CN108359004A - Dog recombinant interferon-λ 1 and the preparation method and application thereof - Google Patents
Dog recombinant interferon-λ 1 and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of dog recombinant interferon λ 1 and the preparation method and application thereof.The nucleotide sequence of dog recombinant interferon λ 1 is as shown in SEQ ID NO.1.The present invention also provides the preparation methods of dog recombinant interferon λ 1, utilize the recombination yeast engineering bacteria of dog recombinant interferon λ 1 gene of the Pichi strain expression system expression containing codon optimization of the present invention, obtain the high activity dog recombinant interferon λ 1 of secreting, expressing, 1 purity of dog recombinant interferon λ that the method for the present invention is prepared is high, reach 0.86mg/ml after purification, the protein yield of 1L recombination yeast engineering bacterias is 42mg.Antiviral specific activity is 2.85 × 107IU/mg can be effectively prevented and treated the communicable diseases such as canine distemper, canine parvovirus, canine parainfluenza virus disease, canine infectious hepatitis, have higher safety, application prospect good.
Description
Technical field
The invention belongs to field of biological product, disclose a kind of dog recombinant interferon-λ 1 of high activity, additionally provide simultaneously
The preparation method of high efficient expression dog recombinant interferon-λ 1 and application.
Background technology
In recent years, the pet industry in China rapidly develops, and pet dog, the quantity of cat are increasing, pet source viral blight
It is the more serious disease of current hazard ratio.The usually harm such as most common canine distemper, canine parvovirus disease is serious, and incidence is high,
Infectiousness is strong, and the death rate is high, is to endanger China's canine farming infectious disease the most serious, seriously constrains the development of pet industry.It passes
The therapeutic scheme of system is difficult clinically to prove effective, therefore the virosis for carrying out active treatment and preventing canine is paid close attention in the industry the most
Problem.
According to the difference of amino acid sequence and specific recognition receptor, IFN points are I types, II types and type III.Wherein I types are dry
It includes IFN-α, IFN-β, IFN- δ, IFN- κ, IFN- ω and IFN- τ to disturb element, and wherein IFN-α has more than 20 hypotypes, various
Structure is similar, and there is only the difference of a small amount of amino acid, IFN-α is current most widely used one of antiviral drugs.IFN-β only has
One hypotype.II types IFN is IFN-γ, has very strong immunoloregulation function；Interferon-λ is 2003 by American scientist
A kind of new forms of interferon that Kotenko etc. and Sheppard etc. have found jointly, since it has both I types interferon and interleukins
The double characteristic of (interleukin, IL) -10 family, therefore its member is named as IFN- λ 1 (IL-29), 2 (IL- of IFN- λ
28A) with IFN- λ 3 (IL-28B), biological effect is more similar to I types IFN, including antiviral, and immunological regulation and inhibition are swollen
The biological activities such as tumor proliferation.IFN- λ with its specific receptor heterodimeric complex (IFN- λ R1/IL-10R2) by tying
It closes, JAK-STAT signal transduction pathways can be mediated, induce the antiviral response of body, to play its antivirus action.Due to
The receptor of type III IFN is mainly distributed on surface epithelial cell, thus it is speculated that type iii interferon is mainly by epidermis and mucous membrane
Skin tissue prevents the invasion of virus from being mainly distributed on stomach, enteron aisle and lung in the tissue, in central nervous system point
Cloth is less, and this distribution can make its targeting more preferably, and reduces the side reaction in Clinical practice.
IFN- λ 1/IL-29 are made of the mature peptide of signal peptide and 178 amino acid containing 22 amino acid, molecular mass
About 20ku~33ku contains 2 disulfide bond, and there are 1 potential N- connections glycosylations on 65~67 amino acids residues
Site.IFN- λ 2/IL-28A and IFN- λ 3/IL-28B by signal peptide and 174 amino acid containing 22 amino acid maturation
Peptide forms, and contains 3 disulfide bond, molecular weight about 22ku, amino acid identity 81%.
Pichia pastoris is a kind of novel heterologous gene expression system, has multiple protein high efficient expression in such a system,
The problem of being difficult to remove there is no the endotoxin of prokaryotic expression system, also be not present mammalian cell expression system virus and
The problems such as mycoplasma contamination, and the post translational processings such as signal peptide shearing and glycosylation that can carry out similar higher eucaryote.Table
Alcohol oxidase promoter AOX1 up to carrier pPICZ α P can strictly be regulated and controled with methanol, and the N-terminal of expression cassette is with guiding secreting, expressing
Band α-factor signal peptide sequences, expression product can be secreted into supernatant, and it is that it is anti-with Zeocin that it, which is also gathered around there are one feature,
Property marker gene, to screening transformant work bring prodigious facility.Furthermore methanotrophic yeast expression system has height
The characteristics of expression, high stable, hypersecretion, host strain pichia pastoris yeast oneself protein secretory volume is few, downstream separation purifying
Operate it is easy, can large scale fermentation production, be a kind of eukaryotic expression system of suitable expression alien gene.
Invention content
The purpose of the present invention is to provide a kind of dog recombinant interferon-λ 1 that activity is high.
Another object of the present invention is to provide the preparation methods of the dog recombinant interferon-λ 1.
Third object of the present invention is to provide the application of dog recombinant interferon-λ 1 made from the above method.
