CN101612389A - One stud bird poultry is used antiviral composition - Google Patents
One stud bird poultry is used antiviral composition Download PDFInfo
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- CN101612389A CN101612389A CN200910041418A CN200910041418A CN101612389A CN 101612389 A CN101612389 A CN 101612389A CN 200910041418 A CN200910041418 A CN 200910041418A CN 200910041418 A CN200910041418 A CN 200910041418A CN 101612389 A CN101612389 A CN 101612389A
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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
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Abstract
Stud bird poultry of the present invention relates to a kind of antiviral agent with antiviral composition, relates in particular to a kind of antiviral agent that is used for domestic birds and animals.It is that 3: 1 blue algae antiviral protein and animal IFN-α forms by mass ratio.The present invention is used for the treatment of blue algae antiviral protein the viral infection of fowl domestic animal first, first tire with the height of the method for gene recombinaton preparation, highly purified animal IFN-α with antivirus action and the blue algae antiviral protein use in conjunction 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.
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, works by inducing intravital other antiviral-mechanism, 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, only the virus 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 needs to 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 class 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 difference between the different genera, 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 now by 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 entering human body, the antibody of the generation follow-up cyanophycin that enters 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 is very restricted 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 present invention is used for the treatment of the viral infection of animal, curative effect ideal to blue algae antiviral protein matter.The trophophase of chicken and duck had only about 6 weeks, and the trophophase of pig and cattle also has only some months; And these fowl poultry often easily suffers from acute viral and infects, 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.
Described fowl poultry is that 3: 1 blue algae antiviral protein and animal IFN-α forms with antiviral composition by mass ratio.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 the oral drug that fowl is raiseeed the usefulness virosis.
Wherein, described blue algae antiviral protein makes by the following method:
(1) extract total RNA from cyanophyceae, by 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, above-mentioned nucleotide sequence is cloned into pET26b (+) carrier, in E.coli BL21 competent cell, expresses;
(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 the following method:
(1) from animal spleen, extracts total RNA, by the genetic fragment of RT-PCR amplification animal IFN-α;
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 the following method:
(1) from pig spleen, extract total RNA, the genetic fragment of the IFN-α by 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, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 the following method:
(1) from cattle spleen, extract total RNA, the genetic fragment of the IFN-α by 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, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 the following method:
(1) from chicken spleen, extract total RNA, the genetic fragment of the IFN-α by 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, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 for the treatment of blue algae antiviral protein the viral infection of fowl domestic animal first, first tire with the height of the method for gene recombinaton preparation, highly purified animal IFN-α with antivirus action and the blue algae antiviral protein use in conjunction 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) extract total RNA from cyanophyceae, by 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, above-mentioned nucleotide sequence is cloned into pET26b (+) carrier, in E.coli BL21 competent cell, expresses;
(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 by 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, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 for the treatment of 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, make the antiviral injectable powder, be used for the treatment of 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〉stud bird 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 (10)
1, stud bird poultry is used antiviral composition, it is characterized in that it is that 3: 1 blue algae antiviral protein and animal IFN-α forms by mass ratio.
2, fowl poultry as claimed in claim 1 is used to prepare the medicine of treatment fowl poultry with virosis with antiviral composition.
3, fowl as claimed in claim 2 poultry is used to prepare the oral drug of treatment fowl poultry with virosis with antiviral composition, and its preparation method 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 blue algae antiviral protein makes by the following method:
(1) extract total RNA from cyanophyceae, by the genetic fragment of RT-PCR amplification blue algae antiviral protein, wherein the aminoacid sequence of blue algae antiviral protein is:
LGKFSQTCYNSAIQGSVLTSTCERTNGGYNTSSIDLNSVIENVDGSLKWQPSNFIETCRNTQLAGS
SELAAECKTRAQQFVSTKINLDDHIANIDGTLKYE。
The nucleotide sequence of blue algae antiviral protein is:
cttggtaaattctcccagacctgctacaactccgctatccagggttccgttctgacctccacctgc
gaacgtaccaacggtggttacaacacctcctccatcgacctgaactccgttatcgaaaacgttgac
ggttccctgaaatggcagccgtccaacttcatcgaaacctgccgtaacacccagctggctggttcc
tccgaactggctgctgaatgcaaaacccgtgctcagcagttcgtttccaccaaaatcaacctggac
gaccacatcgctaacatcgacggtaccctgaaatacgaataa
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into pET26b (+) carrier, in E.coli BL21 competent cell, expresses;
(3) engineering bacteria gets final product with DEAD glucosan ion-exchange chromatography purification through IPTG abduction delivering solubility antiviral protein.
5, fowl poultry as claimed in claim 1 is used antiviral composition, it is characterized in that described animal IFN-α makes by the following method:
(1) from animal spleen, extracts total RNA, by the genetic fragment of RT-PCR amplification animal IFN-α;
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 animal IFN-α is pig IFN-α, and it is to make by the following method:
(1) from pig spleen, extract total RNA, the genetic fragment of the IFN-α by RT-PCR amplification pig, wherein, pig IFN-α aminoacid sequence be:
MAPTSAFLTALVLLSCNAICSLGCDLPQTHSLAHTRALRLLAQMRRISPFSCLDHRRDFGSPHEAF
GGNQVQKAQAMALVHEMLQQTFQLFSTEGSAAAWNESLLHQFCTGLDQQLRDLEACVMQEAGLEGT
PLLEEDSILAVRKYFHRLTLYLQEKSYSPCAWEIVRAEVMRSFSSSRNLQDRLRKKE.
