CN110054680A - One boar, III type interferon receptors IFNLR1 * subunit recombinant protein and its application - Google Patents

Abstract

The embodiment of the invention discloses III type interferon receptors IFNLR1 * subunit recombinant proteins of a boar, the recombinant protein includes extracellular region, transmembrane helical region and intracellular region, and the extracellular region recombinant protein is that amino acid sequence shown in Seq ID No.5 is substituted, lacks and/or increases the identical biological function of albumen one or several amino acid derived and with amino acid sequence such as Seq ID No.5.III type interferon receptors alpha subunit gene genetic evolution conservative of pig provided in an embodiment of the present invention, the immunoregulation that can be used for the structure basis for the signal transduction that pig type iii interferon induces and its mediated is studied and for novel drugs target spot, passes through development enhancing, the prevention and treatment of blocking or antagonist for pig disease.

Description

One boar, III type interferon receptors IFNLR1 * subunit recombinant protein and its application

Technical field

The present invention relates to Animal molecular biology and gene engineering technology fields, and in particular to III type interferon receptor 2 of a boar Body subunit protein and its application.

Background technique

It is a kind of antiviral protein that interferon (Interferons, IFNs), which is originally found,.But have now been found that the albuminoid can Adjusting multiple bioprocess includes cell Proliferation and existence, inflammatory reaction and immune response, and being related to many pathological states includes sense Dye, cancer and autoimmunity.Wherein, I type interferon is the major cytokine of earlier antiviral infection inherent immunity, according to dynamic Species difference includes a variety of hypotypes.People's I type interferon include IFN-α (containing 13 hypotypes), IFN-β, IFN- ω, IFN- κ and IFN- ε, latter four kinds are single hypotype.Similar to people, pig IFN-α is encoded by 17 functional genes, and IFN- δ sample molecule is only in pig It is found in ox body, is named as SPI IFN.IFN-γ is the unique member of II type IFN.Most cell types are for different I type interferon is generated in the reaction of virus, and limited the cell type such as T cell of NK, activation, macrophage and nerve is thin Born of the same parents generate II type interferon.Type III IFNs includes IFN- λ 1, IFN- λ 2, IFN- λ 3 and IFN- λ 4, is newly discovered participation Pathogen infection (such as people T cell leukemia virus Type I, rotavirus and influenza virus virus infection, staphylococcus aureus With the cellularitys disease such as mycobacterium tuberculosis), the immune-regulating factor of tumour and autoimmune disease.

IFN- λ 1, IFN- λ 2 and IFN- λ 3 are referred to as IL-29, IL-28A and IL-28B again, by combining its receptor IFNLR (IFN- λ receptor), a series of signal transduction for activating STAT phosphorylations to rely on induce hundreds of Interferon-stimulated gene expression, form network based on these interferon-induced factors, participate in infectious diseases or itself Immunological regulation in immunity disease generation or development process.And the positive or negative immunoregulation loop that IFN- λ mediates these complicated Basis be its receptor, the different principal element of the immunoregulation effect that this is also IFN- λ and IFN-α/β is mediated.It is first First, IFNLR is not wide expression, and IFNLR is expressed and its existed to the sensibility of IFN- λ apparent especially between immunocyte Difference；Affinity of each member of second, IFN- λ in conjunction with IFNLR is different, is generally lower than IFN-α/β in conjunction with its receptor Affinity；Third participates in the gene genetic variation of IFN- λ signal transduction, and being mainly manifested in gene, there are a systems The single nucleotide polymorphism (single-nucleotide polymorphisms, SNPs) of column includes IFN- λ and its receptor IFNLR。

IFNLR includes α and β Liang Ge subunit, is the heterodimer of IFNLR1 (also name IL28RA) and IL10RB.Wherein IL10RB is expressed in various kinds of cell or tissue, but there are stringent tissue or Cell differentials for IFNLR1 expression, at present lung, intestines, Liver and immunocyte such as B cell, neutrocyte, macrophage and plasmacytoid dendritic cellss mRNA in detect IFNLR1 gene.People's NK cell seems not express IFNLR1, but the NK cells in mice that IL28R is knocked out shows functional defect.By This is as it can be seen that IFNLR is of great significance for the performance of IFN- λ function and host immune response.

