CN106399321A - Production process of fusion expression recombinant chicken interferon alpha - Google Patents
Production process of fusion expression recombinant chicken interferon alpha Download PDFInfo
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- CN106399321A CN106399321A CN201610743781.2A CN201610743781A CN106399321A CN 106399321 A CN106399321 A CN 106399321A CN 201610743781 A CN201610743781 A CN 201610743781A CN 106399321 A CN106399321 A CN 106399321A
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- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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Abstract
The invention discloses a production process of fusion expression recombinant chicken interferon alpha. The process includes the steps of: S1, according to the preference of Escherichia coli codon, conducting codon optimization on a chicken interferon alpha gene sequence published in Genebank, and artificially synthesizing the chicken interferon alpha gene; S2, according to the codon optimized chicken interferon alpha gene, designing three specific primers; S3, constructing recombinant chicken interferon alpha plasmid containing ProS2 dissolution promoting label; S4, transforming and identifying the recombinant expression plasmid; S5, conducting inducible expression of recombinant chicken interferon alpha; S6, extracting an expression product and conducting protein renaturation purification: S61, inclusion body extraction and treatment; S62, inclusion body denaturation; S63, denaturation solution renaturation; and S64, nickel column affinity purification. By means of cell cytopathic inhibition, the invention detects that the interferon has the activity of inhibiting vesicular stomatitis virus proliferation, and the activity unit reaches 7.32*10<7>UI/mg.
Description
Technical field
The present invention relates to gene recombination technology field, particularly to a kind of life preparing amalgamation and expression recombined chicken alpha interferon
Production. art.
Background technology
Interferon is the important a member of body natural immune system, and the antiviral response of its mediation is natural immune system the
One defence line.During poisoning intrusion, body cell can be identified to virus by the pattern recognition receptors of intraor extracellular, then
Activate the natural immunity signal path related to interferon, the expression of inducing interferon gene.The interferon of expression can be with it
Specific receptor on his cell combines, the transduction of antiviral signal in mediated cell, produces antiviral protein, thus effectively supporting
The invasion of antiviral.
Since self-interference element is found, the mankind have carried out invention for many years, deep to the application of interferon.Human interferon
Application development history can be divided into three phases, the first stage is natural prototype interferon, mainly adopts virus induction in vitro
Human leukocyte and people source transformation cell lines cumulative interference element, be then peeled off purifying alpha-interferon.But its shortcoming is leukocyte source
Few；The interferon deriving is the mixture of each hypotype, and curative effect is general；In addition, there being the risk of virus pollution.Second stage
It is recombinant interferon, from the eighties, has been provided with the clone of interferon and the technology of genetic modification, therefore mainly adopt gene
Engineering and recombinant technique prepare human interferon in a large number, improve purity and the potency of product simultaneously, but this recombinant interferon exists
Internal half-life short, is easily eliminated.Phase III is the development of long-acting interferon, on the basis of second stage, using PEG
Change modification, albumin fusion technology preparation, improve pharmacokineticss process, improve curative effect.Current human interferon is
Larger scale clinical is applied to treat multiple viral diseases, such as hepatitis B etc., achieves good curative effect.
Similar with human interferon biological function, restructuring chicken interferon is by reaching to the regulation of self immune system
To the purpose of antiviral, compared with the antivirus veterinary biological product such as traditional chemicalses, vaccine, cause of disease will not be made to produce resistance to
Property of medicine mutation and drug residue, therefore the invention of restructuring chicken interferon is also increasingly taken seriously in recent years, and especially chicken α disturbs
Element shows good effect at aspects such as viral infection resistings.But because the production process of current chicken interferon is more complicated, produce into
This is higher, and the product of birdss recombinant interferon is less on the market, does not also possess the condition of larger scale clinical application.
Content of the invention
The technical problem to be solved in the present invention is the defect overcoming prior art, provides one kind to prepare amalgamation and expression recombination chicken
The production technology of interferon-alpha.
