CN102079784A - Canine interferon-Alpha mutant - Google Patents
Canine interferon-Alpha mutant Download PDFInfo
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- CN102079784A CN102079784A CN2009102501865A CN200910250186A CN102079784A CN 102079784 A CN102079784 A CN 102079784A CN 2009102501865 A CN2009102501865 A CN 2009102501865A CN 200910250186 A CN200910250186 A CN 200910250186A CN 102079784 A CN102079784 A CN 102079784A
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- interferon alpha
- canine interferon
- canine
- mutant
- alpha mutant
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Abstract
The invention relates to a canine interferon-Alpha mutant which is prepared by modifying a natural canine interferon-Alpha by gene engineering means. Compared with the natural canine interferon-Alpha, the canine interferon-Alpha mutant has higher activity in vivo and in vitro. The test result indicates that the protein can be developed into veterinary medicines for treating canine virus diseases.
Description
One. technical field
The present invention relates to genetically engineered recombinant protein field, wherein this engineered protein is a kind of canine interferon alpha mutant.By to the molecular designing of native protein and genetic engineering modified, obtained having more highly active canine interferon alpha mutant.
Two. background technology
Dog in the mankind's life in occupation of important status, but the various virus diseases of dog class are also very serious, as canine parvovirus disease, canine distemper virus disease etc., and some becomes the cause of disease of zoonosis, causes serious problem.But in veterinary applications, also do not have a kind of specific medicament for the treatment of virus disease at present, it seems only have canine interferon alpha to address that need to a certain extent at present, it has the antiviral activity of wide spectrum.Therefore, market demand is developed the high interference of a kind of safe, effective, few side effects, stability Comprehensive Control canine viral disease usually.
Nineteen fifty-seven, Britain scientist Alick Isaacs and Iean Lindenmann find first a kind of can viral interference the material of breeding, be referred to as Interferon, rabbit (interferon, IFN).At present, recombinant human interferon alpha 2 goes on the market already, is widely used in treating viral infections such as hepatitis B, third liver as medical drugs, and has obtained good curative effect.
1987, Himmler etc. at first began the research work of canine interferon alpha (CalNF. α), had reported its gene order (GenBank M28624.1 GI:163973).Make it express canine interferon alpha in intestinal bacteria this gene recombination, recording biological activity on canine cells is 3 * 10
5U/mg.The scientific worker of China has also continuously mutually begun the research work of canine interferon alpha the nineties in last century, and has obtained a series of result of study at aspects such as its gene recombination, clone, expression, and the canine interferon alpha biological activity that obtains also only is 5 * 10
6About U/mg.
At present, the natural canine interferon alpha aminoacid sequence in the bibliographical information is identical, is the albumen that 164 amino acid are formed.In order to improve its biological activity, be applied to virus disease treatment field better, one aspect of the present invention finds that by the computer simulation molecular designing the 128th Tyr of former sequence is mutated into the formed space structure of Phe, more helps this molecule and receptors bind.The present invention adopts the method for genetically engineered rite-directed mutagenesis on the other hand, has obtained a series of single-point and multipoint mutation body, therefrom filters out the very high mutant of a kind of activity, and its former sequence also is that the 128th Tyr replaces with Phe.Then this mutant gene and gst gene are merged, in intestinal bacteria, express, by broken bacterium and purifying, obtained purity greater than 95% canine interferon alpha mutant.Carry out biological activity determination on Madin-Darby canine kidney(cell line), biological activity has reached 3～8 * 10
8U/mg is far above the specific activity level of present bibliographical information.This reduces the clinical consumption of canine interferon alpha greatly, has reduced its toxic side effect, makes the commercialization of this product become possibility, also becomes possibility for veterinary applications provides the high antiviral drug of inexpensive matter.
Three. summary of the invention
One aspect of the present invention relates to a kind of canine interferon alpha mutant, and it is the aminoacid sequence shown in the SEQ ID NO:1.Those of ordinary skills know, and described canine interferon alpha mutant can be by the method preparation of dna recombinant expression.In the present invention, described canine interferon alpha mutant is to obtain by the recombinant expressed method of genetically engineered.In one embodiment of the invention, obtain the canine interferon alpha mutant by the following method, comprise the steps:
1) structure of expression vector
According to known canine interferon alpha mutant aminoacid sequence (SEQ ID NO:1), select intestinal bacteria preference codon Totally-gene-synthesized dog alpha interferon mutant dna sequence dna for use, add restriction enzyme site at 5 ' end and 3 ' end simultaneously corresponding to coded aminoacid sequence, add EK enzyme restriction enzyme site, obtain the nucleotide sequence of encoding canine alpha interferon mutant.To handle well with restriction enzyme as the canine interferon alpha mutant and the screening plasmid pBSK of above-mentioned preparation respectively again, press certain mol proportion as calculated behind each sample concentration and add the T4 linked system.Downcut the target protein gene with Restriction Enzyme, again this gene is connected with the plasmid pGEX of Pharmacia company that handles well with Restriction Enzyme, obtain correct recon through screening.
