CN103290021A - A preparation method for recombinant chicken interferon alpha - Google Patents
A preparation method for recombinant chicken interferon alpha Download PDFInfo
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Abstract
The invention discloses a preparation method for recombinant chicken interferon alpha. The method comprises the following steps of: a, obtaining the gene of the chicken interferon alpha; b, constructing a cloning vector; c, constructing an expression vector; d, expressing a recombinant protein; e, preparing crude recombinant chicken interferon alpha; and f, purifying the crude recombinant chicken interferon-alpha. Compared with methods in the prior art, the recombinant chicken interferon alpha produced by using the method of the invention is a cytokine which can induce chicken cells to produce a variety of broad-spectrum antiviral proteins, and can be used for prevention and treatment of viral diseases such as the Newcastle disease and avian influenza.
Description
Technical field
The invention belongs to the gene recombination technology field, particularly relate to the preparation method of a kind of reorganization chicken interferon α (Recombinant chicken interferon α, rChIFN α).
Background technology
Along with the fast development of global livestock and poultry breeding industry in recent years, China's poultry had developed into society's one big industry by rural households' sideline production already, was that kind or the quantity of bird all is positioned at prostatitis, the world.Yet, also there are a lot of problems in aviculture, as communicable diseases such as bird flu, infectious bursal disease, chicken infectious bronchitis, Marek and newcastle diseases, all can break out in different seasons every year, in case these animal infection morbidities, then anxious, the clinical symptom of morbidity just seriously, is very easily propagated all higher characteristics of diffusion and case fatality rate and mortality ratio, causes the tremendous economic loss to aquaculture; What is more important, chicken contacts closely with human, and some viral infectious such as bird flu etc. are returned human health and are brought potential threat.Vaccine immunity and pharmacological agent are mainly adopted in the control of bird communicable disease at present, because the serotype of vaccine immunity is single, and the serotype complexity of virus, the strain variation is fast, often causes the vaccine immunity failure.Some virus diseases still do not have vaccine at present and can use, and some virus also may directly jeopardize human beings'health.
Pharmacological agent commonly used mainly adopts microbiotic to treat, but in recent years because antibiotic extensive and a large amount of the use causes endurance strain to produce in a large number, and infect to the people by food chain, brings bigger threat to human health.Oneself prohibites some microbiotic and the application of antiseptic-germicide in aquaculture more present countries.Therefore, use and to disturb usually that the virus disease of active treatment and prevention poultry, domestic animal will be the human problems of paying close attention to the most.
The IFN protein with broad-spectrum antiviral, antitumor and immunoregulation effect that to be a class induce that body produces by materials such as virus and lipopolysaccharides, nineteen fifty-seven is at first found by Issacs and Lindeman, it is the multi-functional cytokine of a class, after cell receptor is combined, can induce body to produce multiple specific protein and enzyme, mainly by suppressing that virogene is transcribed and the viral RNA of degrading suppresses the growth and breeding of virus and bringing into play antitumor etc. activity.Generation cell, biochemical character according to IFN reach in the effect difference of bringing into play aspect the immunity of organism, are divided into α, β, three kinds of types of γ.Existing known, interferon alpha can act on virus infected cell in vivo selectively, by the biosynthesizing of the intracellular virus protein that suppresses to be contaminted, and performance wide spectrum and efficient disease-resistance toxic action, but normal host cell is not had effect.But the chicken interferon α that recombinates at present is not reported.
Summary of the invention
Technical problem to be solved by this invention provides the preparation method of a kind of chicken interferon α that recombinates, and described Interferon, rabbit is used for α treatment newcastle disease (ND), bird flu, mark Li Shi disease (MD).
Preparation method of the present invention is: by the design primer, from the chicken liver tissue, extract RNA and carry out the RT-PCR reaction, and behind the T carrier, be connected to the chicken interferon α gene clone that amplifies among the prokaryotic expression carrier pET-32a again, with the host bacterium of escherichia coli BL21 (DE3) as prokaryotic expression, be built into BL21/pET-32a-rChIFN α engineering bacteria, and after identifying by PCR, double digestion, induce it to express chicken interferon α with IPTG, lyophilized injectable powder is made in purified back degerming, packing and freeze-drying.