In order to solve above-mentioned technical problem, the invention provides following technical scheme：
A kind of production technology preparing amalgamation and expression recombined chicken alpha interferon, it comprises the following steps：
S1, password is carried out to the chicken alpha interferon gene sequence delivered in Genebank according to the Preference of e. coli codon
Son optimizes, this chicken alpha interferon gene of synthetic；
S2, according to the chicken alpha interferon gene after codon optimization, design three specific primers：
P1 CGCCATATGTGCAACCATCTGCGCCC；
P2 CGCGAATTCTCAGGTGCGGGTGTTGCCGG；
P3 CGCCCATGGATCACAAAGTGCATCA；
S3, use primer P1 and P2 first, chicken alpha interferon gene as template, obtains chicken interferon by PCR amplification after to optimize
Fragment, purification reclaims PCR primer, with NdeI and EcoRI to the interferon fragment of purification and pCold-ProS2-ChIFN- α carrier
Carry out enzyme action, connect after purification digestion products, convert escherichia coli DH5a bacterial strain, screening positive clone, sequencing confirms, is carried
Body pCold-ProS2-ChIFN- α；
Use primer P3 and P2 afterwards, performing PCR amplification is entered for template with pCold-ProS2-ChIFN- α plasmid, obtains tape label
Chicken interferon fragment, carries out purification to PCR primer, respectively pET28 and PCR primer is entered with NcoI and EcoRI restriction endonuclease afterwards
Row enzyme action, purification is attached after reclaiming, and connection product converts escherichia coli DH5a bacterial strain, screening positive clone, and sequencing confirms,
Obtain purpose carrier pET-28b-ProS2-ChIFN- α；
S4, will be sequenced correct pET-28b-ProS2-ChIFN- α recombinant plasmid transformed BL21 (DE3) competent cell, through ammonia
The screening of benzylpcnicillin resistant panel, the identification of NdeI, EcoRI double digestion, verify this restructuring positive plasmid further；
S5, by the BL21 identifying again (DE3) E.coli/pET-28b-ProS2-ChIFN- α, be inoculated in LB liquid medium,
37 DEG C of concussion and cultivates.When OD value is for 0.6, add the IPTG abduction delivering of final concentration of 1mM in culture.After continuing culture 5h,
Thalline is collected by centrifugation, carries out SDS-PAGE analysis, then the albumen on PAGE gel is transferred to nitrocellulose filter
(NC), Western-blot testing goal albumen.Because destination protein contains His label, so with His tag antibody (1: 2
000) anti-as one, make two with the sheep anti mouse (1: 10 000) of horseradish peroxidase-labeled and resist.
Further, further comprising the steps of：
S6, the extraction of expression product and protein renaturation purification
S61, the extraction of inclusion body and process:
The recombinant bacterium E.coli/pET-28b-ProS2- ChIFN- α of 1L abduction delivering is collected by centrifugation with 10000 r/min
Thalline, by 1:The 5 resuspended thalline of thalline sonication buffer, under conditions of ice bath, abundant ultrasonication, wherein said ultrasonic slow
Rushing liquid is 50mM Tris, 0.5mM EDTA, 50mM NaCl, pH7.0；Then it is centrifuged 15 min in 4 DEG C of 10 000 r/min
Collect precipitation, this precipitation is rough inclusion body；
The inclusion body lavation buffer solution 1 being collected by centrifugation fully suspends, and this lavation buffer solution is 150mM Tris-HCl, and 0.5%
TritonX-100,0.5M NaCl, pH8.0, after magnetic agitation 30min, with 10 000 r/min, 4 DEG C, 20min is centrifuged；
Then with lavation buffer solution 2 and lavation buffer solution 3, inclusion body is washed successively；This lavation buffer solution 2 is 50mM
Tris-HCl, 2M Uera, 1% mercaptoethanol, 2mM EDTA, 0.15M Nacl, pH8.0；This lavation buffer solution 3 is 50mM
Tris- HCl, 4M Uera, 5mM DTT, 0.5M NaCl, pH8.0；
S62, the degeneration of inclusion body
By 1:By the solubilization of inclusion bodies after washing, this denaturing liquid in the denaturing liquid of 8M carbamide is 5 inclusion body degeneration liquid proportional
50mM Tris-HCl, 8M Uera, 5mM DTT, pH8.0；Room temperature magnetic agitation 6 hours, then with 12000rpm, 4 DEG C,
20min is centrifuged, and collects denaturing liquid supernatant, 4 DEG C of preservations；
S63, the renaturation of inclusion body
