CN101289666A - Method for preparing recombination porcine alpha-type interferon - Google Patents
Method for preparing recombination porcine alpha-type interferon Download PDFInfo
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Abstract
The invention discloses a method for preparing porcine recombinant IFN-alpha. The method is to use a special primer - PCR to amplify target genes of the porcine IFN-alpha, to connect the target genes with an expression vector - pET-28a and an expression vector - pGEX-5X, to transform colon bacillus, to make the colon bacillus highly efficiently express the porcine IFN-alpha and to prepare porcine recombinant IFN-alpha freeze-dried injection through the techniques such as high purification, sterilization, split charging, freeze-drying and so on. The method is shown to have obvious treatment effect on porcine virus diarrhea, influenza and so on and be superior to similar products in the aspects of safety and cure rate through safety experiment, IFN efficacy inspection and clinic probation. The preparation technique is simple; industrialized production is easy; transportation and preservation are easy after freeze-drying. After the porcine recombinant IFN-alpha is injected into a porcine body, the porcine recombinant IFN-alpha is characterized by good healing effect, reliable assembly, no side effect like inocuity and so on, can be used for curing diseases like the porcine virus diarrhea, the influenza and so on, and is novel high-efficient safe nonpersistent broad-spectrum antiviral preparation.
Description
Technical field
The present invention relates to a kind of recombination porcine alpha-type interferon (interferon, method of production IFN) and process.System will have porcine alpha-type IFN gene recombination plasmid transformed into escherichia coli, and make it efficiently express porcine alpha-type IFN, and purified back adds the freeze-drying of an amount of porcine blood plasma albumin stablizer makes, and is used for the treatment of virus diseases such as pig virus diarrhoea and influenza.The standard of development and calibrating all adopts the recombination human alpha type IFN (the Chinese biological goods rules Chinese biological standard of articles council compiles 2000 editions) of national SFDA authentication also to implement as reference.
Background technology
The pig virus disease is the main transmissible disease that current serious restriction pig aquaculture develops in a healthy way.Generation is all arranged, as pig virus diarrhoea, influenza etc. the whole year.In case these animal infection morbidities then have onset anxious, clinical symptom is serious, very easily propagates diffusion, and case fatality rate and all higher characteristics of mortality ratio, causes the tremendous economic loss to aquaculture; What is more important, pig contacts closely with human, and some animal virus sexually transmitted disease is returned human health and is brought potential threat.Mainly be by vaccine inoculation and use microbiotic to pig virus prevention and treatment of diseases approach at present.But because breeding environment imperfection, reasons such as virus variation and Abwehrkraft des Koepers variation make traditional prevention and treatment approach be subjected to huge challenge, most of antibiotics and traditional oral antiviral, because the drug residue problem is brought negative impact to human health; And traditional vaccine because its high specific and side effect, can't be resisted the significant damage that the continuous appearance of virus variation and new virus brings for the pig aquaculture.Therefore, active treatment poultry, livestock disease viral disease will be the human problems of paying close attention to the most.
IFN be the infection induced body of a viroid produce have broad-spectrum antiviral, antitumor and have the protein of immunoregulation effect, nineteen fifty-seven Issacs and Lindeman at first find, it is the multi-functional cytokine of a class, after cell receptor combines, 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 being brought into play aspect the immunity of organism, are divided into α, β, γ three classes.Existing known, α type IFN can act on cells infecteds such as virus in vivo selectively, by the biosynthesizing of the intracellular virus protein that suppresses to be contaminted, performance wide spectrum and efficient disease-resistance toxic action.But normal host cell is not had effect or acts on faint.Pig IFN is that a class can induce pig cell to produce the proteic cytokine of multiple broad-spectrum antiviral, is mainly used in the control of virus diseases such as porcine epizootic diarrhea and swine influenza, and when particularly using with other cytokine is collaborative with pig, effect is better.Therefore, IFN is one of important biomolecule preparation that has a extensive future now.