In order to improve the expression quantity of albumen, the present invention carries out the nucleic acid sequence using Pichia pastoris preference codon
Codon optimization.The efficiency of excessively high, mRNA the secondary structure influence translation of G/C content in order to avoid translating the mRNA come, this
Invention using time preference password, connects certain amino acid very much on condition that password is had a preference in this time with most preference password frequency of use
Closely, amino acid sequence original sequence is constant, in the case of certain very special, in order to decrease or increase restriction enzyme site, certain positions
Sequence do sequence appropriate adjustment.Dog interferon-λ 1 genes of optimization design of the present invention codon optimization as a result,
Sequence is as shown in SEQ ID NO.1.
The present invention provides a kind of dog recombinant interferon-λ 1, and amino acid sequence is：
A) amino acid sequence shown in SEQ ID No.2；Or
B) amino acid sequence shown in SEQ ID No.2 is through replacing, lacking and oring add one or several amino acid residues
The amino acid sequence with same function formed.
The present invention provides the gene for encoding the dog recombinant interferon-λ 1, be it is following a) or b)：A) its nucleotide sequence
As shown in SEQ ID No.1 in sequence table；Or
B) nucleotide sequence shown in SEQ ID No.1 is substituted one or several nucleotide, and it is dry to obtain encoding canine recombination
Disturb the nucleotide sequence of element-λ 1.
The present invention provides the recombinant expression carriers containing said gene.
Wherein, above-mentioned recombinant expression carrier contains alcohol oxidase promoter (AOX) or formaldehyde dehydrogenase promoter
(Formate dehydrogenase, FMD).
Preferably, above-mentioned carrier contains alcohol oxidase promoter (AOX).
In an embodiment of the present invention, the recombinant expression carrier of structure is pPICZ α A-CaIFN- λ 1.
The present invention provides the host cells containing the gene or the recombinant expression carrier.
Preferably, the host cell is Pichia pastoris.It is highly preferred that the Pichia pastoris is Pichia pastoris
Strain X -33.
The present invention provides 1 genes of dog interferon-λ of above-mentioned codon optimization or the recombinant expression carriers or described
Application of the host cell in preparing broad-spectrum antiviral medicament.
Further, the virus is canine distemper virus, canine parvovirus, canine parainfluenza virus, dog infectious hepatitis
Poison, vesicular stomatitis virus.
The present invention provides 1 genes of dog interferon-λ of above-mentioned codon optimization or the recombinant expression carriers or described
Application of the host cell in preparing broad-spectrum antiviral preparation.
Further, the virus is canine distemper virus, canine parvovirus, canine parainfluenza virus, dog infectious hepatitis
Poison, vesicular stomatitis virus.
The present invention provides the lifes of 1 genes of dog recombinant interferon-λ containing above-mentioned codon optimization or its coding albumen
Object preparation or drug.
It is by nucleotide sequence such as SEQ ID NO.1 institutes the present invention also provides a kind of method preparing dog interferon-λ 1
Construction recombination plasmid in the 1 gene directed clonings to plasmid of dog interferon-λ shown, recombinant plasmid transformed to Pichia pastoris competence
Cell, Prepare restructuring bacterium, by determining that methanol induction condition, induced concentration, induction time, albumen harvest time etc. optimize yeast
Fermentation, expression, purified, effectively increased using the method for Chelating SFF (Ni) affinity chromatography after ultrafiltration concentration
The yield of product, to obtain the dog recombinant interferon-λ 1 of high yield, high-purity.
Present invention determine that the best induced expression condition of recombination yeast engineering bacteria X-33/pPICZ α A-CaIFN- λ 1 is, it will
The recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 of clone are inoculated in 20-30ml YPD culture mediums, 30 DEG C of 250rpm/min cultures
16~18h or so, until when thalline OD600 values are 6 or so, 1500~3 000g room temperatures centrifuge 5min respectively, discard culture medium, receive
Collect thalline.It is 2,28 DEG C that bacterial sediment, which is resuspended with 20mL BMMY culture mediums to OD600 values, 250rpm/min cultures, and every
Methanol, methanol final concentration 1% are added for 24 hours.After Fiber differentiation 96h, Chelating after culture supernatant is concentrated by ultrafiltration is collected
The method of SFF (Ni) affinity chromatography is purified.