Take gradient dilution method to carry out protein renaturation, adopt dropper hanging drop during renaturation, in three times denaturing liquid is added to pre-cooling
In, this renaturation solution is 50mM Tris-HCl, 0.15M NaCl, 0.3M L-Arg, 1mM GSSG, 5mM GSH, 10% glycerol,
pH7.0；Add rear magnetic agitation 5min every time, make deformation liquid and the ratio of renaturation solution reach 1:10；After the completion of proceed to 4 DEG C of ice
Case, stands renaturation 24h, measures protein concentration by Broadford method, calculates the renaturing inclusion bodies effect of this recombined chicken alpha interferon
Rate；
S64, nickel post affinity purification
After the completion of renaturation, renaturation solution removes L-Arg through dialysis, with reference to QIAGEN company ni-sepharose purification description purification His label
Destination protein, then SDS-PAGE analysis is carried out to purified product.
A kind of chicken alpha interferon optimized gene, its sequence such as SEQ ID NO：Shown in 1.
A kind of aminoacid of chicken alpha interferon, its sequence such as SEQ ID NO：Shown in 2.
For this class of chicken alpha interferon has the biological product of therapeutic effect, its essence is that have the egg of biological activity
White matter, therefore, for the exploitation of this kind biological product, it is it is important that produce bioactive relatively with relatively low cost
The emphasis of high product, therefore this research is exactly that the cost as how relatively low for the research is realized the high efficient expression of chicken interferon and carried
The annealing efficiency of high chicken interferon（Annealing efficiency refers to can after inactive inclusion body reverts to active native conformation
Dissolubility albumen accounts for the ratio of inclusion body protein before renaturation）, thus obtaining the chicken interferon compared with high bioactivity.The present invention uses
Protein S is present in the spore clothing of slime bacteria, is made up of 173 amino acid residues, is a kind of highly stable solubility
Protein.By the N- terminal domains series connection of two Protein S, the effect in conjunction with ProS2 Tag can promote protein
Correct folding, the stability of fusion destination protein and solubility just can be made to be improved.The present invention passes through gene engineering method
Chicken alpha interferon gene is connected with dissolution label ProS2, builds prokaryotic expression carrier pET-28b-ProS2- ChIFN- α, knot
Fruit display, present invention achieves the high efficient expression of chicken interferon, has higher renaturation effect with the interferon of inclusion bodies expression
Rate, annealing efficiency reaches 73%, after ni-sepharose purification interferon purity more than 95%, and the chicken interferon of final gained have higher
Biologic activity, be conducive to commercially producing of chicken interferon, for promote chicken interferon clinical practice have greatly
Commercial significance.
The beneficial effect that the present invention is reached is：
The present invention is carried out to the chicken alpha interferon gene sequence delivered in Genebank according to the Preference of e. coli codon
Codon optimization, this chicken alpha interferon sequence of synthetic.By engineered method, chicken alpha interferon gene and dissolution mark
Sign ProS2 to connect, build prokaryotic expression carrier pET-28b-ProS2- ChIFN- α.After abduction delivering, inclusion body is become
Property, renaturation and nickel post affinity purification, Broadford method measures the inclusion body that protein concentration result shows this recombined chicken alpha interferon
Annealing efficiency reaches 73%, SDS-PAGE testing result and shows that interferon purity is more than 95% after purification.Present invention achieves interference
The high efficient expression of element and purification, reach 7.32 by the antiviral activity that cytopathic-effect inhibition assay determines this recombinant C hIFN- α ×
107UI/mg.The chicken interferon annealing efficiency that the present invention produces is high, and antiviral activity is stronger, prevents for chicken viral infectious disease
Control and be significant.