2) expression of recombinant canine interferon alpha mutant and separation and purification
To be transformed into the expressive host bacterium as above-mentioned gained recon, go out the cance high-expression gene engineering bacteria, then through fermentation, the thalline of centrifugal collection target protein high expression level through expression screening.High-pressure homogeneous crusher machine thalline also centrifugally obtains containing purpose precursor protein supernatant liquor, with sample on the supernatant liquor to the GST affinity column, wash-out purpose precursor protein is further purified sample after also cutting with EK enzyme enzyme again by anion column, make the final purpose purity of protein greater than 98%.
3) the external biological determination of activity of recombinant canine interferon alpha mutant
Use Wish cell strain method to measure the biologic activity of recombinant canine interferon alpha mutant.
4) pharmacodynamics test in the body
Dosage according to 300,000 units/kg is treated the hundstaupe pyreticosis.
Four. embodiment
1. the structure of recombinant canine interferon alpha mutant precursor protein expression plasmid and the acquisition of engineering bacteria
The structure of expression vector
According to known recombinant canine interferon alpha mutant sequence (SEQ ID NO:1), select the dna sequence dna of the synthetic recombinant canine interferon alpha mutant of the full gene of intestinal bacteria preference codon for use, add restriction enzyme site at 5 ' end and 3 ' end simultaneously corresponding to coded aminoacid sequence, add EK enzyme restriction enzyme site, the nucleotide sequence of the recombinant canine interferon alpha mutant that obtains encoding.
GGA?TCC 
UGC?CAC?CUG?CCC?GAC?ACC?CAC?GGC?CUG?CGC
BanHI EK restriction enzyme site
AAC?UAC?CGC?GUG?CUG?ACC?CUC?UUG?GGC?GAG?AUG?CGC?AGG?CUG
AGC?GCC?GGC?AGC?UGC?GAC?CAC?UAC?ACC?AAC?GAC?UUC?GCU?UUC
CCU?AAG?CAG?UUG?UUC?GAU?GGG?GAA?AGG?CUC?GAG?CAA?GCA?GAG
GCC?CUG?UCC?GUG?GUC?CAC?GUU?AUG?ACC?GAG?AAA?GUG?UUU?CAU
CUU?UUC?UGC?CCC?GAU?ACU?UCC?AGC?GCG?CCU?UGG?AAC?AUG?ACA
CUG?CUC?CAA?CAG?CUG?UGC?AGC?GGC?CUU?UCU?CAG?GAG?CUG?GAU
GAC?UUA?CAA?GCC?UGC?CCU?CUA?GAG?CAG?GCC?GGG?CUC?GCU?CAA
ACC?CCU?CUC?AUG?CAC?CAG?GAU?AGC?ACC?CUC?CGC?ACU?UAC?UUC
GAG?CGC?AUC?UCC?CUG?UUU?CUU?GAG?GAC?AGA?AAC?CAU?ACC?CCC
UGC?GCC?UGG?CAG?AUG?GUG?AGG?GCA?CAG?AUC?GGC?AGG?AGC?UUC
UUU?UCC?AGU?ACU?AUU?CUG?GAA?CAA?CGC?AUA?CGU?CGC?AGG?AAG
TAA?TGA?
GAA?TTC
EcoRI
The purpose fragment reclaims small segment respectively behind BamH I, EcoR I double digestion, plasmid PGEX reclaims big fragment behind BamH I, EcoR I double digestion, 14 ℃ in T4 ligase enzyme system two sections target gene fragment connect the recombinant plasmid called after PGEX-IFN that obtains through screening.Use CaCl then
2Method transformed into escherichia coli BL21, be coated on the LB flat board that contains the 100ug/ml penbritin, selecting positive bacteria drops on when being cultured to OD600 for 0.6-0.8 among the LA and to add IPTG 0.1mM and induce 3-4 hour centrifugal collection thalline, occur the protein band about a 44KD behind the broken bacterium of 8M urea during the 15%SDS-PAGE electrophoresis, expression amount is 35% of a thalline soluble proteins.The engineering bacteria that efficiently expresses precursor protein that is obtained is called after PGEX-IFN/BL21 respectively.