But at present it is few for the report of pig IFNLR receptor both at home and abroad, and without complete IFNLR1 gene order for into One step research.Also it is studied without pig IFNLR receptor in novel drugs target spot and its immunoregulation mediated.

Summary of the invention

The object of the present invention is to provide a Tiao ZhuⅢXing interferon receptors subunit pIFNLR1 gene orders, are used for type III Interferon-induced signal transduction, novel drugs target spot and its immunoregulation research mediated.

In order to solve the above technical problems, technical solution provided in an embodiment of the present invention are as follows:

One boar, III type interferon receptors IFNLR1 * subunit recombinant protein, the recombinant protein includes extracellular region, cross-film Helical region and intracellular region, the extracellular region recombinant protein be Seq ID No.5 shown in amino acid sequence be substituted, lack and/or Increase the identical biological function of albumen one or several amino acid derived and with amino acid sequence such as Seq ID No.5.

The embodiment of the present invention provides the gene of the coding III type interferon receptors IFNLR1 extracellular region recombinant protein of pig.

Preferably, the nucleotide sequence of the gene is as shown in Seq ID No.6.

On the other hand the embodiment of the present invention provides the expression vector comprising the gene.

The another aspect of the embodiment of the present invention provides the host cell comprising the expression vector.

The another aspect of the embodiment of the present invention provides a kind of antibody of extracellular region recombinant protein described in combination.

The another aspect of the embodiment of the present invention provides III type interferon receptors subunit of pig recombination egg described in a kind of prepare White method comprising: the expression vector is constructed, the expression vector is transferred to BL21 competent cell, is weighed The process of histone.

The another aspect of the embodiment of the present invention provides further include: according to pig source IFNLR1mRNA primers, extracts Pig pulmonary macrophage 3D4/2 total serum IgE, and utilizing the reverse transcription of M-MLV reverse transcriptase is cDNA, and is carried out by template of cDNA The process of PCR amplification.

Preferably, the primer are as follows: the nucleotide sequence of upstream primer pIFNLR1F as shown in Seq ID No.3,

The sequence of downstream primer pIFNLR1R is as shown in Seq ID No.4.

The embodiment of the present invention also provides application of the recombinant protein in preparation enhancing, blocking or antagonist pharmaceuticals.

Pig IFN- λ receptor 1 sample mRNA sequence (XM_ of the embodiment of the present invention based on GenBank two predictions issued 013999051.1, XM_005674166.1) design primer using the cDNA of pig pulmonary macrophage as template passes through polymerase chain Formula reaction amplification obtains III type interferon receptors subunit pIFNLR1 gene open reading frame of pig, and to its gene order and its The amino acid sequence of coding is compared and phylogenetic analysis, while expressing pIFNLR1 born of the same parents using escherichia expression system Outer region structural domain is lured for the preparation of pIFNLR1 antiserum, polyclonal antibody or single gram of antibody, and then for pig type iii interferon Signal transduction, novel drugs target spot and its immunoregulation research mediated led lay the foundation.

The embodiment of the invention discloses the receptor subunits pIFNLR1 gene orders of an III type interferon of pig.The present invention Embodiment also discloses the source of above-mentioned sequence and the amino acid sequence genetic evolution of complete open reading frame and its coding Relationship.Pig type iii interferon receptor pIFNLR1 subunit sequence based on the embodiment of the present invention, utilizes Bacillus coli expression system System expression pIFNLR1 extracellular region structural domain (ECD), for the preparation of pIFNLR1 antiserum, polyclonal antibody or single gram of antibody, And then it the structure basis of signal transduction for the induction of pig type iii interferon, novel drugs target spot and its is mediated Immunoregulation research.