Brief description
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for description, the reality with the present invention
Apply example and be used for explaining the present invention together, be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 detects the inclusion body of expressing protein for Western-blot, and testing result display destination protein expression is correct；
Fig. 2 detects purifying protein for SDS-PAGE, and testing result shows that this electrophoretic band is single, the purity of interferon 95% with
On；
Fig. 3 measures recombined chicken alpha interferon activity for cytopathic-effect inhibition assay, and result shows that this recombined chicken alpha interferon acts on carefully
The breeding of vesicular stomatitis virus can effectively be suppressed after born of the same parents.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are illustrated it will be appreciated that preferred reality described herein
Apply example to be merely to illustrate and explain the present invention, be not intended to limit the present invention.
A kind of production technology preparing amalgamation and expression recombined chicken alpha interferon, it comprises the following steps：
S1, password is carried out to the chicken alpha interferon gene sequence delivered in Genebank according to the Preference of e. coli codon
Son optimizes, this chicken alpha interferon gene of synthetic；Gene after optimization is closed by Invitrogen (Shanghai) trade Co., Ltd
Become.
S2, according to the chicken alpha interferon gene after codon optimization, design three specific primers：
P1 CGCCATATGTGCAACCATCTGCGCCC；
P2 CGCGAATTCTCAGGTGCGGGTGTTGCCGG；
P3 CGCCCATGGATCACAAAGTGCATCA；
S3, use primer P1 and P2 first, chicken alpha interferon gene as template, obtains chicken interferon by PCR amplification after to optimize
Fragment, purification reclaims PCR primer, with NdeI and EcoRI to the interferon fragment of purification and pCold-ProS2-ChIFN- α carrier
Carry out enzyme action, connect after purification digestion products, convert escherichia coli DH5a bacterial strain, screening positive clone, sequencing confirms, is carried
Body pCold-ProS2-ChIFN- α；
Use primer P3 and P2 afterwards, performing PCR amplification is entered for template with pCold-ProS2-ChIFN- α plasmid, obtains tape label
Chicken interferon fragment, carries out purification to PCR primer, respectively pET28 and PCR primer is entered with NcoI and EcoRI restriction endonuclease afterwards
Row enzyme action, purification is attached after reclaiming, and connection product converts escherichia coli DH5a bacterial strain, screening positive clone, and sequencing confirms,
Obtain purpose carrier pET-28b-ProS2-ChIFN- α；
S4, will be sequenced correct pET-28b-ProS2-ChIFN- α recombinant plasmid transformed BL21 (DE3) competent cell, through ammonia
The screening of benzylpcnicillin resistant panel, the identification of NdeI, EcoRI double digestion, verify this restructuring positive plasmid further；
S5, by the BL21 identifying again (DE3) E.coli/pET-28b-ProS2-ChIFN- α, be inoculated in LB liquid medium,
37 DEG C of concussion and cultivates.When OD value is for 0.6, add the IPTG abduction delivering of final concentration of 1mM in culture.After continuing culture 5h,
Thalline is collected by centrifugation, carries out SDS-PAGE analysis, then the albumen on PAGE gel is transferred to nitrocellulose filter
(NC), Western-blot testing goal albumen.Because destination protein contains His label, so with His tag antibody (1: 2
000) anti-as one, make two with the sheep anti mouse (1: 10 000) of horseradish peroxidase-labeled and resist.
