CN101612389B - Antiviral drug combination for livestock - Google Patents
Antiviral drug combination for livestock Download PDFInfo
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- CN101612389B CN101612389B CN2009100414186A CN200910041418A CN101612389B CN 101612389 B CN101612389 B CN 101612389B CN 2009100414186 A CN2009100414186 A CN 2009100414186A CN 200910041418 A CN200910041418 A CN 200910041418A CN 101612389 B CN101612389 B CN 101612389B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
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Abstract
An antiviral drug combination for livestock in the invention relates to an antiviral agent, in particular to an antiviral agent for poultry and livestock. The drug combination is composed of cyanovirin and animal IFN-alpha in mass ratio of 3:1. The invention initially applies cyanovirin to treating viral infection of poultry and livestock, and animal IFN-alpha which is prepared by gene recombination, is contained in species such as pig, chicken, cattle and the like and has high valence, high purity and antiviral effect and cyanovirin with direct antiviral effect are initially applied in a combining way; meanwhile the effect of cyanovirin is direct and rapid and the antiviral effect of IFN-alpha is wide and permanent, and the effect is better than that of single use of cyanovirin or IFN-alpha.
Description
[technical field]
The present invention relates to a kind of antiviral agent, relate in particular to a kind of antiviral agent that is used for domestic birds and animals.
[background technology]
IFN-α is one and has the cytokine that very strong antiviral activity is, its extensive use in treatment people's viral disease, and become a kind of essential therapy.Such as, in therapy for hepatitis C, IFN-α plays irreplaceable effect.Because the antiviral effect of IFN-α is indirect, be through inducing intravital other antiviral-mechanism and acting, its important feature is not have virus-specific, that is to say that IFN-α has the activity of anti-different virus; But the another one important feature of IFN-α antivirus action is a species specificity, that is to say the IFN-α from some kinds, the virus that only that kind is infected effectively, and to all the other kinds infect viral invalid.Such as, people's IFN-α is only effective to people's viral infection, and other kind is comprised that pig, cattle, chicken etc. are then invalid.The viral infection of anti-other these kinds then need prepare the special IFN-α of kind separately.IFN-α can be stimulated and produces through interferon inducers (such as new castle disease virus, PHA, Radix Astragali extract etc.) by the leukocyte of people or different genera animal.But tiring of this type LeIF is often lower, and all is the semifinished product that does not add purification generally, so general curative effect is relatively poor, also has side effect unavoidably; Have to human in early days, forbid many years ago to human.LeIF for animals is also popular, but preferably prepares gene recombinaton interferon, to guarantee best curative effect, minimum toxic and side effects.Because the IFN-α gene order between the different genera is different, so just need clone respectively and the IFN-α of expression and purification different genera.
Blue algae antiviral protein matter is that of separation and purification has the active phytoprotein of high anti-viral in the cyanophyceae, for the ease of preparing at present through engineered method production in a large number.The initial blue algae antiviral protein of finding has the effect of anti-HIV, has confirmed afterwards that it also had the activity of multiple viruses such as resisiting influenza virus, herpesvirus, hemorrhagic fever virus, hepatitis C virus.But because it is a phytoprotein, can produce antibody response after getting into human body, the antibody of the generation cyanophycin of follow-up entering that can neutralize, thus weaken its antivirus action.That is to say that although cyanophycin matter has a good antiviral activity external, the application of its interior resisting virus receives very big restriction always.Report is to make vaginal suppository with blue algae antiviral protein at present, and prevention and treatment HIV infect, and effect is more sure; The somebody is studying how to reduce its immunogenicity, but practical method is fewer.In view of these characteristics of blue algae antiviral protein, and short characteristic of animal life cycle, the viral infection that the present invention is used to treat animal to blue algae antiviral protein matter, curative effect is desirable.The trophophase of chicken and duck had only about 6 weeks, and the trophophase of pig and cattle also has only some months; And the infection of the often easy trouble acute viral of these fowl poultrys, such as fowl plague, swine fever, foot and mouth disease, reproductive and respiratory syndrome etc.From using blue algae antiviral protein matter often to need one month to producing specific antibody; Producing high specific antibody of tiring then needs repeatedly to use; That is to say the repeatedly use that needs some months, the acute viral disease that is actually treatment fowl domestic animal does not need repeatedly can life-time service yet.Therefore, though use blue algae antiviral protein matter treatment people's viral infection that its limitation is arranged, be well suited for treating the viral infection of fowl domestic animal, especially the acute viral of fowl domestic animal infects.