At present, domestic that be applied to veterinary clinic the earliest is pig leucocyte IFN, but the white corpuscle IFN cost that adopts the isolating method of hemocyte to obtain is higher, and the blood source is easily contaminated, is difficult to carry out suitability for industrialized production.And adopt genetic engineering technique to produce recombination porcine alpha-type IFN except that can avoiding above-mentioned drawback, and the purity height is still arranged, cost is low, and advantage such as be produced on a large scale has vast market prospect.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of recombination porcine alpha-type interferon, be with porcine alpha-type IFN gene recombination plasmid transformed into escherichia coli, make it efficiently express porcine alpha-type IFN, technologies such as purified back degerming, packing and freeze-drying are made, be a kind of broad-spectrum antiviral preparation, cure mainly virus diseases such as pig virus diarrhoea and influenza.
Technical solution of the present invention is as follows:
The preparation method of recombination porcine alpha-type interferon is characterized in that carrying out following steps:
(1), makes up cloning vector
Goal gene is:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Auele Specific Primer: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Cloning vector: pMD18-T simple vector
(2), construction of expression vector
Expression vector: pET-28a, perhaps pGEX-5X
(3), Recombinant Protein Expression
The recombinant expressed bacterium of picking vector construction success is increased, amplification culture, and adds the expression of inductor low temperature induction;
(4), identify recombinant protein
Use anti-alpha-interferon reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain the raw product Interferon, rabbit.
The preparation method of described recombination porcine alpha-type interferon is characterized in that concrete steps are:
(1), makes up cloning vector
Goal gene:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Specific primer sequence: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Behind the goal gene with Auele Specific Primer pcr amplification porcine alpha-type interferon, 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.According to experimental result, PCR and the equal positive plasmid of double digestion are carried out objective gene sequencing;
(2), construction of expression vector
After identifying that the back selects the clone bacterium consistent with goal gene to carry out double digestion, be connected, and correspondingly be converted into the BL-21 intestinal bacteria, cultivate respectively, identify the recombinant expressed bacterium of expression vector establishment success with expression vector pET-28a or pGEX-5X;
(3), Recombinant Protein Expression
The recombinant expressed bacterium that is connected with pET-28a or pGEX-5X expression vector of picking carries out a small amount of amplification, amplification culture respectively, adds isopropylthiogalactoside IPTG, and low temperature induction is expressed, and collects bacterium;
(4), identify recombinant protein
Use anti-alpha-interferon reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain the raw product Interferon, rabbit.
The preparation method of described recombination porcine alpha-type interferon is characterized in that more detailed steps is:
(1), makes up cloning vector
The goal gene of porcine alpha-type IFN is:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Specific primer sequence: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Behind the goal gene with Auele Specific Primer pcr amplification porcine alpha-type IFN, with goal gene clone in pMD18-Tsimple 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.According to experimental result, PCR and the equal positive plasmid of double digestion are carried out objective gene sequencing;
(2), construction of expression vector
After identifying that the back selects the clone bacterium consistent with goal gene to carry out double digestion, be connected with expression vector pET-28a or pGEX-5X, and correspondingly be converted into the BL-21 intestinal bacteria, separate application is in the LB culture medium flat plate that contains kantlex or contain the LB culture medium flat plate overnight incubation of ammonia benzyl green grass or young crops, get the bacterium colony of growing on the aforementioned 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;
(3), Recombinant Protein Expression
The recombinant expressed bacterium that is connected with pET-28a or pGEX-5X expression vector of picking is in the LB substratum that contains kantlex 100 μ g/ml or contain amplification on a small quantity in the LB substratum of the blue or green 100 μ g/ml of ammonia benzyl respectively, after respectively the LB substratum that contains kantlex 100 μ g/ml or contain in the LB substratum of the blue or green 100 μ g/ml of ammonia benzyl amplification culture or fermentation 3h after, to bacterium arrival logarithmic phase, survey bacterium in 600nm OD value during at 0.6-0.8, add isopropylthiogalactoside IPTG, 32 ℃ of low temperature inductions are expressed, final concentration 200 μ g/ml collect bacterium;
(4), identify recombinant protein
Use anti-α type IFN reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain raw product IFN.
The preparation method of described recombination porcine alpha-type interferon, it is characterized in that identifying recombinant protein: with the recombinant plasmid pET-28a/IFN and the pGEX-5X/IFN transformed into escherichia coli BL21 of goal gene, after IPTG induces, intestinal bacteria pET-28a/IFN tropina shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compare with empty bacterium, a dense newly-increased protein band that dyes is arranged about 25KD; Intestinal bacteria pGEX-5X/IFN tropina shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compares with empty bacterium, and a dense newly-increased protein band that dyes is arranged about 45KD.Identify through Western blotting, these two kinds of tropinas all can with anti-α type IFN reference serum generation strong positive reaction.
