CN1887344A - Animal interferon compound and its prepn and application - Google Patents
Animal interferon compound and its prepn and application Download PDFInfo
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- CN1887344A CN1887344A CN 200610103821 CN200610103821A CN1887344A CN 1887344 A CN1887344 A CN 1887344A CN 200610103821 CN200610103821 CN 200610103821 CN 200610103821 A CN200610103821 A CN 200610103821A CN 1887344 A CN1887344 A CN 1887344A
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Abstract
The present invention discloses animal interferon compound and its preparation process and application. The animal interferon compound is prepared through obtaining animal interferon alpha and gamma gene to constitute recombinant cloning vector; selecting and constituting transfer vector or expression vector; transforming or transfecting host bacterium or cell; and identifying, purifying and decomposing the expression product. The animal interferon compound of the present invention has antiviral activity, relatively high immunoregulating activity and superiority complementation of different interferons.
Description
Technical field
The present invention relates to a kind of animal interferon compound and its production and application, this animal interferon compound is the interferon product that obtains through the coexpression of interferon-ALPHA and γ.
Background technology
The outbreak of epidemic of a series of livestock and poultry virus sexually transmitted diseases such as bird flu, foot and mouth disease has caused massive losses for global society and economy, is directly threatening food safety and human health.In order to control and eliminate these Animal diseases, conventional route is by the vaccine immunity prevention and uses antibiotic, but because the feeding environment condition is poor, virus is variation constantly, and meanwhile, the bio-safety of food has been subjected to extensive attention, advocate pollution-free food, food Chinese medicine residue detection etc. is strengthened in the use of restriction antibiotic and antiviral chemicals, makes the method for traditional control livestock and poultry face new problem and challenge.
Interferon is to be produced under the stimulation of derivants such as virus by body cell, has to restrain virus by a type cytokines proteinoid molecule of its activated time multiplexed cell system.Interferon is divided into I type and II type two classes, I type interferon comprises interferon-ALPHA and interferon beta, its main activity be by with the acceptor interaction of target cell and himself, induce and produce antiviral protein 2-5A synzyme and protein kinase PKR etc., thereby suppress virus in time multiplexed cell system, i.e. antiviral activity.II type interferon claims interferon gamma again, and it also has very strong immunoregulatory activity except that having the antiviral activity that the I interferoid had: 1. promote the ADCC effect by raising macrophage IgGFc receptor; 2. activate the NK cell, improve its kill capability, the NK cell that is activated is also secreted interferon gamma; 3. induce the expression of cell mhc class ii molecules such as macrophage, T, B, thereby improve the antigen presentation ability; 4. interferon gamma can be induced the generation of macrophage inducibility nitricoxide synthase, promotes the synthetic of NO, is that interferon gamma kills and wounds intra-cellular pathogens and antitumor and suppresses one of molecular mechanism of virus replication.
But owing to closing rapidly of interferon mRNA instability and its gene, cell induces the interferon amount that produces few by virus or polyinosini, and the persistent period is also short.Therefore stimulate body to produce interference by interferon inducers and usually prevent and treat the animal virus sexually transmitted disease, its effect can be had a greatly reduced quality.Simultaneously, extract interferon by cultured cell, its feasibility is also poor.
Summary of the invention
First purpose of the present invention provides a kind of animal interferon compound.
Another object of the present invention provides the preparation method of this animal interferon compound.
The 3rd purpose of the present invention provides this animal interferon compound in the application that suppresses virus breeding.
In order to achieve the above object, the present invention is by the following technical solutions:
This animal interferon compound makes by following steps:
A, obtain animal interferon α and γ gene, make up recombinant cloning vector;
The selection of B, transfer vector or expression vector and structure;
C, conversion or transfecting host bacterium or cell;
D, expression product evaluation, purification and decomposition.
Described animal interferon α and γ gene have the nucleotide sequence of sequence 1 and sequence 2 in the sequence table respectively.