The lyophilized injectable powder of the reorganization chicken interferon α of the present invention preparation, add the 2ml aseptic double-distilled water after, can be dissolved as even suspension liquid rapidly, specification is: IFN content 〉=20,000 unit/bottles, but 2～8 ℃ of prolonged preservation, room temperature (≤25 ℃) can be preserved 2 years.Can carry out the collunarium inoculation after shaking up gently during use, chick 500 IU//days; Grow up chicken 2000～10000 IU/only/day, once a day, course for the treatment of 3～5 times.When chicken has been fallen ill in treatment, should in time isolate the chicken that do not fall ill, and give the prevention of equivalent relative medicine
The present invention compared with prior art, made reorganization chicken interferon α is that a class can induce chicken cell to produce the cytokine of multiple broad-spectrum disease resistance toxalbumin, can be used in the control of newcastle disease, avian influenza toxicity disease.
Description of drawings
Fig. 1 is the result of chicken interferon α gene RT-PCR amplification.
In Fig. 1,1:DNA Marker DL2000, the 2:RT-PCR amplified production single band occurs at 502bp;
Fig. 2 is recombinant plasmid pMD18-T/ChIFN-α double digestion qualification result;
In Fig. 2,1 is DNA Marker DL2000, and 3,4 is that recombinant plasmid pMD18-T/ChIFN α is through EcoR I and Hind III double digestion result, at the single band of 502bp place appearance.
Fig. 3 recombinant plasmid pMD18-T/ChIFN-α PCR result.
In Fig. 3,5 for being that template PCR product has single band at the 502bp place with recombinant plasmid pMD18-T/ChIFN α.
Fig. 4 is recombinant plasmid pET32/ChIFN-α double digestion qualification result.
In Fig. 4,6,7 cut qualification result for recombinant plasmid pET-32a-rChIFN α EcoR I and Hind III enzyme, and 8 is the pET32 empty plasmid.
Fig. 5 is the PCR result of recombinant plasmid pET32/ChIFN-α.
In Fig. 5,9 are: negative control, 10,11 is with recombinant plasmid pET-32a-rChIFN α template pcr amplification product.
Fig. 6 is 32 ℃ of reorganization chicken interferon α protein SDS-PAGE electrophoresis detection results that induce of 0.5mmol/L IPTG.
In Fig. 6,12 are: albumen marker, 13 is that 32 ℃ of IPTG induce the empty bacterium thalline in back, 14 induce supernatant behind the bacterial cell disruption behind the 5h for reorganization chicken interferon α engineering bacteria, 15 go up the cleer and peaceful 35KD of being deposited in place predominant expression band after inducing bacterial cell disruption postprecipitation behind the 5h, the bacterium of as seen recombinating to induce bacterial cell disruption behind the 5h for reorganization chicken interferon α engineering bacteria.
Fig. 7 is recombinant protein Western Blot qualification result.
In Fig. 7,16 is empty bacterium, and 17 induce broken back supernatant for reorganization chicken interferon α engineering bacteria, and 18 induce broken postprecipitation for reorganization chicken interferon α engineering bacteria.
Fig. 8 is the cytopathogenic restraining effect of the reorganization VSV of chicken interferon α.
In Fig. 8,1 is VSV virus control hole, and 2 is the HEp-2 cell control well;
A3-12 is that the human interferon standard substance of gradient dilution (from right to left) are handled the hole
B3-12 is that the rChIFN α of gradient dilution (from right to left) handles the hole
Fig. 9 is reorganization chicken interferon α engineering bacterial strain gram's staining light microscopic figure (* 1000).
Embodiment
Do detailed explanation below in conjunction with the present invention of embodiment.