The preparation method of described recombination porcine alpha-type interferon is characterized in that purification of recombinant proteins is meant with behind the ultrasonication recombinant protein, obtains the solubility recombinant protein in the supernatant, again through AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography.
The preparation method of described recombination porcine alpha-type interferon, it is characterized in that: purification of recombinant proteins, the concrete steps that obtain raw product IFN are: collect bacterium liquid, 4 ℃ of centrifugal 5min of 12000r/min, abandon supernatant, stay precipitation ,-20 ℃ with room temperature multigelation precipitation 3 times after will precipitate mixing in 9 times of volume PBS liquid, hatch 5min for 4 ℃, ultrasonic degradation obtains recombinant protein, power: 400W, worked 3 seconds, 3 seconds at interval, ultrasonic 6min repeats 3-4 time, can make gram's staining and observe bacterium cracking situation, the centrifugal 10min of 12000r/min, the results supernatant.
The preparation method of described recombination porcine alpha-type interferon is characterized in that: AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography is measured IFN and is tired and specific activity specific activity 〉=1 * 10
6International unit/mg albumen is qualified; Aseptic subpackaged ,-70 ℃ of preservations.
The recombination porcine alpha-type interferon of the present invention's preparation after separation and purification, is made lyophilized injectable powder, after adding the 2ml aseptic double-distilled water, 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 intramuscular injection after will shaking up gently during use, piglet 5000～10,000 units/only/day; Grow up pig 20,000～40,000 units/only/day, inject once every day, course of treatment 3～5 times.
When treating the pig that fallen ill, should in time isolate the pig that do not fall ill, and give the protective inoculation of equivalent relative medicine, when using the recombination porcine alpha-type IFN of the present invention's preparation, should note the comprehensive prevention measures such as sterilization of environment, prevent and treat the recurrence of infection again or eqpidemic disease.
Embodiment:
Recombination porcine alpha-type IFN preparation technology flow process:
1. structure cloning vector:
Porcine alpha-type IFN goal gene is:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCA TGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGA TAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAG CAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGA GCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCA GCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAG AGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATC CAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCC AGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA specific primer sequence: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Behind Auele Specific Primer pcr amplification porcine alpha-type IFN goal gene, with goal gene clone in pMD18-Tsimple 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.According to experimental result, PCR and the equal positive plasmid of double digestion are carried out objective gene sequencing.
2. construction of expression vector:
Select goal gene cloning vector consistent after checking order to carry out behind the double digestion and be connected with expression vector pET-28a or pGEX-5X respectively with Genebank, and being converted into the BL-21 intestinal bacteria, separate application is in the LB culture medium flat plate that contains kantlex or contain the LB culture medium flat plate overnight incubation of ammonia benzyl green grass or young crops.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.
3. Recombinant Protein Expression:
The recombinant expressed bacterium that is connected with pET-28a or pGEX-5X expression vector of picking is in the LB substratum that contains kantlex 100 μ g/ml or contain amplification on a small quantity in the LB substratum of the blue or green 100 μ g/ml of ammonia benzyl respectively, after respectively in two kinds of LB substratum (contain kantlex 100 μ g/ml or contain the blue or green 100 μ g/ml of ammonia benzyl) behind the amplification culture or the 3h that ferments, survey the OD value when 0.6-0.8, add IPTG, 32 ℃ of low temperature inductions are expressed (final concentration 200 μ g/ml) 5h, collect bacterium.
For later on large-scale recombination porcine alpha-type IFN, need in fermentor tank, to express cultivation.The present invention has studied pilot scale fermentation technology simultaneously, and the expression level after the optimization reaches 50mg/L.
4. the preparation of raw product IFN:
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 time, the centrifugal 15min acquisition of centrifugal 12000r/min supernatant.
5. raw product IFN purifying:
5.1AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography.
5.1.1 affinity chromatography:,, collect the IFN peak with Elution buffer (50mMTris-Cl 40mM reductive glutathione pH 8.0) gradient elution with crossing the GST affinity column after the rough albumen filtration treatment.