The preparation method of this animal interferon compound is made up of following steps:
A, obtain animal interferon α and γ gene, make up recombinant cloning vector: synthetic animal interferon α and γ nucleotide sequence, perhaps design Auele Specific Primer, two ends add restriction enzyme site, by RT-PCR animal interferon α and γ gene are come out from amplification through total RNA of inductive peripheral blood lymphocyte or splenocyte extraction, be connected respectively to the T carrier;
B, the selection of transfer vector or expression vector and structure: selection can be by the transfer vector or the expression vector with ampicillin or kanamycin or gentamycin or tetracycline resistance that contain His * 6 labels of Lac Z genescreen, expression vector is selected prokaryotic expression carrier or carrier for expression of eukaryon for use, to from the T carrier that has made up, behind double digestion, be connected to same expression vector respectively with kind animal interferon α and γ gene, place under the same promoter control, the gene reading frame is correct and be 3 integral multiple, and inserts thrombin between two genes or enterokinase is cut the site;
C, conversion or transfecting host bacterium or cell: with transfer vector and the expression vector homologous recombination that builds, Screening and Identification positive expression plasmid, utilize calcium phosphate precipitation method or liposome to infect methods such as method or electrotransformation, with the expression vector that has built, conversion or transfection are in protokaryon or eucaryon host body, by PCR or plasmid enzyme restriction 1% agarose gel electrophoresis or sequencing identify errorless after, carry out the screening of abduction delivering and condition optimizing;
D, expression product evaluation, purification and decomposition: expression product is through nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification, the SDS-PAGE electrophoresis is identified, the purification expression product is through thrombin or enterokinase digestion, a protein molecule is resolved into two parts, promptly get the complex of interferon-ALPHA molecule and interferon gamma molecule.
The application of animal interferon compound in inhibition Avian pneumo-encephalitis virus, bird flu virus or infectious bursa diseases virus duplicate in chick embryo fibroblast.After the undressed cell inoculation virus, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus of chicken interferon α and the compound gene engineering product processing of γ, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Animal interferon compound is suppressing Pseudorabies virus, foot and mouth disease virus, swine fever virus, swine influenza virus, pig parvoviral, pig breathing and breeding syndrome virus or the application of pig circular ring virus in porcine kidney cell or Merc-145 time multiplexed cell system.After the cell inoculation virus of handling without the compound gene engineering product of pig interferon α and γ, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus of pig interferon α and the compound gene engineering product processing of γ, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Animal interferon compound is suppressing hostis pecoris, bovine ephemeral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, the application of bovine parvovirus in bovine kidney cells time multiplexed cell system.After the cell inoculation virus of handling without the compound gene engineering product of Bov IFN α and γ, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus of Bov IFN α and the compound gene engineering product processing of γ, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Animal interferon compound is suppressing canine distemper virus, Canine Parvovirus, canine infectious hepatitis virus, canine parainfluenza virus, canine alphaherpesvirus, the application of canine coronavirus in Madin-Darby canine kidney(cell line) time multiplexed cell system.After the cell inoculation virus of handling without the compound gene engineering product of dog interferon alpha and γ, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus of dog interferon alpha and the compound gene engineering product processing of γ, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
The present invention adopts technique for gene engineering, and animal interferon α and γ gene are cloned, and will connect with kind animal interferon α and γ gene, and the expression vector of recombinating carries out external evokedly, realizes the coexpression of interferon-ALPHA and γ.Make final interferon product both have antiviral activity, have stronger immunoregulatory activity simultaneously again, thereby realize the mutual supplement with each other's advantages of different shaped interferon.
The specific embodiment
Be embodiments of the invention below, described embodiment just is used for illustrating the present invention, and should not be considered to be limitation of the present invention.
Embodiment 1
The preparation method of chicken interferon α and γ is by being undertaken by following steps:
(1), synthetic chicken interferon α and γ nucleotide sequence, two ends add BamH I, Hind III and Sph I restriction enzyme site, are connected respectively to the T carrier.