Detailed description of the invention

It, below will be to embodiment party in order to illustrate more clearly of embodiments of the present invention or technical solution in the prior art Formula or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that the accompanying drawings in the following description is only It is merely exemplary, it for those of ordinary skill in the art, without creative efforts, can also basis The attached drawing of offer, which is extended, obtains other implementation attached drawings.

Fig. 1 is that pIFNLR1 gene PCR expands agarose gel electrophoresis schematic diagram in the embodiment of the present invention 1, wherein M is DL2000marker, A are the PCR amplification electropherogram of pIFNLR1 gene；

Fig. 2 is pEASY-pIFNLR1 recombinant plasmid qualification result figure in the embodiment of the present invention 1, wherein M1 is DL2000marker, M2 are λ-EcoT14I Marker, and A is pEASY-p IFNLR1 (1) plasmid, and B is pEASY-p IFNLR1 (1) plasmid, C are pEASY-p IFNLR1 (2) plasmid, and D is pEASY-p IFNLR1 (2) plasmid BamH I and I double digestion of Sal；

Fig. 3 is the homology analysis figure of pIFNLR1 and reference sequences in the embodiment of the present invention 1, wherein A is nucleotide Horizontal analysis, B. are amino acid levels analysis；

Fig. 4 is SignalP-4.1 in the embodiment of the present invention 2 to pIFNLR1 protein signal peptide prediction result figure；

Fig. 5 is TMHMM and Phobius transmembrane region prediction result figure in the embodiment of the present invention 2；

Fig. 6 is pIFNLR1 phylogenetic analysis result figure in the embodiment of the present invention 3.

Fig. 7 is pEASY-pIFNLR1ECD plasmid enzyme restriction qualification figure in the embodiment of the present invention 4, wherein M1 is λ- EcoT14I Marker, M2 DL5000marker, A are pET28a-pIFNLR1ECD plasmid, B pET28a- BamHI the and XhoI digested plasmid of pIFNLR1ECD.

Fig. 8 is pIFNLR1ECD recombinant protein purification qualification figure in the embodiment of the present invention 5, wherein A is sample, and B is Percolation liquid, C are the sample that 100mM imidazoles affords, and D is the sample that 300mM imidazoles affords, and M is albumen marker.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art. The preparation Yu conversion of competent escherichia coli cell, the extraction of plasmid and digestion with restriction enzyme in embodiment, DNA fragmentation Recycling, the connection of linear DNA fragment, the screening of recombinant plasmid and identification, pcr amplification reaction etc. are referring to Jin Dongyan, Li Mengfeng etc. Translate the progress of " Molecular Cloning:A Laboratory guide " second edition related Sections.

It is huge that III type interferon receptors alpha subunit pIFNLR1 gene of pig provided in an embodiment of the present invention is cloned in pig lung for the first time The cDNA of phagocyte 3D4/2 (ATCC CRL2845), open reading frame overall length 1,578nt, nucleotide sequence such as Seq ID No.1 Shown, the nucleotide sequence of pIFNLR1 extracellular region structural domain is as shown in Seq ID No.6.Pig III provided in an embodiment of the present invention Type interferon receptors alpha subunit pIFNLR1 gene encodes 525 amino acid, amino acid residue sequence such as Seq ID No.2 institute Show, the amino acid sequence of pIFNLR1 extracellular region structural domain is as shown in Seq ID No.5.

1 pig pIFNLR1 gene cloning of embodiment and sequencing

1, the design and synthesis of pig pIFNLR1 gene magnification primer

The pig source IFNLR1mRNA sequence (XM_005674166.1) that prediction is logged according to GenBank, designs following primer (I restriction enzyme site of BamH is added in 5 ' ends, and I restriction enzyme site of Sal, italicized item are added in 3 ' ends), is used for pIFNLR1 base Gene-amplification.