5.1.2 desalination, damping fluid displacement: the IFN of the first step purifying is crossed desalting column,, prepare next step ion exchange chromatography with damping fluid (50mMTris-ClpH6.5) displacement.
5.1.3 ion exchange chromatography: use the good cylinder of Loading buffer (50mMTris-Cl pH6.5) balance respectively, with Elution buffer (50mMTris-Cl 1M NaCl pH6.5) gradient elution, collect the IFN peak behind the last sample.
5.1.4 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.
5.1.5 interpretation of result: detect purity of protein with SDS-page and westen behind each step purifying, Lowry method protein quantification calculates each step purifying yield.
Tire specific activity 〉=1 * 10 and specific activity 5.2 measure IFN
6International unit/mg albumen is qualified;
5.3 aseptic subpackaged ,-70 ℃ of preservations.
6. the evaluation of porcine alpha-type IFN behind the purifying:
6.1 protein quantification detects: use the Lowry method, do standard test with the standard protein of Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
6.2SDS-PAGE electrophoresis detection is compared with empty bacterium, two kinds of reorganization bacterium have a dense newly-increased protein band that dyes respectively about 25KD and 45KD.
6.3 the purity of recombinant porcine alpha IFN behind the efficient liquid phase chromatographic analysis purifying, pig α IFN＞95% behind the purifying.
6.4 western blot test: the sample behind the purifying can with anti-α type IFN reference serum generation strong positive reaction.
7. recombinant porcine alpha IFN tires and the mensuration of specific activity behind the purifying:
7.1IFN titration: recombinant porcine alpha IFN can suppress 100TCID behind the purifying on WISH cell
50The propagation of VSV virus.
7.1.1 on the trace Tissue Culture Plate of 96 holes, cultivate WISH cell, put 5%CO with the DMEM nutritive medium
2Cultivated 24 hours under 37 ℃ of conditions of incubator.
7.1.2 the pig α IFN with various dose handles.
Abandon 7.1.3 inhale after 24 hours, inoculate 100TCID more respectively
50VSV virus.
7.1.4 the result shows that the cytopathy that the recombination porcine alpha-type IFN of acquisition causes VSV 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 two kinds of recombinant porcine alpha IFN processing that obtain, under inverted microscope, observed seven days continuously, cellular form is normal, any pathology do not occur, record to tire＞10
7IU/ml.
7.2 specific activity measuring method: after the detection of the recombination porcine alpha-type IFN work in-process protein quantification behind the purifying, live＞1 * 10 with general in the world WISH/VZV systems measurement ratio
7IU/mg.
8. get supernatant liquor seitz filter pressure filtration, collect bacteria-free filtrate, steriling test and safety verification are carried out in sampling.After qualified after testing, add final concentration contain 5% skim-milk, 5%GS (glucose) and 10% porcine blood plasma albumin as the lyophilized vaccine mixing after, place the Freeze Drying Equipment freeze-drying.
9. recombination porcine alpha-type IFN suppresses the application in the pig parvoviral in porcine kidney cell:
9.1 on 96 holes trace Tissue Culture Plate,, put 5%CO with the DMEM nutrient solution culture nephrocyte (PK-15) of raising pigs
2Cultivated 24 hours under 37 ℃ of conditions of incubator.
9.2 recombination porcine alpha-type IFN function cells with various dose.
Abandon 9.3 inhale after 24 hours, inoculate 100TCID more respectively
50The pig parvoviral suspension.
9.4 the result shows that the cytopathy that the recombination porcine alpha-type IFN of acquisition causes pig parvoviral 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 recombination porcine alpha-type IFN that the obtains processing, under inverted microscope, observed seven days continuously, cellular form is normal, any pathology do not occur.
10. recombination porcine alpha-type IFN safety experiment:
10.1 cavy check:
With 3 of the healthy guinea pigs of body weight 350～400g, each veutro subcutaneous injection recombination porcine alpha-type IFN 2ml observed 10, all strong living.
10.2 small white mouse check:
With 5 of the small white mouses of body weight 18～20g, each tail vein injection recombination porcine alpha-type IFN 0.5ml observed 7, all strong living.
10.3 suckling mouse check:
With 2 age in days suckling mouses, 1 nest (at least 5 are with female mouse to raise), every back subcutaneous injection 0.1ml establishes control group simultaneously, observes 7, all strong living.