Embodiment 1:
Reorganization chicken interferon α preparation technology flow process:
Obtaining of a, chicken interferon α gene:
Adopt the TAKARA RNA of company to extract test kit and from the chicken liver tissue, extract RNA, and utilize primer to carry out the RT-PCR reaction;
Primer sequence: upstream: CCG GAATTC GCCTGC AACCAC CTTCGC
Downstream: CCG AAGCTT AGTGCG CGTGTT GCCTGT
Primer has EcoR I and Hind III restriction enzyme site respectively;
Chicken interferon α goal gene is:
GAATTCGCCTGCAACCACCTTCGCCCCCAGGATGCCACCTTCTCTCACGACAGCCTCCAGCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTGCTCCTTCAACGACACCATCCTGGACACCAGCAACACCCGGCAAGCCGACAAAACCACCCACGACATCCTTCAGCACCTCTTCACAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAACGACAGCCAACGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCAATGCTTGGACAGCAGCGACACGCGCTCCCGGACGCGATGGCCTCGCAACTTCACCTCACCATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAACGATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAACACGCGCACTAAGCTTGC
Extract the RNA reaction conditions: in the total reaction system of 25 μ L, genomic dna 1.5 μ l, each 0.5 μ l of upstream and downstream primer, PCR Mix is 12.5 μ l, adds sterilized water to 25 μ l;
RT-PCR reaction conditions: 95 ℃ of pre-sex change 4 min; 94 ℃ of sex change 45 S, 58 ℃ of annealing 45S, 72 ℃ are extended 1 min, circulate 35 times, and last 72 degree extend 10 min, and the RT-PCR amplified production specific band occurs through agarose gel electrophoresis about 502bp, and its result is as shown in Figure 1.
B, structure cloning vector:
Behind the pcr amplification chicken interferon α goal gene, product reclaims test kit by glue and reclaims, with goal gene clone in pMD18-T simple vector to and transform the JM109 intestinal bacteria and be coated with the LB culture medium flat plate and carry out the screening of blue hickie, the clone bacterium of extracting waste sample carries out the target gene PCR checking, carrying out EcoR I and Hind III double digestion simultaneously identifies, product single band occurs through agarose gel electrophoresis at the 502bp place, its result is shown in Fig. 2,3.
C, construction of expression vector:
Select goal gene cloning vector consistent with Genebank after checking order to carry out behind the double digestion and be connected with expression vector pET-32a respectively, and be converted into the BL21 intestinal bacteria, coat the LB culture medium flat plate overnight incubation that contains penbritin respectively, get the bacterium colony of growing on the LB flat board and identify goal gene through PCR, the capable EcoR I of positive colony bacteria plasmid and Hind III double digestion are identified, be accredited as positive person and represent the expression vector establishment success, pcr amplification and double digestion product single band occurs through agarose gel electrophoresis at the 502bp place, its result is shown in Fig. 4,5.
D, Recombinant Protein Expression:
The reorganization chicken interferon α engineering bacteria that is connected with the pET-32a expression vector of picking amplification on a small quantity in the LB substratum that contains penbritin 100 μ g/ml respectively, the back is in LB substratum (containing penbritin 100 μ g/ml) behind amplification culture 2～3h, survey the OD value at 0.6～0.8 o'clock, add IPTG, 32 ℃ of abduction deliverings (final concentration 100 μ g/ml) 5h, collect bacterium, through the SDS-PAGE electrophoresis detection, the reorganization bacterium goes up the visible predominant expression band in the cleer and peaceful 35KD of being deposited in place after inducing bacterial cell disruption behind the 5h, and its result as shown in Figure 6.