Upstream primer pIFNLR1F (Seq ID No.3):

5′-ggatccatgtcgggggctggctgctgggcctctctactcacgcggggc-3′；

Downstream primer pIFNLR1R (Seq ID No.4):

5′-gtcgactcacctggccaggtaatgccccagtgtcctacctctgacctc-3′。

Above primer commission Jilin provincial treasury U.S. Biotechnology Co., Ltd is synthesized.

2, pig pulmonary macrophage RNA extraction, reverse transcription and PCR

It collects pig pulmonary macrophage 3D4/2 (ATCC CRL2845), extracts cell total rna using Trizol method, it is then sharp It is cDNA, overall length 1,578nt with M-MLV reverse transcriptase reverse transcription.

Using this cDNA as template amplification pIFNLR1 gene, 50 μ L:5 × PCR buffer of total volume, 10 μ L, 20 μm of ol/ are reacted The upper (lower) trip primer of L each 0.5 μ L, 10 μ L of template cDNA, 4 μ L, 2.5U/ μ L of dNTP (each 2.5mmol/L) 1 μ L of FastPfu archaeal dna polymerase, GC buffer 3 μ L, ddH₂O 21μL.PCR response procedures: 95 DEG C of 5min；98℃ 30 Sec, 69 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.

As shown in Figure 1, agarose gel electrophoresis recycles amplified production, and it is cloned into- Blunt Zero clone It is denoted as pEASY-pIFNLR1 on carrier, converts Trans1-T1 competent cell, picking positive bacterium colony and mentioned under screening pressure Plasmid is taken, is identified with BamH I and I double digestion of Sal, as shown in Fig. 2, agarose gel electrophoresis results are shown, obtains recombinant plasmid Carrier pEASY-pIFNLR1.And 2 recombinant plasmids is taken to carry out DNA sequencing to determine nucleotide sequence.

3, the sequence analysis of pig pIFNLR1 gene

DNA sequencing illustrates pig lung the results show that the pIFNLR1 sequencing result in 2 pEASY-pIFNLR1 is completely the same PIFNLR1 cDNA coded sequence contains 1,578nt in macrophage.Nucleotide level, with reference sequences (XM_005674166.1) Homology be 97.4%；As shown in figure 3, amino acid sequence (the XP_ of amino acid levels and reference sequences coding 005674223.1) homology is 96.2%.

2 pig pIFNLR1 signal peptide of embodiment and transmembrane region analysis

The embodiment of the present invention is the amino acid based on the pIFNLR1 cDNA sequence coding obtained in embodiment 1, application The online protein analysis tool of SignalP-4.1 predicts the letter of III interferon receptors alpha subunit pIFNLR1 of pig provided by the invention Number peptide and subcellular localization.As shown in figure 4, prediction result is shown, III interferon receptors alpha subunit of pig provided by the invention PIFNLR1 contains signal peptide, is 1~20 amino acids residue of N-terminal, signal peptide shearing site according to S value predicted signal peptide length For TPG-RL；And protein (XP_005674223.1) no signal peptide of reference sequences coding.

Meanwhile respectively using TMHMM and Phobius on-line analysis software to III interferon receptors α of pig provided by the invention Subunit pIFNLR1 is predicted.As shown in figure 5,2.0 prediction result of TMHMM is shown, amino coded by pIFNLR1 cDNA Acid, it is extracellular region (Outside) that topological structure, which is 1~227, and 228~250aa is transmembrane helical region (Tansmembrane), 251~525aa is intracellular region (Inside)；2.0 prediction result of Phobius and TMHMM is almost the same, and Phobius is predicted Signal peptide area is 1~24 amino acids residue of N-terminal, almost the same with SignalP-4.1 result.

