambda and alpha as defined in claim 1 or 2 for the preparation of antiviral medicaments for chickens.