[summary of the invention]
The present invention aims to provide a kind of fowl poultry that can effectively treat fowl poultry virosis and uses antiviral composition.
It is that 3: 1 blue algae antiviral protein and animal IFN-α forms by mass ratio that described fowl poultry is used antiviral composition.It can be used to prepare the medicine of treatment fowl poultry with virosis.Wherein, animal IFN-α selects the IFN-α of corresponding kind according to the fowl poultry kind of treatment.
After fowl poultry mixes with 5: 2 mass ratio with antiviral composition and the egg powder that contains anti-specificity virus IgY, can be used to prepare and treat fowl and raise oral drug with virosis.
Wherein, described blue algae antiviral protein makes by following method:
(1) from cyanophyceae, extract total RNA, through the genetic fragment of RT-PCR amplification blue algae antiviral protein, the aminoacid sequence of blue algae antiviral protein is:
LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQPSNFIETCRNTQLAGSSELAAECKTRAQQFVSTKINLDDHIANIDGTLKYE。
The nucleotide sequence of blue algae antiviral protein is:
cttggtaaattctcccagacctgctacaactccgctatccagggttccgttctgacctccacctgcgaacgtaccaacggtggttacaacacctcctccatcgacctgaactccgttatcgaaaacgttgacggttccctgaaatggcagccgtccaacttcatcgaaacctgccgtaacacccagctggctggttcctccgaactggctgctgaatgcaaaacccgtgctcagcagttcgtttccaccaaaatcaacctggacgaccacatcgctaacatcgacggtaccctgaaatacgaataa
(2) after order-checking confirms that sequence is errorless, be cloned into pET26b (+) carrier to above-mentioned nucleotide sequence, in E.coli BL21 competent cell, express;
(3) engineering bacteria gets final product with DEAD glucosan ion-exchange chromatography purification through IPTG abduction delivering solubility antiviral protein.
Described animal IFN-α makes by following method:
(1) from animal spleen, extracts total RNA, through the genetic fragment of RT-PCR amplification animal IFN-α;
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
When being used to prepare the virus diseases of pigs medicine, described animal IFN-α is pig IFN-α, and it is to make by following method:
(1) from pig spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification pig, wherein,
Pig IFN-α aminoacid sequence be:
MAPTSAFLTALVLLSCNAICSLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWE IVRAEVMRSFSSSRNLQDRLRKKE.
The nucleotide sequence of pig IFN-α is:
atggccccaacctcagccttcctcacggccctggtgctactcagctgcaatgccatctgctctctgggctgtgacctgcctcagacccacagcctggctcacaccagggccctgaggctcctggcacaaatgaggagaatctctcccttctcctgcctggaccacagaagggactttggatcccctcatgaggcttttgggggcaaccaggtccagaaggctcaagccatggctctggtgcatgagatgctccagcagaccttccagctcttcagcacagagggctcggctgctgcctggaatgagagcctcctgcaccagttctgcactggactggatcagcagctcagggacctggaagcctgtgtcatgcaggaggcggggctggaagggacccccctgctggaggaggactccatcctggctgtgaggaaatacttccacagactcaccctctatctgcaagagaagagctacagcccctgtgcctgggagatcgtcagggcagaagtcatgagatccttctcttcctccagaaacctgcaagacagactcaggaagaaggagtga
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
When being used to prepare cattle disease viral disease medicine, described animal IFN-α is cattle IFN-α, and it is to make by following method:
(1) from cattle spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification cattle, wherein,
Cattle IFN-α aminoacid sequence be:
MAPAWSFLLSLLLLSCNAICSLGCHLPHTHSLANRRVLMLLQQLRRVSPSSCLQDRNDFEFLQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSPATWDKSLLDKLRAALDQQLTDLQACLTQEEGLRGAPLLKEDSSLAVRKYFHRLTLYLQEKRHSPCAWEVVRAEVMRAFSSSTNLQESFRRKD.