Further, further comprising the steps of：
S6, the extraction of expression product and protein renaturation purification
S61, the extraction of inclusion body and process:
The recombinant bacterium E.coli/pET-28b-ProS2- ChIFN- α of 1L abduction delivering is collected by centrifugation with 10000 r/min
Thalline, by 1:The 5 resuspended thalline of thalline sonication buffer, under conditions of ice bath, abundant ultrasonication, wherein said ultrasonic slow
Rushing liquid is 50mM Tris, 0.5mM EDTA, 50mM NaCl, pH7.0；Then it is centrifuged 15 min in 4 DEG C of 10 000 r/min
Collect precipitation, this precipitation is rough inclusion body；
The inclusion body lavation buffer solution 1 being collected by centrifugation fully suspends, and this lavation buffer solution is 150mM Tris-HCl, and 0.5%
TritonX-100,0.5M NaCl, pH8.0, after magnetic agitation 30min, with 10 000 r/min, 4 DEG C, 20min is centrifuged；
Then with lavation buffer solution 2 and lavation buffer solution 3, inclusion body is washed successively；This lavation buffer solution 2 is 50mM
Tris-HCl, 2M Uera, 1% mercaptoethanol, 2mM EDTA, 0.15M Nacl, pH8.0；This lavation buffer solution 3 is 50mM
Tris- HCl, 4M Uera, 5mM DTT, 0.5M NaCl, pH8.0；
S62, the degeneration of inclusion body
By 1:By the solubilization of inclusion bodies after washing, this denaturing liquid in the denaturing liquid of 8M carbamide is 5 inclusion body degeneration liquid proportional
50mM Tris-HCl, 8M Uera, 5mM DTT, pH8.0；Room temperature magnetic agitation 6 hours, then with 12000rpm, 4 DEG C,
20min is centrifuged, and collects denaturing liquid supernatant, 4 DEG C of preservations；
S63, the renaturation of inclusion body
Take gradient dilution method to carry out protein renaturation, adopt dropper hanging drop during renaturation, in three times denaturing liquid is added to pre-cooling
In, this renaturation solution is 50mM Tris-HCl, 0.15M NaCl, 0.3M L-Arg, 1mM GSSG, 5mM GSH, 10% glycerol,
pH7.0；Add rear magnetic agitation 5min every time, make deformation liquid and the ratio of renaturation solution reach 1:10；After the completion of proceed to 4 DEG C of ice
Case, stands renaturation 24h, measures protein concentration by Broadford method, calculates the renaturing inclusion bodies effect of this recombined chicken alpha interferon
Rate；
S64, nickel post affinity purification
After the completion of renaturation, renaturation solution removes L-Arg through dialysis, with reference to QIAGEN company ni-sepharose purification description purification His label
Destination protein carry out, then SDS-PAGE analysis is carried out to purified product.
A kind of chicken alpha interferon optimized gene, its sequence such as SEQ ID NO：Shown in 1.
A kind of aminoacid of chicken alpha interferon, its sequence such as SEQ ID NO：Shown in 2.
Fig. 1 detects the inclusion body of expressing protein for Western-blot, and testing result display destination protein expression is correct；
Fig. 2 detects purifying protein for SDS-PAGE, and testing result shows that this electrophoretic band is single, the purity of interferon 95% with
On.
Wherein, E.coli/DH5 α, BL21 (DE3), VSV virus is real by College of Veterinary Medicine, South China Agricultural University's zoonosis
Test room to preserve；PET-28b, pCold-ProS2 are preserved by College of Veterinary Medicine, South China Agricultural University's zoonosis laboratory.Restriction enzyme
EcoRI, NdeI and T4DNA ligase etc. is purchased in precious biological engineering（Dalian）Company limited.The quick QIAquick Gel Extraction Kit of DNA fragmentation
It is purchased from Tiangeng biochemical technology company limited.