In an embodiment of the present invention, being will be in the gene directed cloning to pPICZ α A of the dog IFN- λ 1 of codon optimization
Construction recombination plasmid pPICZ α A-CaIFN- λ 1, in conversion to Trans5 α competent cells, to obtain recombinant bacterium.To contain
The less salt LB plate screening transformants of Zeocin (25ug/ml), and PCR identifications are carried out, send company to be sequenced.Prepare Pichia yeast
The competent cell of strain X-33, by the electroporated conversion of the linearisation recombinant plasmid of SacI single endonuclease digestions to competent cell, coated plate
In MD tablets, 30 DEG C 2-3 days, choose monoclonal dibbling to MD and MM tablets, screen Mut+ type transformants, be then coated on containing 100 μ
The YPD tablets of g/ml, 250 μ g/ml and 500 μ g/ml Zeocin, the transformant copied using resistance screening height.Picking monoclonal
It is inoculated in BMGY culture mediums, 30 DEG C, 250rpm/min cultivates 18h or so, until thalline OD600 reaches 2~6, bacterium is collected by centrifugation
Body is resuspended with BMMY culture mediums, and it is 2.0 or so, 30 DEG C to be diluted to OD600 values, and 250rpm/min cultures are added every for 24 hours
100% methanol takes 1ml bacterium solutions respectively to final concentration of 0.5%, 1%, 1.5%, 2%, and in 0,24,48,72,96,120h, with
Best methanol induction concentration, the optimum expression time for determining expression, realize the induced expression of recombination yeast engineering bacteria.SDS-PAGE
Analysis and BCA methods quantify the destination protein of expression.Using the destination protein of Western blot identification expression.Using
The antiviral activity of MDCK-VSV system measurement dog recombinant interferons-λ 1.Using the dog recombinant interferon-λ 1 of expression according to 200,000
The sick dog of IU/kg dose subcutaneous injection, continuous injection 6 days, canine distemper, canine parvovirus disease, dog pair for preventing and treating dog
The epidemic diseases such as influenza virus disease, canine infectious hepatitis.
The beneficial effects of the present invention are：Present invention application pichia yeast expression system expresses dog recombinant interferon-λ 1,
The high activity dog recombinant interferon-λ 1 of secreting, expressing is obtained, purity reaches 0.86mg/ml, 1L recombination yeast engineering bacterias after purification
Protein yield be 42mg, antiviral specific activity be 2.85 × 107It is tiny can be effectively prevented and treated canine distemper, dog by IU/mg
The communicable diseases such as virus, canine parainfluenza virus disease, canine infectious hepatitis have higher safety, and can make on a large scale
Standby and purifying, application prospect are good.
Description of the drawings
Fig. 1 is the structure schematic diagram of 1 carriers of pPICZ α A-CaIFN- λ.
Fig. 2 is the 1 plasmid electrophoretograms of pPICZ α A-CaIFN- λ of linearisation, and swimming lane 1 is 1 matter of pPICZ α A-CaIFN- λ in figure
Grain, swimming lane 2 are 1 plasmids of pPICZ α A-CaIFN- λ of SacI linearisations, M:DL 5000plus DNA Marker.
Fig. 3 is the PCR qualification results of recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1.Swimming lane 1-5 is colone genome in figure
The pcr amplification product of DNA, N are the PCR product (negative control) of Pichia pastoris X33 genomic DNAs, and P is pPICZ α A-CaIFN-
The PCR product (positive control) of 1 plasmids of λ, M:DL,2000DNA Marker.
Fig. 4 is the SDS-PAGE qualification results of the expression product of recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1.Swimming lane 1-5
It is negative control (X33 induces culture supernatant when 72h) that the culture supernatant (10 times of concentration) when 72h, N are induced for clone strain, and M is
Molecular weight marker.
Fig. 5 is the Western blot qualification results of the expression product of recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1.Swimming lane
1-5 is the culture supernatant (10 times of concentration) when clone strain induces 72h, and N is negative control (X33 induces culture supernatant when 72h),
P:Anti-His tag western blot positive controls, M are molecular weight marker.
Fig. 6 is 1 protein purification products SDS-PAGE analysis charts of dog recombinant interferon-λ.A:Loading sample, B:Sample is penetrated,
C:50mM imidazole elutions 1, D:50mM imidazole elutions 2, E:500mM imidazole elutions 1, F:500mM imidazole elutions 2, G:
500mM imidazole elutions 3, H:500mM imidazole elutions 2 (non-reduced electrophoresis), M are molecular weight marker.
Fig. 7 is 1 protein purification products Western blot analysis charts of dog recombinant interferon-λ.MK1:Molecular weight marker
(15-170kDa), MK2:Molecular weight marker (20-120kDa) 1:Anti-His tag western blot positive controls, 2:
Western blot negative controls.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art,
Biochemical reagents used in the present invention are commercially available.
Embodiment 1 encodes gene order optimization and the design and synthesis of primer of dog interferon-λ 1
The present invention optimizes codon according to the cDNA sequence of dog interferon-λ 1 in GenBank (No.AB819731.1), closes
At objective gene sequence.The codon of the gene of coding dog interferon-λ 1 has used the codon that Pichia pastoris is most had a preference for.In order to
The efficiency that the G/C content for translating the mRNA come is excessively high, mRNA secondary structure influences translation is avoided, the present invention is to certain amino
Acid is using time preference password, on condition that password is had a preference in this time and most preference password frequency of use is very close, amino acid sequence is former
Sequence is constant, and in the case of certain very special, in order to decrease or increase restriction enzyme site, the sequence of certain positions does sequence appropriate
Row adjustment.Optimization design of the present invention 1 genes of dog interferon-λ (CaIFN- λ 1) as a result, sequence such as SEQ ID NO.1 institutes
Show, encodes the amino acid sequence of albumen as shown in SEQ ID NO.2.5 ' AOX of Pichia pastoris universal primer and 3 ' AOX sequences are shown in
SEQ ID No.3 and SEQ ID No.4.