The nucleotide sequence of cattle IFN-α is:
atggccccagcctggtccttcctgctatccctgttgctgctcagctgcaacgccatctgctctctgggttgccacctgcctcacacccacagcctggccaacaggagggtcctgatgctcctgcaacaactgagaagggtctccccttcctcctgcctgcaggacagaaatgacttcgaattcctccaggaggctctgggtggcagccagttgcagaaggctcaagccatctctgtgctccacgaggtgacccagcacaccttccagctcttcagcacagagggctcgcccgccacgtgggacaagagcctcctggacaagctacgcgctgcgctggatcagcagctcactgacctgcaagcctgtctgacgcaggaggaggggctgcgaggggctcccctgctcaaggaggactccagcctggctgtgaggaaatacttccacagactcactctctatctgcaagagaagagacacagcccttgtgcctgggaggttgtcagagcagaagtcatgagagccttctcttcctcaacaaacttgcaggagagtttcaggagaaaggactga
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
When being used to prepare the chicken virus medicine, described animal IFN-α is chicken IFN-α, and it is to make by following method:
(1) from chicken spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification chicken, wherein,
Chicken IFN-α aminoacid sequence be:
MAVPASPQHPRGYGILLLTLLLKALATTASACNHLRSQDATFSHDSLQLLRDMAPTLPQLCPQHNASCSFNDTILDTSNTRQADKTTHDILQHLFKILSSPSTPAHWNDSQRQSLLNRIHRYTQHLEQCLDSSDTRSRTRWPRNLHLTIKKHFSCLHTFLQDNDYSACAWEHVRLQARAWFLHIHNLTGNTRT
The nucleotide sequence of chicken IFN-α is:
atggctgtgcctgcaagcccacagcacccacgggggtacggcatcctgctgctcacgctccttctgaaagctctcgccaccaccgcctccgcctgcaaccaccttcgctcccaggatgccaccttctctcacgacagcctccagctcctccgggacatggctcccacactaccccagctgtgcccacagcacaacgcgtcttgctccttcaacgacaccatcctggacaccagcaacacccggcaagccgacaaaaccacccacgacatccttcagcacctcttcaaaatcctcagcagccccagcactccagcccactggaacgacagccaacgccaaagcctcctcaaccggatccaccgctacacccagcacctcgagcaatgcttggacagcagcgacacgcgctcccggacgcgatggcctcgcaaccttcacctcaccatcaaaaaacacttcagctgcctccacaccttcctccaagacaacgattacagcgcctgcgcctgggaacacgtccgcctgcaagctcgtgcctggttcctgcacatccacaacctcacaggcaacacgcgcacttag
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
The present invention is used to blue algae antiviral protein to treat the viral infection of fowl domestic animal first, and the height for preparing the method with gene recombinaton is first tired, highly purified animal IFN-α with antivirus action and the blue algae antiviral protein Combined application that direct antivirus action is arranged from different generas such as pig, chicken, cattle; The effect of antiviral protein directly fast, the antivirus action of IFN-α is extensive and long-term, all good than independent use antiviral protein or IFN-α.
In addition, the preparation purification process of blue algae antiviral protein of the present invention, the product purity of gained is up to more than 98%, and output is 0.7 gram up to every liter.The preparation purification process of described animal IFN-α, the product purity of gained are up to 97%, and output is 0.5 gram up to every liter.
[specific embodiment]
Embodiment one
One, preparation blue algae antiviral protein
(1) from cyanophyceae, extract total RNA, through the genetic fragment of RT-PCR amplification blue algae antiviral protein, the aminoacid sequence of blue algae antiviral protein is:
LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQPSNFIETCRNTQLAGSSELAAECKTRAQQFVSTKINLDDHIANIDGTLKYE。
The nucleotide sequence of blue algae antiviral protein is:
cttggtaaattctcccagacctgctacaactccgctatccagggttccgttctgacctccacctgcgaacgtaccaacggtggttacaacacctcctccatcgacctgaactccgttatcgaaaacgttgacggttccctgaaatggcagccgtccaacttcatcgaaacctgccgtaacacccagctggctggttcctccgaactggctgctgaatgcaaaacccgtgctcagcagttcgtttccaccaaaatcaacctggacgaccacatcgctaacatcgacggtaccctgaaatacgaataa
(2) after order-checking confirms that sequence is errorless, be cloned into pET26b (+) carrier to above-mentioned nucleotide sequence, in E.coli BL21 competent cell, express;
(3) engineering bacteria is through IPTG abduction delivering solubility antiviral protein, with DEAD glucosan ion-exchange chromatography purification.