For this class of chicken alpha interferon has the biological product of therapeutic effect, its essence is that have the egg of biological activity
White matter, therefore, for the exploitation of this kind biological product, it is it is important that produce bioactive relatively with relatively low cost
The emphasis of high product, therefore this research is exactly that the cost as how relatively low for the research is realized the high efficient expression of chicken interferon and carried
The annealing efficiency of high chicken interferon, thus obtain the chicken interferon compared with high bioactivity.The Protein S that the present invention uses exists
In the spore clothing of slime bacteria, it is made up of 173 amino acid residues, be a kind of highly stable soluble protein.By two
The N- terminal domains series connection of Protein S, the effect in conjunction with ProS2 Tag can promote the correct folding of protein, just
The stability of fusion destination protein and solubility can be made to be improved.The present invention passes through gene engineering method chicken alpha interferon base
Because being connected with dissolution label ProS2, build prokaryotic expression carrier pET-28b-ProS2- ChIFN- α, the display present invention is real for result
Show the high efficient expression of chicken interferon, Broadford method measures the inclusion body that protein concentration result shows this recombined chicken alpha interferon
Annealing efficiency reaches 73%, SDS-PAGE testing result and shows that interferon purity is more than 95% after purification.And the chicken of final gained
Interferon has higher biologic activity, is conducive to commercially producing of chicken interferon, for the clinic promoting chicken interferon
Application has great commercial significance.
The present invention enters to the chicken alpha interferon gene sequence delivered in Genebank according to the Preference of e. coli codon
Go codon optimization, this chicken alpha interferon sequence of synthetic.By engineered method, chicken alpha interferon gene and rush
Molten label ProS2 connects, and builds prokaryotic expression carrier pET-28b-ProS2- ChIFN- α.After abduction delivering, inclusion body is carried out
Degeneration, renaturation and nickel post affinity purification, Western-blot, SDS-PAGE result show present invention achieves interferon efficient
Expression and purification, the renaturing inclusion bodies efficiency of this recombined chicken alpha interferon reaches 73%, and interferon purity is more than 95% after purification.Logical
Cross cytopathic-effect inhibition assay and determine the antiviral activity of this recombinant C hIFN- α and reach 7.32 × 107UI/mg.This technique productions
Chicken interferon annealing efficiency is high, and antiviral activity is stronger, and the preventing and treating for chicken viral infectious disease is significant.
Measure the activity of recombinant interferon by cytopathic-effect inhibition assay：
DF-1 cell is inoculated in 96 porocyte culture plates, at 37 DEG C, trains in the saturation carbon dioxide steam incubator of 5% CO2
Support cell and grow up to monolayer.Each column adds the 4 times of recombinant interferon being serially diluted every hole 100 μ L, sets virus control and thin simultaneously
Born of the same parents compare, and after culture 12h, every hole adds the vesicular stomatitis virus of 1000TCID50, and this vesicular stomatitis virus is on DF-1
TCID50 is 7.32.Concurrently set the cell controls being not added with virus.Cultivate to 24h and start observation of cell pathological changes, treat that the positive goes out
Now carry out violet staining during obvious pathological changes.Abandon cell culture fluid, washed 1 time with aseptic PBS, add violet staining in every hole
Liquid 100 μ L, room temperature places 30min；Abandon dyestuff, distilled water rinses does not have illuminating colour, then add destaining solution 100 μ L in every hole,
Room temperature places 10min decolouring；Measure OD570 value with microplate reader and record；Data processing：Can suppress 50% cytopathic
Interference cellulose content is defined as an active unit, calculates interferon potency with Reed-Muench method, calculates recombinant interferon
Active unit is 7.32 × 107UI/mg.
In Fig. 3,1,2,3,4, respectively 410,411,412,413After the chicken interferon of dilution acts on cell again, to cell
Antiviral effect figure；5,6, respectively positive-virus comparison, negative control.The DF-1 cytopathy being led to according to vesicular stomatitis virus
Change may determine that：1,2,3, there is not cytopathy in cell, illustrate 410,411,412The chicken interferon of dilution acts on cell again
Cell anti-virus effect is significant afterwards；The 4 obvious cytopathys of appearance, illustrate 413This dilution interferon is to cell resistance again
Virus function is inconspicuous；5 positive controls are set up（There is cytopathy）；6 negative control group are set up（There is not cytopathy）.