The structure of 2 pPICZ α A-CaIFN- λ of embodiment, 1 carriers
1 genetic fragments of CaIFN- λ of embodiment 1 and carrier pPICZ α A are subjected to double digestion with XhoI and NotI, recycling is double
Endonuclease bamhi, 1 genetic fragments of CaIFN- λ press 3 with carrier pPICZ α A：14 DEG C of connections of molar ratio overnight, are converted to Escherichia coli
Competent cell DH5 α, for coated plate on the less salt LB tablets containing Zeocin (25ug/ml), 37 DEG C are cultivated 16-24h.Picking single bacterium
It falls, extracts plasmid, carry out PCR identifications and the sequencing of Song Sheng works company.The structure schematic diagram of 1 carriers of pPICZ α A-CaIFN- λ is shown in figure
1。
The screening of the Electroporation Transformation of 3 yeast cells of embodiment and high expression quantity transformant
Picking is enlarged culture through PCR and the correct bacterium colony of sequencing, chooses in the plasmid in the green skies and measures extraction agent box
(Plasmid Midi Preparation Kit) carries out taking out in plasmid to specifications.By Invitrogen Pichia
Expression Kit operating guidances prepare pichia pastoris X-33 competent cell.Using Sac I to pPICZ α A-CaIFN- λ 1
Plasmid is linearized.Linearisation system is shown in Table 1.
Table 1
37 DEG C overnight.1 plasmids of pPICZ α A-CaIFN- λ of linearisation carry out gel electrophoresis, and result sizes are consistent with expection,
As a result see Fig. 2.
The recombinant plasmid pPICZ alpha A-CaIFN- λ 1 for taking 200 μ l competent cells and 20 μ l (10~15 μ g) SacI to linearize
Mixing, injects in the 0.2cm pole cups of precooling, after placing several minutes on ice, in Bio-Rad Gene Pulser electroporations
Electrotransformation under the conditions of 1680V, 25pF, 3000 Ω, 5ms.After electrotransformation, the 1M D-glucitols of 1ml precoolings are added immediately, gently
For several times, the yeast cells after electricity is turned is transferred in the EP pipes of 1.5ml, is placed on ice several minutes for piping and druming.EP pipes are put into 30 DEG C
It is incubated 1h in incubator, 100 μ l bacterium solutions is taken to be coated on the YPD containing 100 μ g/ml, 250 μ g/ml and 500 μ g/ml Zeocin resistances
On tablet, 30 DEG C are inverted culture 3 days.
PCR identifications are done from picking individual colonies on YPD tablets, are inoculated in the YPD culture mediums containing Zeocin, 30 DEG C of shaking tables
In, 250r/min cultures behind left and right, take 1ml bacterium solution conservations for 24 hours.It is another to take extraction yeast X33 bases in 1ml bacterium solutions to 1.5ml EP pipes
Because of group, the specific steps are：Yeast cells X33 is collected by centrifugation, is resuspended with 200 μ l TE Buffer, 1% (V/V) E sulfydryl second is added
Alcohol and 1% (V/V) Proteinase K, 50 DEG C of water-bath digestion 3h or so, during which rock frequently.200 μ l chloroforms and 200 μ l phenol are added,
It is vortexed the several seconds.12000rpm centrifuges 5min, carefully draws supernatant liquid, and 2 times of volume absolute ethyl alcohol precipitation recycling are added.
5 ' AOX and 3 ' AOX universal primers are selected to be identified (shown in SEQ ID NO.3-4).Reaction system is shown in Table 2：
Table 2
PCR reaction conditions：Pre-degeneration, 96 DEG C of 5min；94 DEG C of 20s are denaturalized, anneal 55 DEG C of 20s, extends 72 DEG C of 20s, and totally 35
A cycle；72℃5min.
By after 5 μ L PCR products and 1 μ L Loading buffer mixings, with 1.0% Ago-Gel nucleic acid electrophoresis,
120V, 35min observe electrophoresis result under ultraviolet gel imager.As shown in figure 3, result show 1-5 clone PCR product with
It is expected that in the same size.The PCR product of 1-5 clones is sequenced, is 100% with target gene homology.
The copy screening of 4 yeast height of embodiment
High copy screening is carried out with Zeocine, the yeast-positive single bacterium colony on YPDS tablets is transferred to containing 800 μ g/ml
On the YPD plates of Zeocine, 30 DEG C culture 3 days after carry out expression and positive identification.
The induced expression of 5 recombination yeast engineering bacteria of embodiment and identification
By the recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 and empty carrier transformed yeast bacterium X-33/ of 5 positive colonies
PPICZ α A monoclonals are inoculated in respectively in 20-30ml YPD culture mediums, and 30 DEG C of 250rpm/min cultivate 16~18h or so, until bacterium
Body OD600 values are 2-6, and 1500~3000g, room temperature centrifugation 5min discard culture medium, collect thalline.Bacterial sediment 20mL
BMMY culture mediums are resuspended, and OD600 values are 2 or so, 28,00 DEG C, and 250rpm/min cultivates 4~5d, every adding methanol for 24 hours to end
A concentration of 1%.After Fiber differentiation, collects culture supernatant and be concentrated by ultrafiltration, sample is then added isometric 2 ×
Loading buffer, 100 DEG C are boiled 10min, take 10 μ l sample loadings, carry out SDS-PAGE electrophoresis and western blot inspections
It surveys.SDS-PAGE (Fig. 4) and Western blot (Fig. 5) testing result show：Supernatant is secreted after ultrafiltration concentration, on the left sides 22KD
A right visible apparent purpose band, destination protein obtain secreting, expressing.According to 1-5 clonal expression amounts, it is seen that No. 3 clone's tools
There is higher expression, therefore, selectes No. 3 clones and be used for subsequent experimental.