The preparation of e, reorganization chicken interferon α raw product:
Collect bacterium ,-20 ℃ with room temperature multigelation precipitation 3 times, 4 ℃ of ultrasonic degradation bacterial precipitations, power: 400W, work 3S, 3S at interval, ultrasonic 6min repeats 3～4 times, the centrifugal 15min acquisition of centrifugal 12000 r/min supernatant, precipitation obtain rough albumen;
F, reorganization chicken interferon α raw product purifying:
F.1 AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography:
F.1.1 affinity chromatography:
To cross the GST affinity column after the rough albumen filtration treatment, with Elution buffer(50mMTris-Cl 40mM reductive glutathione pH 8.0) gradient elution, collect the IFN peak;
F.1.2 desalination, damping fluid displacement:
The IFN of the first step purifying is crossed desalting column, with damping fluid (50mMTris-Cl pH6.5) displacement, prepare next step ion exchange chromatography;
F.1.3 ion exchange chromatography:
Use Loading buffer(50mMTris-Cl pH6.5 respectively) the good cylinder of balance, with Elution buffer (50mMTris-Cl 1M NaCl pH6.5) gradient elution, collect the IFN peak behind the last sample;
F.1.4 sieve chromatography:
The IFN that ion exchange chromatography is collected crosses molecular sieve chromatography, Elution buffer (50mMNa
2HPO
40.15MNaCl pH7.0) collect the IFN peak;
Measure IFN and tire and specific activity specific activity 〉=1.0 * 10
6IU/mg albumen is qualified; Aseptic subpackaged ,-70 ℃ of preservations.
The evaluation of reorganization chicken interferon α:
Protein quantification detects:
Use the Lowry method, do standard test with the standard protein of Chinese food pharmaceutical biological product calibrating institute.
The SDS-PAGE electrophoresis detection:
Compare with empty bacterium, the reorganization bacterium has a dense newly-increased protein band that dyes at 35KD.
Western Blot result:
Being primary antibodie with abcam company mouse-anti chicken alpha-interferon (1:1000 dilution), is two anti-(1:2000 dilutions) with goat anti-mouse IgG-HRP.Reorganization chicken interferon α sample can with anti-chicken interferon alpha monoclonal antibodies generation specific reaction, specific band appears in the 35KD place, as shown in Figure 7.
Embodiment 3:
Reorganization chicken interferon α detections of tiring: according to micro-cytopathic-effect inhibition assay, adopt HEp-2 cell/VSV virus system: with DMEM nutritive medium cultivation Hep-2 cell, put 5%CO at 96 holes trace Tissue Culture Plate
2Cultivated 24 hours under 37 ℃ of conditions of incubator, add the reorganization chicken interferon α of various dose, inhale after 24 hours and abandon, inoculate 100 TCID more respectively
50VSV virus.The result shows that the cytopathy that the VSV of reorganization chicken interferon α of acquisition causes has the obvious suppression effect.Cell rounding all occurs after the undressed cell inoculation virus, come off, pathology such as disintegration.And after the cell inoculation virus after the reorganization chicken interferon α that the obtains processing, under inverted microscope, observe continuously, cellular form is normal, any pathology do not occur, record to tire＞1.0 * 10
6IU/ml, as shown in Figure 8.
Claims (8)
1. the preparation method of reorganization chicken interferon α is characterized in that: may further comprise the steps: the obtaining of a, chicken interferon α gene; B, structure cloning vector; C, construction of expression vector; D, Recombinant Protein Expression; The preparation of e, reorganization chicken interferon α raw product; F, reorganization chicken interferon α raw product purifying.
2. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that: described
Chicken interferon α goal gene is:
gaattcgcct gcaaccacct tcgcccccag gatgccacct tctctcacga cagcctccag 60
ctcctccggg acatggctcc cacactaccc cagctgtgcc cacagcacaa cgcgtcttgc 120
tccttcaacg acaccatcct ggacaccagc aacacccggc aagccgacaa aaccacccac 180
gacatccttc agcacctctt cacaatcctc agcagcccca gcactccagc ccactggaac 240
gacagccaac gccaaagcct cctcaaccgg atccaccgct acacccagca cctcgagcaa 300
tgcttggaca gcagcgacac gcgctcccgg acgcgatggc ctcgcaactt cacctcacca 360
tcaaaaaaca cttcagctgc ctccacacct tcctccaaga caacgattac agcgcctgcg 420
cctgggaaca cgtccgcctg caagctcgtg cctggttcct gcacatccac aacctcacag 480
gcaacacgcg cactaagctt gc 502。
3. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
Obtaining of described a, chicken interferon α gene:
Adopt the TAKARA RNA of company to extract test kit and from the chicken liver tissue, extract RNA, and utilize primer to carry out the RT-PCR reaction;
Primer sequence: upstream: CCG GAATTC GCCTGC AACCAC CTTCGC
Downstream: CCG AAGCTT AGTGCG CGTGTT GCCTGT
Primer has EcoR I and HindIII restriction enzyme site respectively;
Extract the RNA reaction conditions: in the total reaction system of 25 μ L, genomic dna 1.5 μ l, each 0.5 μ l of upstream and downstream primer, PCR Mix is 12.5 μ l, adds sterilized water to 25 μ l;
RT-PCR reaction conditions: 95 ℃ of pre-sex change 4 min; 94 ℃ of sex change 45 S, 58 ℃ of annealing 45S, 72 ℃ are extended 1 min, circulate 35 times, and last 72 degree extend 10 min, and the RT-PCR product reclaims test kit by glue and reclaims.
4. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
Described b, structure cloning vector:
Behind the pcr amplification chicken interferon α goal gene, with goal gene clone in pMD18-T simple vector to and transform the JM109 intestinal bacteria and be coated with the LB culture medium flat plate and carry out the screening of blue hickie, the clone bacterium of extracting waste sample carries out the target gene PCR checking, carries out double digestion simultaneously and identifies.
5. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
Described c, construction of expression vector:
Select goal gene cloning vector consistent with Genebank after checking order to carry out behind the double digestion and be connected with expression vector pET-32a respectively, and be converted into the BL21 intestinal bacteria, coat the LB culture medium flat plate overnight incubation that contains penbritin respectively, get the bacterium colony of growing on the LB flat board and identify goal gene through PCR, the capable double digestion of positive colony bacteria plasmid is identified, is accredited as positive person and represents the expression vector establishment success.
6. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
Described d, Recombinant Protein Expression:
The recombinant expressed bacterium that is connected with the pET-32a expression vector of picking is increased in the LB substratum that contains penbritin 100 μ g/ml respectively, surveys OD value at 0.6～0.8 o'clock, adds IPTG, 32 ℃ of abduction deliverings (final concentration 100 μ g/ml) 5h, collection bacterium.
7. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
The preparation of described e, reorganization chicken interferon α raw product:
Collect bacterium ,-20 ℃ with room temperature multigelation precipitation 3 times, 4 ℃ of ultrasonic degradation bacterial precipitations, power: 400W, work 3S, 3S at interval, ultrasonic 6min repeats 3～4 times, the centrifugal 15min acquisition of centrifugal 12000 r/min supernatant, precipitation obtain rough albumen.
8. the preparation method of reorganization chicken interferon α according to claim 1 is characterized in that:
Described f, reorganization chicken interferon α raw product purifying:
F.1 AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography:
F.1.1 affinity chromatography:
To cross the GST affinity column after the rough albumen filtration treatment, with Elution buffer(50mMTris-Cl 40mM reductive glutathione pH 8.0) gradient elution, collect the IFN peak;
F.1.2 desalination, damping fluid displacement:
The IFN of the first step purifying is crossed desalting column, with damping fluid (50mMTris-Cl pH6.5) displacement, prepare next step ion exchange chromatography;
F.1.3 ion exchange chromatography:
Use Loading buffer(50mMTris-Cl pH6.5 respectively) the good cylinder of balance, with Elution buffer (50mMTris-Cl 1M NaCl pH6.5) gradient elution, collect the IFN peak behind the last sample;
F.1.4 sieve chromatography:
The IFN that ion exchange chromatography is collected crosses molecular sieve chromatography, Elution buffer (50mMNa
2HPO
40.15MNaCl pH7.0) collect the IFN peak;
Measure IFN and tire and specific activity specific activity 〉=1 * 10
6International unit/mg albumen is qualified; Aseptic subpackaged ,-70 ℃ of preservations.