Two, preparation pig IFN-α
(1) from pig spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification pig, wherein,
Pig IFN-α aminoacid sequence be:
MAPTSAFLTALVLLSCNAICSLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSRNLQDRLRKKE.
The nucleotide sequence of pig IFN-α is:
atggccccaacctcagccttcctcacggccctggtgctactcagctgcaatgccatctgctctctgggctgtgacctgcctcagacccacagcctggctcacaccagggccctgaggctcctggcacaaatgaggagaatctctcccttctcctgcctggaccacagaagggactttggatcccctcatgaggcttttgggggcaaccaggtccagaaggctcaagccatggctctggtgcatgagatgctccagcagaccttccagctcttcagcacagagggctcggctgctgcctggaatgagagcctcctgcaccagttctgcactggactggatcagcagctcagggacctggaagcctgtgtcatgcaggaggcggggctggaagggacccccctgctggaggaggactccatcctggctgtgaggaaatacttccacagactcaccctctatctgcaagagaagagctacagcccctgtgcctgggagatcgtcagggcagaagtcatgagatccttctcttcctccagaaacctgcaagacagactcaggaagaaggagtga
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, the collection culture fluid, from supernatant through deae dextran ion-exchange chromatography purification.
Three, blue algae antiviral protein and pig IFN-α are pressed mass ratio: mix at 3: 1.
Four, oral antivirus test
(1) oral antivirus test:
The antiviral composition of step 3 gained is mixed with the mass ratio of the egg powder that contains anti-specificity virus IgY by 5: 2, forms antiviral oral formulations (fast), be used to treat the pig that suffers from Schweineseuche to call the power of exempting from the following text:
30 of the pigs that catches a foot and mouth disease are divided 3 groups, every group each 10.Do not do any special treatment for first group, as negative control; Second group with bacterium KUAIKE spice (consumption is 5 gram/kilogram feedstuffs), as positive control; The 3rd group with the fast spice of the power of exempting from (consumption is 5 gram/kilogram feedstuffs), as the treatment group.After 5 days as a result, first group all dead, only deposits 2 for second group, all survives for the 3rd group.
(2) injection antivirus test:
With the antiviral composition lyophilizing of step 3 gained, process the antiviral injectable powder, be used to treat the pig that suffers from Schweineseuche:
30 of the pigs that catches a foot and mouth disease are divided 3 groups, every group each 10.Do not do any special treatment for first group, as negative control; Second group with foot and mouth disease antiserum 0.5ml/kg body weight subcutaneous injection, as positive control; The 3rd group of subcutaneous injection antiviral injectable powder (consumption is the 0.1g/kg body weight) is as the treatment group.After 3 days as a result, first group all dead, only deposits 4 for second group, all survives for the 3rd group.
Sequence table
< 110>Guangzhou Rosson Bioscience Co., Ltd.