In order to determine methanol induction condition, the recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 that No. 3 are cloned are inoculated in 20-
In 30ml YPD culture mediums, 30 DEG C of 250rpm/min cultivate 16~18h or so, until when thalline OD600 values are 6 or so, respectively
1500~3 000g room temperatures centrifuge 5min, discard culture medium, collect thalline.Bacterial sediment with 20mL BMMY culture mediums be resuspended to
OD600 values are respectively 1,2,28 DEG C, 250rpm/min cultures, and every adding methanol, methanol final concentration 1% for 24 hours.Fiber differentiation
It after 96h, collects culture supernatant and is concentrated by ultrafiltration, sample is then added isometric 2 × loading buffer, 100
DEG C 10min is boiled, take 10 μ l sample loadings, carries out SDS-PAGE electrophoresis and gray analysis.
Table 3
In order to determine methanol induction concentration, the recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 that No. 3 are cloned are inoculated in 20-
In 30ml YPD culture mediums, 30 DEG C of 250rpm/min cultivate 16~18h or so, until when thalline OD600 values are 6 or so, respectively
1500~3 000g room temperatures centrifuge 5min, discard culture medium, collect thalline.Bacterial sediment with 20mL BMMY culture mediums be resuspended to
OD600 values are 2,28 DEG C, 250rpm/min cultures, and every adding methanol for 24 hours, methanol final concentration is respectively 0.5%, 1%,
1.5%, 2%.After Fiber differentiation 96h, collects culture supernatant and be concentrated by ultrafiltration, sample is then added isometric 2 ×
Loading buffer, 100 DEG C are boiled 10min, take 10 μ l sample loadings, carry out SDS-PAGE electrophoresis and gray analysis.
Table 4
In order to determine the best harvest time of albumen, the recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 that No. 3 are cloned are inoculated in
In 20-30ml YPD culture mediums, 30 DEG C of 250rpm/min cultivate 16~18h or so, until when thalline OD600 values are 6 or so, respectively
1500~3000g room temperatures centrifuge 5min, discard culture medium, collect thalline.Bacterial sediment with 20mL BMMY culture mediums be resuspended to
OD600 values are 2,28 DEG C, 250rpm/min cultures, and every adding methanol, methanol final concentration of 1% for 24 hours.Fiber differentiation 48h,
After 72h, 96h, 120h, collects culture supernatant and be concentrated by ultrafiltration, then sample is added to 2 isometric × loading
Buffer, 100 DEG C are boiled 10min, take 10 μ l sample loadings, carry out SDS-PAGE electrophoresis and gray analysis.
Table 5
Conditions above optimum results show by bacterial sediment with 20mL BMMY culture mediums be resuspended to OD600 values be 2 when it is excellent
In 1 it is therefore preferable that 2.The induced concentration of methanol difference at 1% and 1.5% and 2% is not notable, is better than 0.5%, therefore preferably
1%.Albumen difference when 96h and 120h is harvested is not notable, is better than 48h and 72h, therefore preferably 96h is the best harvest of albumen
Time.
Described in synthesis, the best induced expression condition of recombination yeast engineering bacteria X-33/pPICZ α A-CaIFN- λ 1 is, by 3
The recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 of number clone are inoculated in 20-30ml YPD culture mediums, 30 DEG C of 250rpm/min training
16~18h or so is supported, until when thalline OD600 values are 6 or so, 1500~3 000g room temperatures centrifuge 5min respectively, discard culture medium,
Collect thalline.It is 2,28 DEG C that bacterial sediment, which is resuspended with 20mL BMMY culture mediums to OD600 values, 250rpm/min cultures, and every
Methanol, methanol final concentration 1% are added for 24 hours.After Fiber differentiation 96h, collects culture supernatant and be concentrated by ultrafiltration.6 weight of embodiment
The purifying of group pPICZ α P-CaIFN- λ 1 and BCA method protein quantifications
No. 3 clone recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 are inoculated in 20-30ml YPD culture mediums, 30 DEG C
250rpm/min cultivates 16~18h or so, until when thalline OD600 values are 6 or so, 1500~3 000g room temperatures centrifugation respectively
5min discards culture medium, collects thalline.It is 2,28 DEG C that bacterial sediment, which is resuspended with 20mL BMMY culture mediums to OD600 values,
250rpm/min is cultivated, and every adding methanol, methanol final concentration 1% for 24 hours.After Fiber differentiation 96h, 4 DEG C of 12000rpm/min
10min is centrifuged, culture supernatant is harvested, pipe, which is concentrated by ultrafiltration, with 3K is concentrated about 10 times, after 0.45 μm of filter filtering, is used
Chelating SFF (Ni) affinity column carries out affinity chromatography, the specific steps are：
Balanced gel medium：With the equilibration buffer Buffer A of 10 column volumes (20mM PB, 150mM NaCl,
PH7.4 pillar) is balanced；
Supernatant is dialysed to Buffer A, is added in purification column；
Nucleic acid-protein detector is adjusted, adjustment flow velocity is 2mg/ml；
The buffer in pillar is emptied, collection flows through liquid；
Pillar is cleaned with the equilibration buffer of 5 times of column volumes；
Adjustment flow velocity is 1mg/ml；Using eluent (the 20mM PB, 150mM of different imidazole concentrations (50mM, 500mM)
NaCl, 500mM Imidazole, pH7.4) carry out gradient elution.Foreign protein is eluted, flow point is collected；
After protein purification, pillar is balanced into 20min with equilibration buffer Buffer A, trickle is finally used
20% ethyl alcohol cleans pillar.