Finally it should be noted that：The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention,
Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
To modify to the technical scheme described in foregoing embodiments, or equivalent is carried out to wherein some technical characteristics.
All any modification, equivalent substitution and improvement within the spirit and principles in the present invention, made etc., should be included in the present invention's
Within protection domain.
Claims (4)
1. a kind of production technology preparing amalgamation and expression recombined chicken alpha interferon is it is characterised in that comprise the following steps：
S1, password is carried out to the chicken alpha interferon gene sequence delivered in Genebank according to the Preference of e. coli codon
Son optimizes, this chicken alpha interferon gene of synthetic；
S2, according to the chicken alpha interferon gene after codon optimization, design three specific primers：
P1CGCCATATGTGCAACCATCTGCGCCC；
P2CGCGAATTCTCAGGTGCGGGTGTTGCCGG；
P3CGCCCATGGATCACAAAGTGCATCA；
S3, use primer P1 and P2 first, chicken alpha interferon gene as template, obtains chicken interferon by PCR amplification after to optimize
Fragment, purification reclaims PCR primer, with NdeI and EcoRI to the interferon fragment of purification and pCold-ProS2-ChIFN- α carrier
Carry out enzyme action, connect after purification digestion products, convert escherichia coli DH5a bacterial strain, screening positive clone, sequencing confirms, is carried
Body pCold-ProS2-ChIFN- α；
Use primer P3 and P2 afterwards, performing PCR amplification is entered for template with pCold-ProS2-ChIFN- α plasmid, obtains tape label
Chicken interferon fragment, carries out purification to PCR primer, respectively pET28 and PCR primer is entered with NcoI and EcoRI restriction endonuclease afterwards
Row enzyme action, purification is attached after reclaiming, and connection product converts escherichia coli DH5a bacterial strain, screening positive clone, and sequencing confirms,
Obtain purpose carrier pET-28b-ProS2-ChIFN- α；
S4, will be sequenced correct pET-28b-ProS2-ChIFN- α recombinant plasmid transformed BL21 (DE3) competent cell, through ammonia
The screening of benzylpcnicillin resistant panel, the identification of NdeI, EcoRI double digestion, verify this restructuring positive plasmid further；
S5, by the BL21 identifying again (DE3) E.coli/pET-28b-ProS2-ChIFN- α, be inoculated in LB liquid medium,
37 DEG C of concussion and cultivates.When OD value is for 0.6, add the IPTG abduction delivering of final concentration of 1mM in culture.After continuing culture 5h,
Thalline is collected by centrifugation, carries out SDS-PAGE analysis, then the albumen on PAGE gel is transferred to nitrocellulose filter
(NC), Western-blot testing goal albumen.Because destination protein contains His label, thus with His tag antibody (1:
2000) anti-as one, make two with the sheep anti mouse (1: 10000) of horseradish peroxidase-labeled and resist.