Sequence (the XP_ of more existing two predictions of pIFNLR1 receptor sequence provided in an embodiment of the present invention 013854505.1, XP_005674223.1) it is more complete.

3 pig pIFNLR1 gene genetic evolutionary analysis of embodiment

The embodiment of the present invention is passed through based on the amino acid of the pIFNLR1 cDNA sequence coding obtained in embodiment 1 III interferon receptors alpha subunit pIFNLR1 of pig provided in an embodiment of the present invention is carried out amino acid by NCBI BLAST online software It compares, 5 Molecular Evolutionary Genetics of IFN- λ receptor application MEGA point of selected part and other higher species of pIFNLR1 homology Analysis, carries out the building and analysis of systematic evolution tree.As a result as shown in fig. 6, III interferon receptors alpha subunit IFNLR1 of pig evolve compared with Be it is conservative, have now been found that pig IFNLR1 there are two predict mRNA copy, wherein pIFNLR1 provided in an embodiment of the present invention with Wild boar 1 closely clusters, and 2 branch length of wild boar is distal to wild boar 1 and pIFNLR1 gene provided in an embodiment of the present invention shows that this is copied The protein sequence of shellfish coding is changed.

III type interferon receptors alpha subunit gene genetic evolution conservative of pig provided in an embodiment of the present invention, can be used for pig III New drug is studied and are used for the structure basis of the interferon-induced signal transduction of type and its immunoregulation mediated Object target spot, the prevention and treatment for being used for pig disease by developing enhancing, blocking or antagonist.

The building of embodiment 4pIFNLR1 extracellular region prokaryotic expression carrier

The embodiment of the present invention expands pIFNLR1 extracellular region (ECD) genetic fragment (1-681bp nucleosides by PCR method Acid), and it is cloned into pEASY-Blunt simple carrier, after sequencing and digestion identification are correct, pass through BamHI and XhoI digestion Building obtains recombinant plasmid by conventional plasmid construction, extraction and identification method to prokaryotic expression carrier pET28a The map of pET28a-pIFNLR1ECD, the expression vector recombinant plasmid are as shown in Figure 7.

The Prokaryotic expression, purification and renaturation of embodiment 5pIFNLR1 extracellular region recombinant protein

The e. coli bl21 (DE3) for carrying pIFNLR1ECD genetic fragment is seeded in self-induction culture medium, 37 DEG C Shake culture 12h, thalline were collected by centrifugation, and every 100mg thallus (weight in wet base) plus PBS buffer solution 1-5mL are resuspended, while adding appropriate egg White enzyme inhibitor (10 μ l/mL) and nuclease (3 U/mL), for ultrasonication thallus until liquid is clarified, 5,000g are centrifuged 5min, Supernatant, supernatant 10 are collected, 000g is centrifuged 20min, collects precipitating.It uses equilibration buffer (8M urea, 5mM imidazoles, pH 8.0) Precipitating, 37 DEG C of concussion 30min, centrifuging and taking supernatant is resuspended.It is purified using Ni affinity column by AKTApurifier, It is eluted using the equilibration buffer of the imidazoles containing 300mM.It is the Escherichia coli of pIFNLR1ECD genetic fragment as shown in Fig. 8 BL21 (DE3) expresses the Purification figure of albumen, and the protein sample after elution carries out renaturation using the method for dialysis.

Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.

Sequence table

<110>Academy of Military Sciences's military medical research institute military affairs veterinary institute

III type interferon receptors IFNLR1 * subunit recombinant protein of<120>one boar and its application

<130> GG18125221A

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 525

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Met Ser Gly Ala Gly Cys Trp Ala Ser Leu Leu Leu Cys Leu Leu Gln