2. the fermentation of recombinant canine interferon alpha mutant
1). shake bottle and express: the PGEX-IFN/BL21 that contains recombinant canine interferon alpha mutant precursor protein gene, in containing the LB substratum of 100ug/ml penbritin, shake bottle and spend the night (37 ℃, 200rpm), contain in the LB substratum of 100ug/ml penbritin by inoculation in 1: 30 again, cultivate after 3 hours for 37 ℃, add 0.1mMIPTG and induced 4 hours.Collect thalline through the SDS-PAGE electrophoretic analysis, the precursor protein of finding to contain the recombinant canine interferon alpha mutant (44KD) is based on solubility expression, and expression amount accounts for 30% of thalline soluble proteins.
2) zymotechnique:
A. substratum:
A) seed liquor substratum (LA):
Tryptones: 10 grams per liters, yeast powder: 5 grams per liters, sodium-chlor: 10 grams per liters, penbritin: 100 mcg/ml.
B) substratum (15 liters):
Sodium phosphate dibasic: 375 grams, potassium primary phosphate: 80 grams, sodium-chlor: 10. restrain,
Ammonium chloride: 45 grams, Tryptones: 50 grams.
Above composition is sterilized in jar together; After sterilizing separately, following composition adds fermentor tank.
Sal epsom: 15 grams, glucose: 150 grams,
Feed supplement (500 grams per liter): glucose
B. fermenting process:
(a) seed culture:
Draw from the plate identified and to get bacterial classification, be inoculated among 50 milliliters of (250 milliliters triangular flasks) LA, behind 37 degree, 200 rev/mins, 8 hours these 50 milliliters of seed liquor are transferred among 700 milliliters of LA, 36 degree, 200 rev/mins spend the night.
(b) fermenting process:
Cultured seed liquid (0D600=3-4) is added in the jar, mix up each parameter, 37 degree, 150 change, dissolved oxygen 100%, begin fermentation; Beginning adds 2 hours with 2.5 fast streams after 5 hours, and the IPTG that adds final concentration 0.3mM induces (five hours).
3. the purifying of recombinant canine interferon alpha mutant:
(1) affinity chromatography
Adopt GST Fast Flow chromatography media (GE company), balance liquid adopts 25mM Tris-HCl, pH8, thalline is through broken and centrifuging and taking supernatant, after the last sample balance, split Glutathione Sepharose chromatography column on the centrifugal supernatant of bacterium, wash fusion rotein with the elutriant that contains 10mM reduced glutathion (GSH) after the balance.
(2) enzymolysis
The elutriant of previous step adds 37 ℃ of enzymes of EK enzyme (5NIHU/mL) and cut 20 hours.
(3) anion-exchange chromatography
Adopt the Source30Q chromatography media, balance liquid is 20mM PB, pH7, and the samples with water dilution that previous step obtains is gone up sample for 1 times, adopts 20mM PB after the balance, pH7,0-1M NaCl, the gradient elution of 20 column volumes is collected the target protein elution peak.
4. detect
The recombinant canine interferon alpha mutant that obtains detects by SDS-PAGE purity detecting and reversed-phase HPLC, and purity is all greater than 95%.
5. external activity detects
Interferon, rabbit can stimulate some indicator cells (as people's amniotic epithelial cells Wish strain, people's laryngeal cancer cell strain Hep-2 and people's embryo flesh skin or lung monolayer cell) to produce antiviral protein; thereby make cell avoid the attack of vesicular stomatitis virus (VSV); according to the different dilution protective capabilities of testing sample, calculate interferon biological activity unit.The plain extent of dilution of high interference of avoiding the virus infringement with protection half (50%) cell is 1 interferon activity unit.Also can from typical curve, try to achieve the IFN activity unit of testing sample.
The result:
| Grouping | Specific activity (IU/mg) |
| The canine interferon alpha group | 5X10 6 |
| Recombinant canine interferon alpha mutant group | 5X10 8 |
As can be seen from the above table, use recombinant canine interferon alpha mutant exobiology activity apparently higher than natural canine interferon alpha.
The treatment canine distemper: 24 of test dogs are divided into 2 groups, 12 every group at random.A organizes negative control group, and the B group is reorganization dog interferon mutant treatment group.The A group is injecting normal saline behind 48h behind the inoculation canine distemper.Injection reorganization dog interferon mutant behind the 48h behind the B winding kind canine distemper, injected dose is 300,000 units/kg.Observed 7 days and write down clinical symptom, the statistics death toll.
| Group | The morbidity number of elements | Dead number of elements |
| A | 11 | 11 |
| B | 8 | 0 |
As can be seen from the above table, with recombinant canine interferon alpha mutant treatment canine distemper, can obviously improve the survival rate of disease dog.