Nucleotide sequence when chicken interferon α expresses in prokaryotic expression carrier sees also the sequence 1 in the appended sequence table.
Nucleotide sequence when chicken interferon gamma is expressed in prokaryotic expression carrier sees also the sequence 2 in the appended sequence table.
(2), the selection of expression vector and structure: selection can be by the prokaryotic expression carrier with ampicillin or kanamycin or gentamycin or tetracycline resistance that contains His * 6 labels of Lac Z genescreen, chicken interferon α and γ gene are connected to expression vector through same enzyme action respectively behind double digestion from the T carrier that has made up, place under the control of same promoter, and insert thrombin between two genes or enterokinase is cut the site;
(3), transform the host bacterium: utilize the calcium phosphate precipitation method to transform the host bacterium expression vector that builds, by blue white macula and resistance screening, PCR and plasmid double digestion are identified the positive expression plasmid, sequencing further identify errorless after, carry out the screening of IPTG abduction delivering and condition optimizing;
(4), expression product evaluation, purification and preparation: collect thalline, through ultrasonic treatment, degeneration, nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification expression product, the SDS-PAGE electrophoresis is identified, the purification expression product becomes two parts with the expressed protein molecule from thrombin or enterokinase restriction enzyme site digest and decompose, i.e. the complex of chicken interferon alpha molecule and interferon gamma molecule through thrombin or enterokinase digestion.
Embodiment 2
The preparation of pig interferon α and γ specifically is made up of following steps:
(1), obtains pig interferon α and γ gene, make up recombinant cloning vector: the design Auele Specific Primer, two ends add Not I, EcoR I and Sal I restriction enzyme site, by the RT-PCR technology pig interferon α and γ gene are come out from amplification through total RNA of ConA or inductive peripheral blood lymphocyte of PHA or splenocyte extraction, be connected respectively to the T carrier.
(2), the selection of transfer vector or expression vector and structure: selection can be by the transfer vector and the expression vector with ampicillin or kanamycin or gentamycin or tetracycline resistance that contain His * 6 labels of Lac Z genescreen, pig interferon α and γ gene are connected to expression vector through same enzyme action respectively behind double digestion from the T carrier that has made up, series connection places under the same promoter control, two gene reading frames are correct and be 3 integral multiple, insertion thrombin or enterokinase are cut the site between two genes, by PCR or plasmid enzyme restriction or sequencing evaluation.
(3), conversion or transfecting host bacterium or cell: with transfer vector and the expression vector homologous recombination that builds, expression vector can be selected prokaryotic expression carrier or carrier for expression of eukaryon for use, Screening and Identification positive expression plasmid, utilize the method that electricity transforms or liposome transforms, with the expression vector that has built, conversion or transfection are carried out the screening of abduction delivering and condition optimizing in host.
(4), expression product evaluation, purification and preparation: SDS-PAGE electrophoresis or fluorescent antibody staining microscopy are identified expression product, nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification, the purification expression product is through thrombin or enterokinase digestion, the expressed protein molecule is become two parts through thrombin or enterokinase restriction enzyme site digest and decompose, i.e. the complex of pig interferon alpha molecule and interferon gamma molecule.
Embodiment 3
The preparation of Bov IFN α and γ specifically is made up of following steps:
(1), obtains Bov IFN α and γ gene, make up recombinant cloning vector: the design Auele Specific Primer, two ends add Not I, EcoR I and Sal I restriction enzyme site, by the RT-PCR technology Bov IFN α and γ gene are come out from amplification through total RNA of ConA or inductive peripheral blood lymphocyte of PHA or splenocyte extraction, be connected respectively to the T carrier.