1 5 10 15

Ser Thr Pro Gly Arg Leu His Leu Ala Pro Pro Gln Asn Val Thr Leu

20 25 30

Leu Ser Arg Asp Phe Gly Val Tyr Leu Thr Trp Leu Pro Gly Pro Gly

35 40 45

Asn Pro Gln Asn Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Ala Thr

50 55 60

Pro Lys Arg Trp Gln Arg Val Lys Met Cys Ala Arg Thr Lys Glu Leu

65 70 75 80

Val Cys Ser Leu Met Cys Leu Glu Lys Gln Asp Leu Cys Asn Lys Phe

85 90 95

Lys Gly Arg Val Gln Ala Val Ser Pro Ser Ala Arg Ser Pro Trp Val

100 105 110

Glu Ser Lys Ser Met Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro

115 120 125

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

130 135 140

Tyr Gln Leu Pro His Cys Val Pro Gln Pro Asp Leu Asn Tyr Glu Val

145 150 155 160

Asp Phe Trp Lys Glu Gly Thr Arg Asn Lys Thr Arg Phe Pro Ala Thr

165 170 175

Pro His Gly Gln Pro Val Gln Ile Pro Leu Gln Pro Ala Thr Arg Gly

180 185 190

Pro His Cys Leu Ser Gly Arg Thr Ile Tyr Thr Phe Gly Asp Pro Lys

195 200 205

Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Phe Leu Glu Gly Pro Gly

210 215 220

Pro Asn Trp Ala Phe Leu Val Leu Leu Pro Phe Leu Leu Pro Leu Leu

225 230 235 240

Leu Val Ile Ala Ala Gly His Val Ile Trp Lys Ser Val Thr Gly Asn

245 250 255

Tyr Trp Phe Gln Gln Ala Lys Met Pro Gln Ala Leu Asp Phe Pro Gly

260 265 270

His Arg Pro Pro Gly Ala Thr Phe Arg Pro Ser Gly Pro Glu Cys Leu

275 280 285

Asp Asp Leu Ser Leu Tyr Pro Gln Lys Glu Leu Ala Ile Arg Val Arg

290 295 300

Leu Met Pro Arg Val Arg Ala Pro Ala Thr Ile Gln Ala Arg Ser Glu

305 310 315 320

Lys Asn Arg Ala Glu Glu Glu Lys Asn Glu Glu Gly Asp Thr Asp Glu

325 330 335

Asp Thr Asp Asp His Val Ile Phe Gln Pro Tyr Met Glu Pro Pro Pro

340 345 350

Leu Leu Gly Leu Glu His Gln Thr Pro Cys His Ala Glu Ala Glu Gly

355 360 365

Pro Gln Ser Pro Leu Val Gln Val Glu Gly Ser Ser Val Cys Asp Ser

370 375 380

Ser Asp Arg Ser Trp Thr Ser Thr Ala Gly Ser Ser Pro Trp Asp Glu

385 390 395 400

Ala Glu Ser Ser Gly Tyr Leu Ala Lys Lys Gly Pro Gly Arg Gly Pro

405 410 415

Asp Gly Glu Glu Cys Gln Lys Pro Leu Pro Pro Pro Glu Phe Ser Glu

420 425 430

Asp Leu Ser Ser Leu Glu Glu Pro Pro Lys Asp Asn Leu Ser Trp Ala

435 440 445

Ser Trp Gly Phe Ser Ser Pro Arg Leu Asn Leu Val Pro Arg Glu Pro

450 455 460

Pro Val Ser Leu Arg Thr Leu Thr Leu Cys Trp Asp Ser Ser Pro Glu

465 470 475 480

Glu Asp Glu Glu Glu Glu Glu Glu Glu Gly Gly Ser Glu Ser Glu Ser

485 490 495

Glu Asp Ser Gly Ala Gly Thr Trp Glu Thr Lys Ser Leu Glu Ser Thr