The component collected in purification process is analyzed by SDS-PAGE, the dog recombination interference that display the present embodiment obtains
1 albumen of element-λ is purified well, sees that Fig. 6, swimming lane E, F, G are 1 albumen of dog recombinant interferon-λ of Ni enrichments.
E500 elution fractions (swimming lane E, F, G) are merged, concentrates, carries out dialysis PBS, in pH7.4 buffer systems,
Thermo BCA method protein quantifications, 1 a concentration of 0.43mg/ml of albumen of dog recombinant interferon-λ after purification.
SDS-PAGE testing results are shown：1 molecular weight of albumen of dog recombinant interferon-λ of recombinant bacterial strain expression is on the left sides 22KD
The right side, molecular size range and expected destination protein are in the same size, and analysis result table is scanned using gel imaging system software
The expression quantity of bright destination protein accounts for the 32% of bacterial protein, has preferable expression quantity.
The Western blot identifications of 7 expression product of embodiment
Isometric 2 × loading buffer are added in the dog recombinant interferon-λ 1 of embodiment 6 after purification, 100 DEG C are boiled
10min takes 10 μ l to boil rear sample loading, selects 15% PAGE gel electrophoresis, the polyacrylamide gel after electrophoresis
By the wet transfer system (Bio.Rad) of electricity with 300mA, on the parameter electrotransfer to pvdf membrane of 60min.Electricity turns to finish, and uses TBST
It washes film to be subsequently placed in a closed polybag, 37 DEG C of closing 2h in the TBSA containing 5% skimmed milk power is added；Film is washed with TBST,
Anti-His monoclonal antibodies, 4 DEG C of overnight incubations are added；Film is washed with TBST, is added and contains 1:5000 diluted HRP- goats resist small
37 DEG C of incubation 1h of monoclonal antibody of mouse；Film is washed with TBST, the colour developing of TMB one-component developing solutions waits for that the color of protein band reaches and wants
(about 10-30min) is asked, is reacted, is photographed to record with tap water color development stopping.As shown in Figure 7.
Western-blot results are shown：Expression product can be combined with monoclonal antibody specificity, and have at 22KD compared with
It is consistent with SDS-PAGE results significantly to react band, illustrate that the expression product has immunoreactivity well.
The antiviral activity of 8 dog recombinant interferon-λ 1 of embodiment detects
Method is inhibited using few cells lesion, it is dry that the dog recombination that the purifying of embodiment 6 obtains is measured in MDCK-VSV systems
Disturb the antiviral activity of element-λ 1.By mdck cell with 2 × 104The density in the holes cells/ is laid in 96 porocyte culture plates, and 37
DEG C, 5%CO2Under the conditions of cultivate 4-6h, after cell is adherent, be separately added into measure culture solution (DMEM containing 7%FBS) multiple proportions
Dog recombinant interferon-λ 1 after dilution and empty carrier expression albumen do negative control, blank control, parallel three repetitions, 37 DEG C,
5%CO2Under the conditions of after culture 18-24h, discard supernatant, PBS is washed three times, and malicious culture solution (DMEM containing 2%FBS) is dilute with attacking
VSV is released, by VSV with 100TCID50Attack toxic dose infection mdck cell, per 100 μ l of hole, at the same be arranged positive control (only plus
VSV), protein control (only plus after dog recombinant interferon-λ 1 changes complete culture solution, 37 DEG C, 5%CO into2Under the conditions of cultivate for 24 hours, see
Cell half lesion quantity is examined, is an interference primitive unit cell (IU) to there is the highest dilution of 50% cytopathy.According to
Reed-muench methods calculate the activity (IU/mg) of dog recombinant interferon-λ 1.The result shows that dog recombinant interferon-λ's 1 is antiviral
Activity is 8 × 106U/ml, specific activity are 2.85 × 107IU/mg.It can be seen that the dog recombinant interferon-λ 1 of the present invention has preferably
Anti-viral activity in vitro.
The amplification of 9 recombination yeast engineering bacteria of embodiment is expressed
No. 3 clone recombinant bacterium X-33/pPICZ α A-CaIFN- λ 1 are inoculated in the BMGY culture mediums of 25mL, 30 DEG C,
250rpm/min cultivates 16-18h or so, until thalline OD600 values are 2-6,1500-3 000g, room temperature centrifuges 5min and collects thalline.
Bacterial sediment is resuspended with 20mL BMMY culture mediums, and OD600 values are 2 or so, 28-30 DEG C, and 250rpm/min cultivates 16~18h.