(2), the selection of transfer vector or expression vector and structure: selection can be by the transfer vector and the expression vector with ampicillin or kanamycin or gentamycin or tetracycline resistance that contain His * 6 labels of Lac Z genescreen, Bov IFN α and γ gene are connected to expression vector through same enzyme action respectively behind double digestion from the T carrier that has made up, series connection places under the same promoter control, two gene reading frames are correct and be 3 integral multiple, insertion thrombin or enterokinase are cut the site between two genes, by PCR or plasmid enzyme restriction or sequencing evaluation.
(3), conversion or transfecting host bacterium or cell: with transfer vector and the expression vector homologous recombination that builds, expression vector can be selected prokaryotic expression carrier or carrier for expression of eukaryon for use, Screening and Identification positive expression plasmid, utilize the method that electricity transforms or liposome transforms, with the expression vector that has built, conversion or transfection are carried out the screening of abduction delivering and condition optimizing in host.
(4), expression product evaluation, purification and preparation: SDS-PAGE electrophoresis or fluorescent antibody staining microscopy are identified expression product, nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification, the purification expression product is through thrombin or enterokinase digestion, the expressed protein molecule is become two parts through thrombin or enterokinase restriction enzyme site digest and decompose, i.e. the complex of Bov IFN alpha molecule and interferon gamma molecule.
Embodiment 4
The preparation of dog interferon alpha and γ specifically is made up of following steps:
(1), obtains dog interferon alpha and γ gene, make up recombinant cloning vector: the design Auele Specific Primer, two ends add Not I, EcoR I and Sal I restriction enzyme site, by the RT-PCR technology dog interferon alpha and γ gene are come out from amplification through total RNA of ConA or inductive peripheral blood lymphocyte of PHA or splenocyte extraction, be connected respectively to the T carrier.
(2), the selection of transfer vector or expression vector and structure: selection can be by the transfer vector and the expression vector with ampicillin or kanamycin or gentamycin or tetracycline resistance that contain His * 6 labels of Lac Z genescreen, dog interferon alpha and γ gene are connected to expression vector through same enzyme action respectively behind double digestion from the T carrier that has made up, series connection places under the same promoter control, two gene reading frames are correct and be 3 integral multiple, insertion thrombin or enterokinase are cut the site between two genes, by PCR or plasmid enzyme restriction or sequencing evaluation.
(3), conversion or transfecting host bacterium or cell: with transfer vector and the expression vector homologous recombination that builds, expression vector can be selected prokaryotic expression carrier or carrier for expression of eukaryon for use, Screening and Identification positive expression plasmid, utilize the method that electricity transforms or liposome transforms, with the expression vector that has built, conversion or transfection are carried out the screening of abduction delivering and condition optimizing in host.
(4), expression product evaluation, purification and preparation: SDS-PAGE electrophoresis or fluorescent antibody staining microscopy are identified expression product, nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification, the purification expression product is through thrombin or enterokinase digestion, the expressed protein molecule is become two parts through thrombin or enterokinase restriction enzyme site digest and decompose, i.e. the complex of dog interferon alpha molecule and interferon gamma molecule.