500 505 510

Glu Val Arg Gly Arg Thr Leu Gly His Tyr Leu Ala Arg

515 520 525

<210> 2

<211> 1578

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

atgtcggggg ctggctgctg ggcctctcta ctcctatgcc tgcttcagtc cactccaggg 60

aggctccacc tggcccctcc ccagaacgtg acgctgctct ccagggactt cggtgtgtac 120

ctgacgtggc tcccagggcc tggcaacccc cagaatgtga cctattttgt ggcctatcaa 180

agctcagcaa cccctaaacg gtggcaaaga gtgaaaatgt gtgcaagaac caaagagctg 240

gtgtgttctc tgatgtgcct ggagaaacaa gacctgtgca acaagttcaa ggggcgagtg 300

caagcagttt ctcccagcgc caggtccccc tgggtggagt ccaagtccat ggattacctt 360

tttgaagtgg agccggcccc accagttctg gtattcaacc ggacagagga gatcctcagc 420

gtcaatgcca cataccagct accccactgt gtgccccagc ccgatctgaa ctatgaggtg 480

gatttctgga aggaggggac caggaacaag acccgatttc cagccactcc ccatggccag 540

ccagtccaga ttcccctcca gccagccacc cgcggacccc actgtctcag cggcagaacc 600

atctacacct tcggtgaccc taaatacagc aagttctcca agcctacctg cttcttcctg 660

gagggcccag gccccaactg ggcattcctg gttctcctac catttctgct gccgctgctg 720

ttggtcattg ccgcagggca tgtgatctgg aagagcgtca caggaaacta ctggtttcag 780

caggcaaaga tgccacaggc cctggacttc ccaggacaca gaccccctgg ggcaaccttt 840

cggcccagtg gcccagaatg cctggatgac ttgagcctct atccccagaa ggaactggct 900

ataagggtca ggctgatgcc tagagtcagg gccccagcca ccatccaggc aagatcagag 960

aagaacaggg ctgaggagga gaagaatgag gagggggaca cagacgagga cacagacgac 1020

catgtcatct tccagcccta catggagcca ccccccctcc tggggctgga gcaccagacc 1080

ccgtgccatg ctgaagcaga agggccccag tcacctctgg tccaggtcga aggctcctct 1140

gtttgcgatt cttcagacag aagctggacc agcactgcgg gctcctcccc ctgggatgag 1200

gctgagtcct cgggctattt ggccaagaag gggccaggcc gagggccaga tggggaggag 1260

tgccagaagc ctctgccacc accggaattc tccgaggact tgagttccct ggaagagccc 1320

cccaaagaca acctctcctg ggccagctgg ggcttctcat caccacggct gaacctggtc 1380

cccagggagc ccccagtttc tctgcggaca ctgaccctct gctgggacag cagccctgag 1440

gaggacgagg aggaggagga agaagagggt gggagcgaat cggaaagcga ggacagcggt 1500

gctggcacct gggagactaa gagcctcgag agcaccgagg tcagaggtag gacactgggg 1560

cattacctgg ccaggtga 1578

<210> 3

<211> 48

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ggatccatgt cgggggctgg ctgctgggcc tctctactca cgcggggc 48

<210> 4

<211> 48

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gtcgactcac ctggccaggt aatgccccag tgtcctacct ctgacctc 48

<210> 5

<211> 227

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Met Ser Gly Ala Gly Cys Trp Ala Ser Leu Leu Leu Cys Leu Leu Gln