2. the production technology preparing amalgamation and expression recombined chicken alpha interferon according to claim 1 is it is characterised in that also wrap
Include following steps：
S6, the extraction of expression product and protein renaturation purification
S61, the extraction of inclusion body and process:
The recombinant bacterium E.coli/pET-28b-ProS2-ChIFN- α of 1L abduction delivering is collected by centrifugation thalline with 10000r/min,
By 1:The 5 resuspended thalline of thalline sonication buffer, under conditions of ice bath, abundant ultrasonication, wherein said sonication buffer
For 50mM Tris, 0.5mM EDTA, 50mM NaCl, pH7.0；Then it is centrifuged 15min in 4 DEG C of 10000r/min and collect precipitation,
This precipitation is rough inclusion body；
The inclusion body lavation buffer solution 1 being collected by centrifugation fully suspends, and this lavation buffer solution is 150mMTris-HCl, and 0.5%
TritonX-100,0.5M NaCl, pH8.0, after magnetic agitation 30min, with 10000r/min, 4 DEG C, 20min is centrifuged；
Then with lavation buffer solution 2 and lavation buffer solution 3, inclusion body is washed successively；This lavation buffer solution 2 is 50mM
Tris-HCl, 2M Uera, 1% mercaptoethanol, 2mM EDTA, 0.15M Nacl, pH8.0；This lavation buffer solution 3 is 50mM
Tris-HCl, 4M Uera, 5mM DTT, 0.5M NaCl, pH8.0；
S62, the degeneration of inclusion body
By 1:By the solubilization of inclusion bodies after washing, this denaturing liquid in the denaturing liquid of 8M carbamide is 5 inclusion body degeneration liquid proportional
50mM Tris-HCl, 8M Uera, 5mM DTT, pH8.0；Room temperature magnetic agitation 6 hours, then with 12000rpm, 4 DEG C,
20min is centrifuged, and collects denaturing liquid supernatant, 4 DEG C of preservations；
S63, the renaturation of inclusion body
Take gradient dilution method to carry out protein renaturation, adopt dropper hanging drop during renaturation, in three times denaturing liquid is added to pre-cooling
In, this renaturation solution is 50mM Tris-HCl, 0.15M NaCl, 0.3M L-Arg, 1mM GSSG, 5mM GSH, 10% glycerol,
pH7.0；Add rear magnetic agitation 5min every time, make deformation liquid and the ratio of renaturation solution reach 1:10；After the completion of proceed to 4 DEG C of ice
Case, stands renaturation 24h；
S64, nickel post affinity purification
After the completion of renaturation, renaturation solution removes L-Arg through dialysis, with reference to QIAGEN company ni-sepharose purification description purification His label
Destination protein, then SDS-PAGE analysis is carried out to purified product.
3. a kind of chicken alpha interferon optimized gene, its sequence such as SEQ ID NO：Shown in 1.
4. a kind of aminoacid of chicken alpha interferon, its sequence such as SEQ ID NO：Shown in 2.
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| CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
| CN108864300A (en) * | 2017-08-09 | 2018-11-23 | 安徽九川生物科技有限公司 | Chicken Albumin-interferon-' alpha '-interleukin-22 fusion protein, preparation method and its encoding gene, a breeder long-acting interferon |
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| CN109608535A (en) * | 2018-12-29 | 2019-04-12 | 广州市微生物研究所 | A kind of the chicken alpha interferon peptide chain and its recombinant expression engineered strain of optimization |
| CN111138523A (en) * | 2019-12-10 | 2020-05-12 | 天津生机集团股份有限公司 | Method for purifying and preparing recombinant chicken interferon α from recombinant chicken interferon α renaturation solution |
| CN111455006A (en) * | 2020-04-10 | 2020-07-28 | 天津生机集团股份有限公司 | Recombinant chicken interferon α product expressed by escherichia coli and preparation method and application thereof |
| CN113045670A (en) * | 2019-12-27 | 2021-06-29 | 华南农业大学 | Soluble chicken alpha interferon fusion protein and production method and application thereof |
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| CN109371050A (en) * | 2018-12-20 | 2019-02-22 | 天津瑞普生物技术股份有限公司 | A kind of preparation method of dog interferon |
| CN109371050B (en) * | 2018-12-20 | 2022-02-11 | 天津瑞普生物技术股份有限公司 | Preparation method of canine interferon |
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| CN111138523A (en) * | 2019-12-10 | 2020-05-12 | 天津生机集团股份有限公司 | Method for purifying and preparing recombinant chicken interferon α from recombinant chicken interferon α renaturation solution |
| CN113045670A (en) * | 2019-12-27 | 2021-06-29 | 华南农业大学 | Soluble chicken alpha interferon fusion protein and production method and application thereof |
| CN111455006A (en) * | 2020-04-10 | 2020-07-28 | 天津生机集团股份有限公司 | Recombinant chicken interferon α product expressed by escherichia coli and preparation method and application thereof |
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