Embodiment 5
The propagation of chicken interferon α that is obtained by embodiment 1 and the compound gene engineering product of γ can suppress 100 TCID50 on chick embryo fibroblast Avian pneumo-encephalitis virus, bird flu virus, infectious bursa diseases virus.On the trace Tissue Culture Plate of 96 holes, use RPMI1640 culture medium culturing chick embryo fibroblast, 5% CO
2Cultivated 24 hours under 37 ℃ of conditions of incubator, chicken interferon α and the compound gene engineering product of γ with various dose are handled, inhale after 24 hours and abandon, inoculate Avian pneumo-encephalitis virus, bird flu virus, the infectious bursa diseases virus of 100 TCID50 more respectively.It is all inhibited that the result shows that chicken interferon α and the compound gene engineering product of γ infect caused cytopathy to Avian pneumo-encephalitis virus, bird flu virus, infectious bursa diseases virus.After the undressed cell inoculation virus, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus that chicken interferon α that embodiment 1 obtains and the compound gene engineering product of γ are handled, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Embodiment 6
Pig interferon α that is obtained by embodiment 2 and the compound gene engineering product of γ can suppress 100 TCID50 on porcine kidney cell (PK) and Merc145 cell Pseudorabies virus, foot and mouth disease virus, swine fever virus, swine influenza virus, pig parvoviral, pig breathe the propagation with breeding syndrome virus, pig circular ring virus.On the trace Tissue Culture Plate of 96 holes, use DMEM culture medium culturing porcine kidney cell (PK) and Merc145 cell, 5% CO
2After being cultured to cell under 37 ℃ of conditions of incubator and growing up to monolayer, pig interferon α and the compound gene engineering product of γ with various dose are handled, inhale after 24 hours and abandon, the Pseudorabies virus, foot and mouth disease virus, swine fever virus, swine influenza virus, pig parvoviral, the pig that inoculate 100 TCID50 more respectively breathe and breeding syndrome virus, pig circular ring virus.The result show this product to Pseudorabies virus, foot and mouth disease virus, swine fever virus, swine influenza virus, pig parvoviral, pig breathe with the breeding syndrome virus, that pig circular ring virus infects caused cytopathy is all inhibited.After the cell inoculation virus that the compound gene engineering product of pig interferon α that obtains without embodiment 2 and γ is handled, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus that pig interferon α that embodiment 2 obtains and the compound gene engineering product of γ are handled, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Embodiment 7
The propagation of Bov IFN α that is obtained by embodiment 3 and the compound gene engineering product of γ can suppress 100 TCID50 on bovine kidney cells (MDBK) hostis pecoris, bovine ephemeral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, bovine parvovirus.On the trace Tissue Culture Plate of 96 holes, use DMEM culture medium culturing bovine kidney cells (MDBK), 5% CO
2After being cultured to cell under 37 ℃ of conditions of incubator and growing up to monolayer, Bov IFN α and the compound gene engineering product of γ with various dose are handled, inhale after 24 hours and abandon, inoculate hostis pecoris, bovine ephemeral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, the bovine parvovirus of 100 TCID50 more respectively.It is all inhibited that the result shows that this product infects caused cytopathy to hostis pecoris, bovine ephemeral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, bovine parvovirus.After the cell inoculation virus that the compound gene engineering product of Bov IFN α that obtains without embodiment 3 and γ is handled, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus that Bov IFN α that embodiment 3 obtains and the compound gene engineering product of γ are handled, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
Embodiment 8
The propagation of dog interferon alpha that is obtained by embodiment 4 and the compound gene engineering product of γ can suppress 100 TCID50 on Madin-Darby canine kidney(cell line) (MDCK) canine distemper virus, Canine Parvovirus, canine infectious hepatitis virus, canine parainfluenza virus, canine alphaherpesvirus, canine coronavirus.On the trace Tissue Culture Plate of 96 holes, use DMEM culture medium culturing Madin-Darby canine kidney(cell line) (MDCK), 5% CO
2After being cultured to cell under 37 ℃ of conditions of incubator and growing up to monolayer, dog interferon alpha and the compound gene engineering product of γ with various dose are handled, inhale after 24 hours and abandon, inoculate canine distemper virus, Canine Parvovirus, canine infectious hepatitis virus, canine parainfluenza virus, canine alphaherpesvirus, the canine coronavirus of 100 TCID50 more respectively.The result shows that this product is all inhibited to canine distemper virus, Canine Parvovirus, canine infectious hepatitis virus, canine parainfluenza virus, canine alphaherpesvirus, the caused cytopathy of canine Coronavirus Infection.After the cell inoculation virus that the compound gene engineering product of dog interferon alpha that obtains without embodiment 4 and γ is handled, cell rounding all occurs, come off, pathological changes such as disintegrate.And after the cell inoculation virus that dog interferon alpha that embodiment 4 obtains and the compound gene engineering product of γ are handled, under inverted microscope, observed 7 days continuously, cellular morphology is normal, any pathological changes do not occur.