1500-3 000g room temperatures centrifuge 5min, collect thalline.Bacterial sediment is resuspended with 1L BMMY culture mediums, and OD600 values are 2 or so,
28-30 DEG C, 250rpm/min cultivates 4~5d, every adding methanol for 24 hours to final concentration of 1%.4 DEG C after culture 96h
12000rpm/min centrifuges 10min and harvests culture supernatant, after being concentrated using 3K ultrafiltration concentration cups, the filter mistake through 0.45um
After filter, affinity chromatography is carried out with AKTA protein purifications instrument, albumen after purification passes through SDS-PAGE purity analysis, Thermo BCA
Method protein quantification, dog recombinant interferon-λ 1 after purification is 0.86mg/ml, and the protein yield of 1L recombination yeast engineering bacterias is
42mg。
10 dog recombinant interferon-λ 1 of embodiment prevents and treatment effectiveness evaluation
It carries out injecting disease according to 200,000 IU/kg dose subcutaneous using the dog recombinant interferon-λ 1 of the expression of embodiment 9, purifying
Dog, it is continuous to inject 6 days as a treatment course, it is evaluated by Symptom Observation, clinical detection etc. and is used to prevent and treat the hundstaupe of dog
The effect of the epidemic diseases such as heat, canine parvovirus disease, canine parainfluenza virus disease, canine infectious hepatitis, as a result shows dog recombinant interferon-
λ 1 can effectively prevent and treat the epidemic diseases such as canine distemper, canine parvovirus disease, the canine parainfluenza virus disease of dog, canine infectious hepatitis
Disease, obviously there is symptom mitigation in injection dog, appetite is restored, and the death rate reduces, and toxin expelling is reduced.
Table 6 is to epidemic diseases clinical cases such as canine distemper, canine parvovirus disease, canine parainfluenza virus disease, canine infectious hepatitis
Sick dog is injected according to 200,000 IU/kg dose subcutaneous using dog recombinant interferon-λ 1, after continuous injection is treated for 6 days, observation disease
Symptom, the appetite of dog, toxin expelling situation, the death rate.
6 dog recombinant interferon-λ of table, 1 treatment effectiveness evaluation tables
Table 7 is using canine distemper virus, canine parvovirus, canine parainfluenza virus, canine infectious hepatitis virus artificial challenge
60-70 age in days experimental dogs, start for 24 hours before infection, are injected according to 200,000 IU/kg dose subcutaneous using dog recombinant interferon-λ 1
Sick dog, after continuous injection is prevented for 6 days, symptom, the appetite of malicious dog, toxin expelling situation, the death rate are attacked in observation.
7 dog recombinant interferon-λ of table, 1 preventive effect evaluation tables
Canine distemper symptom is given a mark：Body temperature raising/ups and downs, diphasic fever, spirit is depressed, and appetite is poor, and eye conjunctiva is rubescent, eye
Eyelid swelling secrets out of watery secretion, and nose is dry, flows out watery secretion.The visible vola Keratoderma transition of a small number of cases
Venereal disease becomes.It coughs, the appearance vomiting of stomach type infection, diarrhea, there are about 10~30% sick dogs nervous symptoms (convulsion occurs
Contraction, epilepsy, twitch etc.).According to symptom from again to gently marking is 10,9,8,7,6,5,4,3,2,1,0 successively.
Enteritis canine parvovirus symptom is given a mark：Enteritis disease dog initial stage spirit is depressed, apocleisis, accidental fever, soft stool or
Slight vomiting then develops into frequently vomiting and violent diarrhea.Originally excrement gray, yellow or milky, band g., jelly-like
The soy sauce sample or tomato juice sample bloody stool of stench is discharged thereafter in mucus.Sick dog rapid dehydration, becomes thin, and proscopiny, hair is in disorder,
Skin is nonelastic, ear nose, cold limbs, and spirit height is depressed, and shock is dead.According to symptom from again to gently successively marking for 10,
9、8、7、6、5、4、3、2、1、0。
Canine parainfluenza virus disease symptoms are given a mark：Fever, cough, runny nose, sick dog is sluggish, body temperature be increased to 40 DEG C with
On, sick dog hind leg can support body, but cannot walk.According to symptom from again to gently successively marking for 10,9,8,7,6,5,4,3,
2、1、0。
Canine infectious hepatitis symptom is given a mark：Sick dog spirit is depressed, and flowing water sample or purulence nose liquid, conjunctiva inflammation, photophobia are shed tears.
Oral cavity and gingiva bleeding gingival hemorrhage are shown in blutpunkte, and body temperature increases, accelerated breathing, rapid heart beat, irregularity of pulse.Cough.The sick dog vomiting having
Or passage of loose stools.According to symptom from again to gently marking is 10,9,8,7,6,5,4,3,2,1,0 successively.
Appetite scoring criterion：Normal 5 points of appetite, slightly worse 4 points of appetite, appetite poor 3, appetite less 2, substantially without appetite 1
Point, appetite is given up exhausted 0 point.
In summary dog recombinant interferon-λ 1 is sick, canine infectious to canine distemper, canine parvovirus disease, canine parainfluenza virus
The epidemic diseases such as hepatitis clinical prevention/treatment, dog recombinant interferon-λ 1 inject dog and symptom mitigation, appetite recovery obviously occur, extremely
Rate reduction is died, toxin expelling is reduced.Show dog recombinant interferon-λ 1 to canine distemper, canine parvovirus disease, canine parainfluenza virus disease, dog
The epidemic diseases such as catarrhal jaundice have preferable preventing/treating effect, can carry out Clinical practice.