< 120>one stud birds poultry is used antiviral composition
<160>4
<210>1
<211>306
<212>DNA
< 213>blue algae antiviral protein
<221>CDS
<222>(1)...(306)
<400>1
ctt ggt aaa ttc tcc cag acc tgc tac aac tcc gct atc cag ggt 45
Leu Gly Lys Phe Ser Gln Thr Cys Tyr Asn Ser Ala Ile Gln Gly
1 5 10 15
tcc gtt ctg acc tcc acc tgc gaa cgt acc aac ggt ggt tac aac 90
Ser Val Leu Thr Ser Thr Cys Glu Arg Thr Asn Gly Gly Tyr Asn
20 25 30
acc tcc tcc atc gac ctg aac tcc gtt atc gaa aac gtt gac ggt 135
Thr Ser Ser Ile Asp Leu Asn Ser Val Ile Glu Asn Val Asp Gly
35 40 45
tcc ctg aaa tgg cag ccg tcc aac ttc atc gaa acc tgc cgt aac 180
Ser Leu Lys Trp Gln Pro Ser Asn Phe Ile Glu Thr Cys Arg Asn
50 55 60
acc cag ctg gct ggt tcc tcc gaa ctg gct gct gaa tgc aaa acc 225
Thr Gln Leu Ala Gly Ser Ser Glu Leu Ala Ala Glu Cys Lys Thr
65 70 75
cgt gct cag cag ttc gtt tcc acc aaa atc aac ctg gac gac cac 270
Arg Ala Gln Gln Phe Val Ser Thr Lys Ile Asn Leu Asp Asp His
80 85 90
atc gct aac atc gac ggt acc ctg aaa tac gaa taa 306
Ile Ala Asn Ile Asp Gly Thr Leu Lys Tyr Glu ＊
95 100
<210>2
<211>570
<212>DNA
< 213>pig IFN-α
<221>CDS
<222>(1)...(570)
<400>2
atg gcc cca acc tca gcc ttc ctc acg gcc ctg gtg cta ctc agc 45
Met Ala Pro Thr Ser Ala Phe Leu Thr Ala Leu Val Leu Leu Ser
1 5 10 15
tgc aat gcc atc tgc tct ctg ggc tgt gac ctg cct cag acc cac 90
Cys Asn Ala Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln Thr His
20 25 30
agc ctg gct cac acc agg gcc ctg agg ctc ctg gca caa atg agg 135
Ser Leu Ala His Thr Arg Ala Leu Arg Leu Leu Ala Gln Met Arg
35 40 45
aga atc tct ccc ttc tcc tgc ctg gac cac aga agg gac ttt gga 180
Arg Ile Ser Pro Phe Ser Cys Leu Asp His Arg Arg Asp Phe Gly
50 55 60
tcc cct cat gag gct ttt ggg ggc aac cag gtc cag aag gct caa 225
Ser Pro His Glu Ala Phe Gly Gly Asn Gln Val Gln Lys Ala Gln
65 70 75
gcc atg gct ctg gtg cat gag atg ctc cag cag acc ttc cag ctc 270
Ala Met Ala Leu Val His Glu Met Leu Gln Gln Thr Phe Gln Leu
80 85 90
ttc agc aca gag ggc tcg gct gct gcc tgg aat gag agc ctc ctg 315
Phe Ser Thr Glu Gly Ser Ala Ala Ala Trp Asn Glu Ser Leu Leu
95 100 105
cac cag ttc tgc act gga ctg gat cag cag ctc agg gac ctg gaa 360
His Gln Phe Cys Thr Gly Leu Asp Gln Gln Leu Arg Asp Leu Glu
110 115 120
gcc tgt gtc atg cag gag gcg ggg ctg gaa ggg acc ccc ctg ctg 405
Ala Cys Val Met Gln Glu Ala Gly Leu Glu Gly Thr Pro Leu Leu
125 130 135
gag gag gac tcc atc ctg gct gtg agg aaa tac ttc cac aga ctc 450
Glu Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe His Arg Leu
140 145 150
acc ctc tat ctg caa gag aag agc tac agc ccc tgt gcc tgg gag 495
Thr Leu Tyr Leu Gln Glu Lys Ser Tyr Ser Pro Cys Ala Trp Glu
155 160 165
atc gtc agg gca gaa gtc atg aga tcc ttc tct tcc tcc aga aac 540
Ile Val Arg Ala Glu Val Met Arg Ser Phe Ser Ser Ser Arg Asn
170 175 180
ctg caa gac aga ctc agg aag aag gag tga 570
Leu Gln Asp Arg Leu Arg Lys Lys Glu ＊
185
<210>3
<211>534
<212>DNA
< 213>cattle IFN-α
<221>CDS
<222>(1)...(534)
<400>3
atg gcc cca gcc tgg tcc ttc ctg cta tcc ctg ttg ctg ctc agc tgc 48
Met Ala Pro Ala Trp Ser Phe Leu Leu Ser Leu Leu Leu Leu Ser Cys
1 5 10 15
aac gcc atc tgc tct ctg ggt tgc cac ctg cct cac acc cac agc ctg 96
Asn Ala Ile Cys Ser Leu Gly Cys His Leu Pro His Thr His Ser Leu
20 25 30
gcc aac agg agg gtc ctg atg ctc ctg caa caa ctg aga agg gtc tcc 144