More than animal interferon compound provided by the present invention and its production and application is described in detail, used specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof; Simultaneously, for one of ordinary skill in the art, according to thought of the present invention, the part that all can change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.
Sequence table
＜110〉Zhou Jichen
＜120〉animal interferon compound and its production and application
<160>2
<210>1
<211>582
<212>DNA
＜213〉artificial sequence
<400>1
atggctgtgc?ctgcaagccc?acagcaccca?cggggctacg?gcatcctgct?gctcaccctc 60
cttctgaaag?ctctcgccac?caccgcctcc?gcctgcaacc?accttcgccc?acaggatgcc 120
accttctctc?acgacagcct?ccagctcttc?cgggacatgg?ctccaacact?gctccagctg 180
tgcccacagc?acaacgcgtc?ttgctccttc?aacgacacca?tcctggacac?cagcaacacc 240
cagcaagccg?acaaaaccac?ccacgacatc?cttcagcacc?tcttcaaaat?cctcagcagc 300
ccaagcactc?cagcccactg?gaacgacagc?caacgccaaa?gcctcctcaa?ccgcatccac 360
cgctacaccc?agcacctcga?gcaatgcttg?gacagcagcg?acacccgctc?ccggacccgc 420
tggcctcgca?accttcacct?caccatcaaa?aaacacttca?gctgcctcca?caccatcctc 480
caagacaacg?attacagcgc?ctgcgcctgg?gaacacgtcc?gcctgcaagc?tcgtgcctgg 540
ttcctgcaca?tccacaacct?cacaggcaac?acccgcactt?aa 582
<210>2
<211>495
<212>DNA
＜213〉artificial sequence
<400>2
atgacttgcc?agacttacaa?cttgtttgtt?ctgtctgtca?tcatgattta?ttatggccat 60
actgcaagta?gtctgaatct?tgttcaactt?caagatgata?ttgacaaact?gaaagctgac 120
tttaactcaa?gtcattcaga?tgtagctgac?ggtggcccta?ttattgtaga?gaaactgaag 180
aactggacag?agcgcaatga?gaaacgcatc?attctgagcc?agattgtttc?gatgtacttg 240
gaaatgcttg?aaaacactga?caagtcaaag?ccgcacatca?aacacatttc?tgaggagctc 300
tatactctga?aaaacaacct?tcctgatggc?gtgaagaagg?tgaaagatat?catggacctg 360
gccaagctcc?cgatgaacga?cttgcgcatc?cagcgcaaag?ccgcgaatga?actcttcagc 420
atcttacaga?agctggtgga?tcctccgagt?ttcaaacgca?aacgcagcca?gtctcagcgc 480
cgctgcaatt?gctaa 495
Claims (7)
1. an animal interferon compound is characterized in that, makes by following steps:
A, obtain animal interferon α and γ gene, make up recombinant cloning vector;
The selection of B, transfer vector or expression vector and structure;
C, conversion or transfecting host bacterium or cell;
D, expression product evaluation, purification and decomposition.
2. animal interferon compound as claimed in claim 1 is characterized in that, described animal interferon α and γ gene have the nucleotide sequence of sequence 1 and sequence 2 in the sequence table respectively.