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| CN103789302A (en) * | 2013-12-05 | 2014-05-14 | 东北林业大学 | Universal primers for cloning avian alpha-interferon and application thereof |
| CN104531690A (en) * | 2014-12-20 | 2015-04-22 | 安徽九川生物科技有限公司 | Primers for obtaining genes of sheep interferon tau and preparation method for recombinant sheep interferon tau |
| CN104531691A (en) * | 2014-12-20 | 2015-04-22 | 安徽九川生物科技有限公司 | Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha |
| CN105039474A (en) * | 2015-08-25 | 2015-11-11 | 安徽九川生物科技有限公司 | Preparation method of recombinant chicken interferon-alpha standard substance |
| CN105132497A (en) * | 2015-08-25 | 2015-12-09 | 安徽九川生物科技有限公司 | Preparation process of recombinant poultry interferon alpha product lyophilized preparation |
| CN106362140A (en) * | 2016-08-25 | 2017-02-01 | 芜湖英特菲尔生物制品产业研究院有限公司 | Preparation method for recombinant pig IL-2 finish product freeze-dried preparation |
| CN106892976A (en) * | 2017-02-14 | 2017-06-27 | 华南农业大学 | A kind of recombination chicken interferon lambda（rChIFN‑λ）Clonal expression of gene and its preparation method and application |
| CN108840937A (en) * | 2017-08-09 | 2018-11-20 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma and preparation method thereof and a kind of recombination chicken long-acting interferon γ |
| CN109134641A (en) * | 2017-06-19 | 2019-01-04 | 杭州俊丰生物工程有限公司 | A kind of preparation method of chicken interferon-α |
| CN110013545A (en) * | 2019-03-12 | 2019-07-16 | 西北农林科技大学 | A kind of preparation method and applications of Cytokines Drug |
| CN115850514A (en) * | 2022-10-26 | 2023-03-28 | 山东迅达康兽药有限公司 | Recombinant chicken interferon alpha protein, preparation method and application thereof |
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| CN103789302A (en) * | 2013-12-05 | 2014-05-14 | 东北林业大学 | Universal primers for cloning avian alpha-interferon and application thereof |
| CN103789302B (en) * | 2013-12-05 | 2016-01-27 | 东北林业大学 | The universal primer of clone fowl alpha-interferon and application thereof |
| CN104531690A (en) * | 2014-12-20 | 2015-04-22 | 安徽九川生物科技有限公司 | Primers for obtaining genes of sheep interferon tau and preparation method for recombinant sheep interferon tau |
| CN104531691A (en) * | 2014-12-20 | 2015-04-22 | 安徽九川生物科技有限公司 | Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha |
| CN105039474A (en) * | 2015-08-25 | 2015-11-11 | 安徽九川生物科技有限公司 | Preparation method of recombinant chicken interferon-alpha standard substance |
| CN105132497A (en) * | 2015-08-25 | 2015-12-09 | 安徽九川生物科技有限公司 | Preparation process of recombinant poultry interferon alpha product lyophilized preparation |
| CN106362140A (en) * | 2016-08-25 | 2017-02-01 | 芜湖英特菲尔生物制品产业研究院有限公司 | Preparation method for recombinant pig IL-2 finish product freeze-dried preparation |
| CN106892976A (en) * | 2017-02-14 | 2017-06-27 | 华南农业大学 | A kind of recombination chicken interferon lambda（rChIFN‑λ）Clonal expression of gene and its preparation method and application |
| CN109134641A (en) * | 2017-06-19 | 2019-01-04 | 杭州俊丰生物工程有限公司 | A kind of preparation method of chicken interferon-α |
| CN108840937A (en) * | 2017-08-09 | 2018-11-20 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of fusion protein being made of Chicken Albumin and chicken interferon gamma and preparation method thereof and a kind of recombination chicken long-acting interferon γ |
| CN110013545A (en) * | 2019-03-12 | 2019-07-16 | 西北农林科技大学 | A kind of preparation method and applications of Cytokines Drug |
| CN115850514A (en) * | 2022-10-26 | 2023-03-28 | 山东迅达康兽药有限公司 | Recombinant chicken interferon alpha protein, preparation method and application thereof |
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Application publication date: 20130911 |