Ala Asn Arg Arg Val Leu Met Leu Leu Gln Gln Leu Arg Arg Val Ser
35 40 45
cct tcc tcc tgc ctg cag gac aga aat gac ttc gaa ttc ctc cag gag 192
Pro Ser Ser Cys Leu Gln Asp Arg Asn Asp Phe Glu Phe Leu Gln Glu
50 55 60
gct ctg ggt ggc agc cag ttg cag aag gct caa gcc atc tct gtg ctc 240
Ala Leu Gly Gly Ser Gln Leu Gln Lys Ala Gln Ala Ile Ser Val Leu
65 70 75 80
cac gag gtg acc cag cac acc ttc cag ctc ttc agc aca gag ggc tcg 288
His Glu Val Thr Gln His Thr Phe Gln Leu Phe Ser Thr Glu Gly Ser
85 90 95
ccc gcc acg tgg gac aag agc ctc ctg gac aag cta cgc gct gcg ctg 336
Pro Ala Thr Trp Asp Lys Ser Leu Leu Asp Lys Leu Arg Ala Ala Leu
100 105 110
gat cag cag ctc act gac ctg caa gcc tgt ctg acg cag gag gag ggg 384
Asp Gln Gln Leu Thr Asp Leu Gln Ala Cys Leu Thr Gln Glu Glu Gly
115 120 125
ctg cga ggg gct ccc ctg ctc aag gag gac tcc agc ctg gct gtg agg 432
Leu Arg Gly Ala Pro Leu Leu Lys Glu Asp Ser Ser Leu Ala Val Arg
130 135 140
aaa tac ttc cac aga ctc act ctc tat ctg caa gag aag aga cac agc 480
Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Arg His Ser
145 150 155 160
cct tgt gcc tgg gag gtt gtc aga gca gaa gtc atg aga gcc ttc tct 528
Pro Cys Ala Trp Glu Val Val Arg Ala Glu Val Met Arg Ala Phe Ser
165 170 175
tcc tca aca aac ttg cag gag agt ttc agg aga aag gac tga 570
Ser Ser Thr Asn Leu Gln Glu Ser Phe Arg Arg Lys Asp ＊
180 185
<210>4
<211>546
<212>DNA
< 213>chicken IFN-α
<221>CDS
<222>(1)...(546)
<400>4
atg gct gtg cct gca agc cca cag cac cca cgg ggg tac ggc atc ctg 48
Met Ala Val Pro Ala Ser Pro Gln His Pro Arg Gly Tyr Gly Ile Leu
1 5 10 15
ctg ctc acg ctc ctt ctg aaa gct ctc gcc acc acc gcc tcc gcc tgc 96
Leu Leu Thr Leu Leu Leu Lys Ala Leu Ala Thr Thr Ala Ser Ala Cys
20 25 30
aac cac ctt cgc tcc cag gat gcc acc ttc tct cac gac agc ctc cag 144
Asn His Leu Arg Ser Gln Asp Ala Thr Phe Ser His Asp Ser Leu Gln
35 40 45
ctc ctc cgg gac atg gct ccc aca cta ccc cag ctg tgc cca cag cac 192
Leu Leu Arg Asp Met Ala Pro Thr Leu Pro Gln Leu Cys Pro Gln His
50 55 60
aac gcg tct tgc tcc ttc aac gac acc atc ctg gac acc agc aac acc 240
Asn Ala Ser Cys Ser Phe Asn Asp Thr Ile Leu Asp Thr Ser Asn Thr
65 70 75 80
cgg caa gcc gac aaa acc acc cac gac atc ctt cag cac ctc ttc aaa 288
Arg Gln Ala Asp Lys Thr Thr His Asp Ile Leu Gln His Leu Phe Lys
85 90 95
atc ctc agc agc ccc agc act cca gcc cac tgg aac gac agc caa cgc 336
Ile Leu Ser Ser Pro Ser Thr Pro Ala His Trp Asn Asp Ser Gln Arg
100 105 110
caa agc ctc ctc aac cgg atc cac cgc tac acc cag cac ctc gag caa 384
Gln Ser Leu Leu Asn Arg Ile His Arg Tyr Thr Gln His Leu Glu Gln
115 120 125
tgc ttg gac agc agc gac acg cgc tcc cgg acg cga tgg cct cgc aac 432
Cys Leu Asp Ser Ser Asp Thr Arg Ser Arg Thr Arg Trp Pro Arg Asn
130 135 140
ctt cac ctc acc atc aaa aaa cac ttc agc tgc ctc cac acc ttc ctc 480
Leu His Leu Thr Ile Lys Lys His Phe Ser Cys Leu His Thr Phe Leu
145 150 155 160
caa gac aac gat tac agc gcc tgc gcc tgg gaa cac gtc cgc ctg caa 528
Gln Asp Asn Asp Tyr Ser Ala Cys Ala Trp Glu His Val Arg Leu Gln
165 170 175
gct cgt gcc tgg ttc ctg cac atc cac aac ctc aca ggc aac acg cgc 576
Ala Arg Ala Trp Phe Leu His Ile His Asn Leu Thr Gly Asn Thr Arg
180 185 190
act tag 582
Thr ＊
193
Claims (9)