1 5 10 15

Ser Thr Pro Gly Arg Leu His Leu Ala Pro Pro Gln Asn Val Thr Leu

20 25 30

Leu Ser Arg Asp Phe Gly Val Tyr Leu Thr Trp Leu Pro Gly Pro Gly

35 40 45

Asn Pro Gln Asn Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Ala Thr

50 55 60

Pro Lys Arg Trp Gln Arg Val Lys Met Cys Ala Arg Thr Lys Glu Leu

65 70 75 80

Val Cys Ser Leu Met Cys Leu Glu Lys Gln Asp Leu Cys Asn Lys Phe

85 90 95

Lys Gly Arg Val Gln Ala Val Ser Pro Ser Ala Arg Ser Pro Trp Val

100 105 110

Glu Ser Lys Ser Met Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro

115 120 125

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

130 135 140

Tyr Gln Leu Pro His Cys Val Pro Gln Pro Asp Leu Asn Tyr Glu Val

145 150 155 160

Asp Phe Trp Lys Glu Gly Thr Arg Asn Lys Thr Arg Phe Pro Ala Thr

165 170 175

Pro His Gly Gln Pro Val Gln Ile Pro Leu Gln Pro Ala Thr Arg Gly

180 185 190

Pro His Cys Leu Ser Gly Arg Thr Ile Tyr Thr Phe Gly Asp Pro Lys

195 200 205

Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Phe Leu Glu Gly Pro Gly

210 215 220

Pro Asn Trp

225

<210> 6

<211> 681

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

atgtcggggg ctggctgctg ggcctctcta ctcctatgcc tgcttcagtc cactccaggg 60

aggctccacc tggcccctcc ccagaacgtg acgctgctct ccagggactt cggtgtgtac 120

ctgacgtggc tcccagggcc tggcaacccc cagaatgtga cctattttgt ggcctatcaa 180

agctcagcaa cccctaaacg gtggcaaaga gtgaaaatgt gtgcaagaac caaagagctg 240

gtgtgttctc tgatgtgcct ggagaaacaa gacctgtgca acaagttcaa ggggcgagtg 300

caagcagttt ctcccagcgc caggtccccc tgggtggagt ccaagtccat ggattacctt 360

tttgaagtgg agccggcccc accagttctg gtattcaacc ggacagagga gatcctcagc 420

gtcaatgcca cataccagct accccactgt gtgccccagc ccgatctgaa ctatgaggtg 480

gatttctgga aggaggggac caggaacaag acccgatttc cagccactcc ccatggccag 540

ccagtccaga ttcccctcca gccagccacc cgcggacccc actgtctcag cggcagaacc 600

atctacacct tcggtgaccc taaatacagc aagttctcca agcctacctg cttcttcctg 660

gagggcccag gccccaactg g 681

Claims (10)

1. III type interferon receptors IFNLR1 * subunit recombinant protein of a boar, which is characterized in that

The recombinant protein includes extracellular region, transmembrane helical region and intracellular region, and the extracellular region recombinant protein is Seq ID No.5 Shown amino acid sequence is substituted, lacks and/or increases one or several amino acid derived and has amino acid sequence such as Seq The identical biological function of the albumen of ID No.5.

2. encoding the gene of III type interferon receptors IFNLR1 extracellular region recombinant protein of pig described in claim 1.

3. gene as claimed in claim 2, which is characterized in that the nucleotide sequence of the gene is as shown in Seq ID No.6.

4. the expression vector comprising gene described in Claims 2 or 3.

5. the host cell comprising expression vector described in claim 4.

6. a kind of antibody of combination extracellular region recombinant protein described in claim 1.

7. a kind of method for preparing III type interferon receptors * subunit recombinant protein of pig described in claim 1, it is characterised in that Include:

Expression vector described in claim 4 is constructed, the expression vector is transferred to BL21 competent cell, obtains recombinant protein Process.

8. the method for claim 7, it is characterised in that further include:

According to pig source IFNLR1 mRNA sequence design primer, pig pulmonary macrophage 3D4/2 total serum IgE is extracted, and utilizes M-MLV Reverse transcriptase reverse transcription is cDNA, and the process of PCR amplification is carried out using cDNA as template.

9. method according to claim 8, which is characterized in that

The primer are as follows: the nucleotide sequence of upstream primer pIFNLR1F as shown in Seq ID No.3,

The sequence of downstream primer pIFNLR1R is as shown in Seq ID No.4.

10. application of the recombinant protein described in claim 1 in preparation enhancing, blocking or antagonist pharmaceuticals.