Sequence table
＜110〉Li Weimin
＜120〉a kind of canine interferon alpha mutant
<160>1
<210>1
<211>164
<212>PRT
<213>SEQ?ID?NO：1
<400>1
Cys?His?Leu?Pro?Asp?Thr?His?Gly?Leu?Arg
1 5 10
Asn?Trp?Arg?Val?Leu?Thr?Leu?Leu?Gly?Gln
15 20
Met?Arg?Arg?Leu?Ser?Ala?Gly?Ser?Cys?Asp
25 30
His?Tyr?Thr?Asn?Asp?Phe?Ala?Phe?Pro?Lys
35 40
Glu?Leu?Phe?Asp?Gly?Gln?Arg?Leu?Gln?Glu
45 50
Ala?Gln?Ala?Leu?Ser?Val?Val?His?Val?Met
55 60
Thr?Gln?Lys?Val?Phe?His?Leu?Phe?Cys?Pro
65 70
Asp?Thr?Ser?Ser?Ala?Pro?Trp?Asn?Met?Thr
75 80
Leu?Leu?Glu?Glu?Leu?Cys?Ser?Gly?Leu?Ser
85 90
Glu?Gln?Leu?Asp?Asp?Leu?Glu?Ala?Cys?Pro
95 100
Leu?Gln?Glu?Ala?Gly?Leu?Ala?Glu?Thr?Pro
105 110
Leu?Met?His?Glu?Asp?Ser?Thr?Leu?Arg?Thr
115 120
Tyr?Phe?Gln?Arg?Ile?Ser?Leu?Phe?Leu?Gln
125 130
Asp?Arg?Asn?His?Ser?Pro?Cys?Ala?Trp?Glu
135 140
Met?Val?Arg?Ala?Glu?Ile?Gly?Arg?Ser?Phe
145 150
Phe?Ser?Ser?Thr?Ile?Leu?Gln?Glu?Arg?Ile
155 160
Arg?Arg?Arg?Lys
164
Claims (4)
1. a canine interferon alpha mutant (SEQ ID NO:1) is characterized in that the 128th Tyr replacement becoming Phe with natural canine interferon alpha aminoacid sequence.
1?chlpdthglr?nwrvltllgq?mrrlsagscd?hytndfafpk?elfdgqrlqe?aqalsvvhvm
61?tqkvfhlfcp?dtssapwnmt?lleelcsgls?eqlddleacp?lqeaglaetp?lmhedstlrt
121?yfqrislflq?drnhspcawe?mvraeigrsf?fsstilqeri?rrrk
2. a polynucleotide sequence (SEQ ID NO:2), the described canine interferon alpha mutant of its coding claim 1.
3. the canine interferon alpha mutant in the claim 1 uses intestinal bacteria to be host cell, and the method for using gene engineering is expressed.
4. a method for the treatment of the dog disease viral disease comprises to the canine interferon alpha mutant described in the claim 1 of described study subject administering therapeutic significant quantity.
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| CN2009102501865A CN102079784A (en) | 2009-11-30 | 2009-11-30 | Canine interferon-Alpha mutant |
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| CN2009102501865A CN102079784A (en) | 2009-11-30 | 2009-11-30 | Canine interferon-Alpha mutant |
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Family
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108117595A (en) * | 2018-02-28 | 2018-06-05 | 中国科学院微生物研究所 | The preparation and its application of a kind of canine interferon alpha |
| CN108912222A (en) * | 2018-08-02 | 2018-11-30 | 中国农业科学院北京畜牧兽医研究所 | A kind of recombination dog interferon CaIFN- λ and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376885A (en) * | 2008-10-07 | 2009-03-04 | 浙江大学 | Method for producing recombinant canine interferon alpha from cultivated silkworm |
-
2009
- 2009-11-30 CN CN2009102501865A patent/CN102079784A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376885A (en) * | 2008-10-07 | 2009-03-04 | 浙江大学 | Method for producing recombinant canine interferon alpha from cultivated silkworm |
Non-Patent Citations (1)
| Title |
|---|
| JUNZHI LI ETAL.: "Interferon-τand Interferon-αInterferon-αwith the Same Receptors in Bovine Endometrium", 《THE JOURNAL OF BIOUGICALC. HEMISTRY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108117595A (en) * | 2018-02-28 | 2018-06-05 | 中国科学院微生物研究所 | The preparation and its application of a kind of canine interferon alpha |
| CN108117595B (en) * | 2018-02-28 | 2020-06-26 | 中国科学院微生物研究所 | Preparation and application of canine α interferon |
| CN108912222A (en) * | 2018-08-02 | 2018-11-30 | 中国农业科学院北京畜牧兽医研究所 | A kind of recombination dog interferon CaIFN- λ and its application |
| CN108912222B (en) * | 2018-08-02 | 2020-06-19 | 中国农业科学院北京畜牧兽医研究所 | Recombinant canine interferon CaIFN-lambda and application thereof |
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Application publication date: 20110601 |