1. stud bird poultry use antiviral composition, it is characterized in that it is that 3: 1 blue algae antiviral protein and fowl is raiseeed IFN-α and forms by mass ratio, and wherein the aminoacid sequence of blue algae antiviral protein is:
LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSS IDLNSVIENVDGSLKWQPSNFIETCRNTQLAGSSELAAECKTRAQQFVSTKINLDDHIANIDGTLKYE。
2. one kind is used to treat the medicine that fowl is raiseeed virosis, it is characterized in that: it contains the described fowl poultry of claim 1 uses antiviral composition.
3. one kind prepares the method that is used to treat the medicine of fowl poultry virosis as claimed in claim 2, it is characterized in that described method for preparing is: the fowl poultry is mixed getting final product with 5: 2 mass ratio with antiviral composition and the egg powder that contains anti-specificity virus IgY.
4. fowl poultry as claimed in claim 1 is used antiviral composition, it is characterized in that described fowl poultry IFN-α makes by following method:
(1) from animal spleen, extracts total RNA, through the genetic fragment of RT-PCR amplification fowl poultry IFN-α;
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
5. fowl poultry as claimed in claim 1 is used antiviral composition, it is characterized in that described fowl poultry IFN-α is pig IFN-α, and it is to make by following method:
(1) from pig spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification pig, wherein,
Pig IFN-α aminoacid sequence be:
MAPTSAFLTALVLLSCNAICSLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGSPHEAFGGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGTPLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSRNLQDRLRKKE
The nucleotide sequence of pig IFN-α is:
atggccccaacctcagccttcctcacggccctggtgctactcagctgcaatgccatctgctctctgggctgtgacctgcctcagacccacagcctggctcacaccagggccctgaggctcctggcacaaatgaggagaatctctcccttctcctgcctggaccacagaagggactttggatcccctcatgaggcttttgggggcaaccaggtccagaaggctcaagccatggctctggtgcatgagatgctccagcagaccttccagctcttcagcacagagggctcggctgctgcctggaatgagagcctcctgcaccagttctgcactggactggatcagcagctcagggacctggaagcctgtgtcatgcaggaggcggggctggaagggacccccctgctggaggaggactccatcctggctgtgaggaaatacttccacagactcaccctctatctgcaagagaagagctacagcccctgtgcctgggagatcgtcagggcagaagtcatgagatccttctcttcctccagaaacctgcaagacagactcaggaagaaggagtga
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
6. fowl poultry as claimed in claim 1 is used antiviral composition, it is characterized in that described fowl poultry IFN-α is cattle IFN-α, and it is to make by following method:
(1) from cattle spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification cattle, wherein,
Cattle IFN-α aminoacid sequence be:
MAPAWSFLLSLLLLSCNAICSLGCHLPHTHSLANRRVLMLLQQLRRVSPSSCLQDRNDFEFLQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSPATWDKSLLDKLRAALDQQLTDLQACLTQEEGLRGAPLLKEDSSLAVRKYFHRLTLYLQEKRHSPCAWEVVRAEVMRAFSSSTNLQESFRRKD
The nucleotide sequence of cattle IFN-α is:
atggccccagcctggtccttcctgctatccctgttgctgctcagctgcaacgccatctgctctctgggttgccacctgcctcacacccacagcctggccaacaggagggtcctgatgctcctgcaacaactgagaagggtctccccttcctcctgcctgcaggacagaaatgacttcgaattcctccaggaggctctgggtggcagccagttgcagaaggctcaagccatctctgtgctccacgaggtgacccagcacaccttccagctcttcagcacagagggctcgcccgccacgtgggacaagagcctcctggacaagctacgcgctgcgctggatcagcagctcactgacctgcaagcctgtctgacgcaggaggaggggctgcgaggggctcccctgctcaaggaggactccagcctggctgtgaggaaatacttccacagactcactctctatctgcaagagaagagacacagcccttgtgcctgggaggttgtcagagcagaagtcatgagagccttctcttcctcaacaaacttgcaggagagtttcaggagaaaggactga