Although the present invention is described in detail with a general description of the specific embodiments for above-mentioned word,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>Dog recombinant interferon-λ 1 and the preparation method and application thereof
<130> KHP171119241.7
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 537
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggaccagttc ctacttctaa gcctactatg gcttggagag gatgtgatat tggtagattt 60
aagtccttgt cacctagaga attggaggct ttcaagaagg ctaaggatgc tttggaatac 120
tccttgaaga actggtcctg taactccaga ttgtttccaa gaaacagaga tttgagacaa 180
ttgcaagttt gggaaagacc agttgctttg gaagctgaat tggctttgac tttgaaggtc 240
ttggaaacta tggctgatag atccttgggt gatatcttgg atcaaccatt gcatactttg 300
agacatattc attctcaatt gcaagcgtgt gtttccgctc aaccacctgc tggtccacaa 360
cctagaggta gattgcatcc gtggttgcat agattgcatg aggcatctaa gaaggaatcc 420
caaggatgtt tggaggcctc cgtcttgttt aacttgttta gattgttgaa gaaggatttg 480
gaatgtgttg ctgttggtga tttgtgtgtt ggttcacatc atcatcatca tcattaa 537
<210> 2
<211> 178
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Gly Pro Val Pro Thr Ser Lys Pro Thr Met Ala Trp Arg Gly Cys Asp
1 5 10 15
Ile Gly Arg Phe Lys Ser Leu Ser Pro Arg Glu Leu Glu Ala Phe Lys
20 25 30
Lys Ala Lys Asp Ala Leu Glu Tyr Ser Leu Lys Asn Trp Ser Cys Asn
35 40 45
Ser Arg Leu Phe Pro Arg Asn Arg Asp Leu Arg Gln Leu Gln Val Trp
50 55 60
Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val
65 70 75 80
Leu Glu Thr Met Ala Asp Arg Ser Leu Gly Asp Ile Leu Asp Gln Pro
85 90 95
Leu His Thr Leu Arg His Ile His Ser Gln Leu Gln Ala Cys Val Ser
100 105 110
Ala Gln Pro Pro Ala Gly Pro Gln Pro Arg Gly Arg Leu His Pro Trp
115 120 125
Leu His Arg Leu His Glu Ala Ser Lys Lys Glu Ser Gln Gly Cys Leu
130 135 140
Glu Ala Ser Val Leu Phe Asn Leu Phe Arg Leu Leu Lys Lys Asp Leu
145 150 155 160
Glu Cys Val Ala Val Gly Asp Leu Cys Val Gly Ser His His His His
165 170 175
His His
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gactggttcc aattgacaag c 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gcaaatggca ttctgacatc c 21
Claims (10)
1. a kind of dog recombinant interferon-λ 1, which is characterized in that its amino acid sequence is：
A) amino acid sequence shown in SEQ ID No.2；Or
B) amino acid sequence shown in SEQ ID No.2 is formed through replacing, lacking and oring add one or several amino acid residues
The amino acid sequence with same function.
2. encoding the gene of dog recombinant interferon-λ 1 described in claim 1, which is characterized in that be it is following a) or b)：
A) in its nucleotide sequence such as sequence table shown in SEQ ID No.1；Or
B) nucleotide sequence shown in SEQ ID No.1 is substituted one or several nucleotide, obtains encoding canine recombinant interferon-
The nucleotide sequence of λ 1.
3. the recombinant expression carrier containing gene described in claim 2.
4. the host cell containing recombinant expression carrier described in gene described in claim 2 or claim 3.
5. host cell as claimed in claim 4 is Pichia pastoris.
6. the host cell described in recombinant expression carrier or claim 4 described in the gene or claim 3 described in claim 2
Application in preparing broad-spectrum antiviral medicament.
7. the host cell described in recombinant expression carrier or claim 4 described in the gene or claim 3 described in claim 2
Application in preparing broad-spectrum antiviral preparation.
8. application as claimed in claims 6 or 7, which is characterized in that the virus is canine distemper virus, canine parvovirus, dog
Parainfluenza virus, canine infectious hepatitis virus, vesicular stomatitis virus.
9. biological agent or drug containing gene described in claim 2 or its coding albumen.
10. a kind of method preparing dog recombinant interferon-λ 1, which is characterized in that by nucleotide sequence as shown in SEQ ID NO.1
1 gene directed clonings to plasmid of dog interferon-λ in construction recombination plasmid, recombinant plasmid transformed is thin to Pichia pastoris competence
Born of the same parents, Prepare restructuring bacterium, by determining that methanol induction condition, induced concentration, induction time, albumen harvest time etc. optimize yeast
Fermentation, expression, are purified using the method for Chelating SFF-Ni affinity chromatographys after ultrafiltration concentration, effectively increase product
Yield, obtain high yield, high-purity dog recombinant interferon-λ 1.
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| CN113980972A (en) * | 2021-12-07 | 2022-01-28 | 山东农业大学 | Codon-optimized canine lambda 1 interferon coding gene and application thereof in preparation of canine lambda 1 interferon |
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