3. the preparation method of an animal interferon compound is characterized in that, is made up of following steps:
A, obtain animal interferon α and γ gene, make up recombinant cloning vector: synthetic animal interferon α and γ nucleotide sequence, perhaps design Auele Specific Primer, two ends add restriction enzyme site, by RT-PCR animal interferon α and γ gene are come out from amplification through total RNA of inductive peripheral blood lymphocyte or splenocyte extraction, be connected respectively to the T carrier;
B, the selection of transfer vector or expression vector and structure: selection can be by the transfer vector or the expression vector with ampicillin or kanamycin or gentamycin or tetracycline resistance that contain His * 6 labels of Lac Z genescreen, expression vector is selected prokaryotic expression carrier or carrier for expression of eukaryon for use, to from the T carrier that has made up, behind double digestion, be connected to same expression vector respectively with kind animal interferon α and γ gene, place under the same promoter control, the gene reading frame is correct and be 3 integral multiple, and inserts thrombin between two genes or enterokinase is cut the site;
C, conversion or transfecting host bacterium or cell: with transfer vector and the expression vector homologous recombination that builds, Screening and Identification positive expression plasmid, utilize calcium phosphate precipitation method or liposome to infect methods such as method or electrotransformation, with the expression vector that has built, conversion or transfection are in protokaryon or eucaryon host body, by PCR or plasmid enzyme restriction 1% agarose gel electrophoresis or sequencing identify errorless after, carry out the screening of abduction delivering and condition optimizing;
D, expression product evaluation, purification and decomposition: expression product is through nickel affinity chromatography or monoclonal antibody coupling carrier granule chromatography purification, the SDS-PAGE electrophoresis is identified, the purification expression product is through thrombin or enterokinase digestion, a protein molecule is resolved into two parts, promptly get the complex of interferon-ALPHA molecule and interferon gamma molecule.
4. claim 1 or the 2 described animal interferon compounds application in inhibition Avian pneumo-encephalitis virus, bird flu virus or infectious bursa diseases virus duplicate in chick embryo fibroblast.
5. the described animal interferon compound of claim 1 is suppressing Pseudorabies virus, foot and mouth disease virus, swine fever virus, swine influenza virus, pig parvoviral, pig breathing and breeding syndrome virus, the application of pig circular ring virus in porcine kidney cell or Merc-145 time multiplexed cell system.
6. the application of the described animal interferon compound of claim 1 in inhibition bovine ephemeral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, bovine parvovirus duplicate in bovine kidney cells.
7. the application of the described animal interferon compound of claim 1 in inhibition canine distemper virus, Canine Parvovirus, canine infectious hepatitis virus, canine parainfluenza virus, canine alphaherpesvirus, canine coronavirus duplicate in Madin-Darby canine kidney(cell line).
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|---|---|---|---|
| CN 200610103821 CN1887344A (en) | 2006-07-27 | 2006-07-27 | Animal interferon compound and its prepn and application |
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| CN 200610103821 CN1887344A (en) | 2006-07-27 | 2006-07-27 | Animal interferon compound and its prepn and application |
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| CN1887344A true CN1887344A (en) | 2007-01-03 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7682619B2 (en) | 2006-04-06 | 2010-03-23 | Cornell Research Foundation, Inc. | Canine influenza virus |
| CN108276486A (en) * | 2015-04-23 | 2018-07-13 | 中国农业科学院生物技术研究所 | 2 interferon mutants of cat ω and its preparation method and application |
| CN117050158A (en) * | 2023-10-10 | 2023-11-14 | 云南农业大学 | Application of red mouth gull IFN-gamma gene and recombinant protein encoded by same |
-
2006
- 2006-07-27 CN CN 200610103821 patent/CN1887344A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7682619B2 (en) | 2006-04-06 | 2010-03-23 | Cornell Research Foundation, Inc. | Canine influenza virus |
| CN108276486A (en) * | 2015-04-23 | 2018-07-13 | 中国农业科学院生物技术研究所 | 2 interferon mutants of cat ω and its preparation method and application |
| CN108276486B (en) * | 2015-04-23 | 2021-06-15 | 中国农业科学院生物技术研究所 | Cat omega 2 interferon mutant and preparation method and application thereof |
| CN117050158A (en) * | 2023-10-10 | 2023-11-14 | 云南农业大学 | Application of red mouth gull IFN-gamma gene and recombinant protein encoded by same |
| CN117050158B (en) * | 2023-10-10 | 2023-12-29 | 云南农业大学 | Application of red mouth gull IFN-gamma gene and recombinant protein encoded by same |
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