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
7. fowl poultry as claimed in claim 1 is used antiviral composition, it is characterized in that described fowl poultry IFN-α is chicken IFN-α, and it is to make by following method:
(1) from chicken spleen, extract total RNA, the genetic fragment of the IFN-α through RT-PCR amplification chicken, wherein,
Chicken IFN-α aminoacid sequence be:
MAVPASPQHPRGYGILLLTLLLKALATTASACNHLRSQDATFSHDSLQLLRDMAPTLPQLCPQHNASCSFNDTILDTSNTRQADKTTHDILQHLFKILSSPSTPAHWNDSQRQSLLNRIHRYTQHLEQCLDSSDTRSRTRWPRNLHLTIKKHFSCLHTFLQDNDYSACAWEHVRLQARAWFLHIHNLTGNTRT
The nucleotide sequence of chicken IFN-α is:
atggctgtgcctgcaagcccacagcacccacgggggtacggcatcctgctgctcacgctccttctgaaagctctcgccaccaccgcctccgcctgcaaccaccttcgctcccaggatgccaccttctctcacgacagcctccagctcctccgggacatggctcccacactaccccagctgtgcccacagcacaacgcgtcttgctccttcaacgacaccatcctggacaccagcaacacccggcaagccgacaaaaccacccacgacatccttcagcacctcttcaaaatcctcagcagccccagcactccagcccactggaacgacagccaacgccaaagcctcctcaaccggatccaccgctacacccagcacctcgagcaatgcttggacagcagcgacacgcgctcccggacgcgatggcctcgcaaccttcacctcaccatcaaaaaacacttcagctgcctccacaccttcctccaagacaacgattacagcgcctgcgcctgggaacacgtccgcctgcaagctcgtgcctggttcctgcacatccacaacctcacaggcaacacgcgcacttag
(2) after order-checking confirms that sequence is errorless, be cloned into the pIC9K carrier to above-mentioned nucleotide sequence, in Pichia pastoris carrier, express;
(3) through methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
8. fowl poultry as claimed in claim 5 is used antiviral composition, it is characterized in that it is used to prepare the medicine of treating virus diseases of pigs.
9. fowl poultry as claimed in claim 9 is used antiviral composition, it is characterized in that it is used to prepare the medicine of treating Schweineseuche.
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| CN107254482A (en) * | 2017-07-18 | 2017-10-17 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving Recombinant Swine interferon alpha fusion protein antiviral activity |
| CN111494604B (en) * | 2020-05-11 | 2022-05-06 | 中国药科大学 | Application of blue algae antiviral protein N in preparation of anti-inflammatory drugs |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962668A (en) * | 1995-04-27 | 1999-10-05 | The United States Of America As Represented By The Department Of Health And Human Services | Nucleic acids encoding antiviral proteins and peptides fused to effector proteins |
| CN101104851A (en) * | 2006-07-14 | 2008-01-16 | 金宁一 | Preparation for antiviral protein CV-N |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962668A (en) * | 1995-04-27 | 1999-10-05 | The United States Of America As Represented By The Department Of Health And Human Services | Nucleic acids encoding antiviral proteins and peptides fused to effector proteins |
| CN101104851A (en) * | 2006-07-14 | 2008-01-16 | 金宁一 | Preparation for antiviral protein CV-N |
Non-Patent Citations (1)
| Title |
|---|
| 庞峰等.蓝藻抗病毒蛋白N的研究进展.《中国海洋药物杂志》.2006,第25卷(第6期),第51-55页. * |
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