The nucleotide sequence of pig IFN-α is:
atggccccaacctcagccttcctcacggccctggtgctactcagctgcaatgccatctgctctctg
ggctgtgacctgcctcagacccacagcctggctcacaccagggccctgaggctcctggcacaaatg
aggagaatctctcccttctcctgcctggaccacagaagggactttggatcccctcatgaggctttt
gggggcaaccaggtccagaaggctcaagccatggctctggtgcatgagatgctccagcagaccttc
cagctcttcagcacagagggctcggctgctgcctggaatgagagcctcctgcaccagttctgcact
ggactggatcagcagctcagggacctggaagcctgtgtcatgcaggaggcggggctggaagggacc
cccctgctggaggaggactccatcctggctgtgaggaaatacttccacagactcaccctctatctg
caagagaagagctacagcccctgtgcctgggagatcgtcagggcagaagtcatgagatccttctct
tcctccagaaacctgcaagacagactcaggaagaaggagtga
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 animal IFN-α is cattle IFN-α, and it is to make by the following method:
(1) from cattle spleen, extract total RNA, the genetic fragment of the IFN-α by RT-PCR amplification cattle, wherein, cattle IFN-α aminoacid sequence be:
MAPAWSFLLSLLLLSCNAICSLGCHLPHTHSLANRRVLMLLQQLRRVSPSSCLQDRNDFEFLQEAL
GGSQLQKAQAISVLHEVTQHTFQLFSTEGSPATWDKSLLDKLRAALDQQLTDLQACLTQEEGLRGA
PLLKEDSSLAVRKYFHRLTLYLQEKRHSPCAWEVVRAEVMRAFSSSTNLQESFRRKD.
The nucleotide sequence of cattle IFN-α is:
atggccccagcctggtccttcctgctatccctgttgctgctcagctgcaacgccatctgctctctg
ggttgccacctgcctcacacccacagcctggccaacaggagggtcctgatgctcctgcaacaactg
agaagggtctccccttcctcctgcctgcaggacagaaatgacttcgaattcctccaggaggctctg
ggtggcagccagttgcagaaggctcaagccatctctgtgctccacgaggtgacccagcacaccttc
cagctcttcagcacagagggctcgcccgccacgtgggacaagagcctcctggacaagctacgcgct
gcgctggatcagcagctcactgacctgcaagcctgtctgacgcaggaggaggggctgcgaggggct
cccctgctcaaggaggactccagcctggctgtgaggaaatacttccacagactcactctctatctg
caagagaagagacacagcccttgtgcctgggaggttgtcagagcagaagtcatgagagccttctct
tcctcaacaaacttgcaggagagtttcaggagaaaggactga
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by 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 1 is used antiviral composition, it is characterized in that described animal IFN-α is chicken IFN-α, and it is to make by the following method:
(1) from chicken spleen, extract total RNA, the genetic fragment of the IFN-α by RT-PCR amplification chicken, wherein, chicken IFN-α aminoacid sequence be:
MAVPASPQHPRGYGILLLTLLLKALATTASACNHLRSQDATFSHDSLQLLRDMAPTLPQLCPQHNA
SCSFNDTILDTSNTRQADKTTHDILQHLFKILSSPSTPAHWNDSQRQSLLNRIHRYTQHLEQCLDS
SDTRSRTRWPRNLHLTIKKHFSCLHTFLQDNDYSACAWEHVRLQARAWFLHIHNLTGNTRT
The nucleotide sequence of chicken IFN-α is:
atggctgtgcctgcaagcccacagcacccacgggggtacggcatcctgctgctcacgctccttctg
aaagctctcgccaccaccgcctccgcctgcaaccaccttcgctcccaggatgccaccttctctcac
gacagcctccagctcctccgggacatggctcccacactaccccagctgtgcccacagcacaacgcg
tcttgctccttcaacgacaccatcctggacaccagcaacacccggcaagccgacaaaaccacccac
gacatccttcagcacctcttcaaaatcctcagcagccccagcactccagcccactggaacgacagc
caacgccaaagcctcctcaaccggatccaccgctacacccagcacctcgagcaatgcttggacagc
agcgacacgcgctcccggacgcgatggcctcgcaaccttcacctcaccatcaaaaaacacttcagc
tgcctccacaccttcctccaagacaacgattacagcgcctgcgcctgggaacacgtccgcctgcaa
gctcgtgcctggttcctgcacatccacaacctcacaggcaacacgcgcacttag
(2) after order-checking confirms that sequence is errorless, above-mentioned nucleotide sequence is cloned into the pIC9K carrier, in Pichia pastoris carrier, expresses;
(3) by methanol induction, cultivated 5 days, collect culture fluid, from supernatant, get final product through deae dextran ion-exchange chromatography purification.
9, fowl poultry as claimed in claim 6 is used to prepare the medicine for the treatment of virus diseases of pigs with antiviral composition.
10, fowl poultry as claimed in claim 9 is used to prepare the medicine for the treatment of Schweineseuche with antiviral composition.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548881A (en) * | 2013-10-23 | 2014-02-05 | 鞠志国 | Biological antiviral agent and preparation method thereof |
| 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 |
| CN111494604A (en) * | 2020-05-11 | 2020-08-07 | 中国药科大学 | Application of blue algae antiviral protein N in preparation of anti-inflammatory drugs |
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| 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 |
-
2009
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548881A (en) * | 2013-10-23 | 2014-02-05 | 鞠志国 | Biological antiviral agent and preparation method thereof |
| CN103548881B (en) * | 2013-10-23 | 2016-01-20 | 鞠志国 | Biological antiviral agent and preparation method thereof |
| 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 |
| CN111494604A (en) * | 2020-05-11 | 2020-08-07 | 中国药科大学 | Application of blue algae antiviral protein N in preparation of anti-inflammatory drugs |
| 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 |
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