10.4 pig check:
With 4 of the healthy susceptible weanling pigs that detects no swine fever maternal antibody through the neutralization test method (observed 5～7 before the injection, every day at the upper and lower noon is respectively surveyed body temperature 1 time, selects body temperature, spirit, the normal person's use of appetite).Every intramuscular injection finished product 6ml, inoculation back every day at the upper and lower noon respectively surveys body temperature and observes 1 time, observes 21.Compare before body temperature, spirit, appetite and injection this product and do not have considerable change; Or fervescence surpasses 0.5 ℃, but is no more than 1 ℃, delays to be no more than 2; Or subtract the food be no more than 1.Assay, sample are all qualified.
11. recombination porcine alpha-type IFN efficacy test:
Check with piglet:
According to " People's Republic of China's veterinary biologics rules " regulation, the test of selection is the weanling pig of healthy susceptible 21 ages in days with piglet, and every crowd of IFN injects 8.During test, each intramuscular injection IFN 20,000 U/ only (only is total to about 60,000 U/) for three days on end.24h after medication first, per os feeding pig parvoviral suspension (hemagglutinative titer 〉=1: 1280; TCID
50〉=1.0 * 10
-9/ ml) 0.1ml (200TCID
50).Establish simultaneously virus control group and normal control group equally.Attack poison back 12h, IFN protection group and all behaviors of normal control group test piglet are normal, and clinical symptom then appears in virus control group piglet.Cut open extremely after observing 96h, IFN protection group and normal control group intestinal tissue do not have obvious pathological change, and virus is separated negative; Obvious intestinal tissue pathological change then appears in the virus control group, and virus is separated positive.
12. clinical trial result:
The IFN of development in the Hefei City, Anhui Province, Duo Jia pig farms (family) such as Mingguang City and Wuhe County are on probation, the result proves that reorganization IFN is 86.5% to treating pig virus diarrhoea efficient, curative ratio is 67.9%; It is 88.53% that the treatment porcine parvovirus has rate, and curative ratio is 69.93%, and quality is better than existing products in markets.
＜110〉Wang Mingli
＜120〉preparation method of recombination porcine alpha-type interferon
<160>1
<210>1
<211>501
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>CDS
<222>(1)...(501)
<400>1
tcactccttc?ttcctgagtc?tgtcttgcag?gtttgtggag?gaagagaagg?ctctcatgac?60
ttctgccctg?acgatctccc?aggcacaggg?gctgtagctc?ttctcttgca?gatagagggt?120
gagtctgtgg?aagtatttcc?tcacagccag?gatggagtcc?tcctccagca?ggggggtccc?180
ttccagcccg?gcctcctgca?tgacacaggc?ttccaggtcc?ctgagctgct?gatccagtcc?240
agtgcagaac?tggtgcagga?ggctctcatc?ccaggcagca?gccgagccct?ctgtgctgaa?300
gagctggaag?gtctgctgga?gcatctcatg?caccagagcc?atggcttgag?ccttctggac?360
ctggttgccc?cccaaggcct?cttgggggaa?tccaaagtcc?cttctgtggt?ccaggcagga?420
gaagggggag?attctcctca?tttgtgccag?gagcctcagg?gccctggtgt?gagccaggct?480
gtgggtctga?ggcaggtcgc?a?501
Claims (7)
1, the preparation method of recombination porcine alpha-type interferon is characterized in that carrying out following steps:
(1), makes up cloning vector
Goal gene is:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Auele Specific Primer: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Cloning vector: pMD18-T simple vector
(2), construction of expression vector
Expression vector: pET-28a, perhaps pGEX-5X
(3), Recombinant Protein Expression
The recombinant expressed bacterium of picking vector construction success is increased, amplification culture, and adds the expression of inductor low temperature induction;
(4), identify recombinant protein
Use anti-alpha-interferon reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain the raw product Interferon, rabbit.
2, the preparation method of recombination porcine alpha-type interferon according to claim 1 is characterized in that concrete steps are:
(1), makes up cloning vector
Goal gene:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Specific primer sequence: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Behind the goal gene with Auele Specific Primer pcr amplification porcine alpha-type interferon, 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.According to experimental result, PCR and the equal positive plasmid of double digestion are carried out objective gene sequencing;
(2), construction of expression vector
After identifying that the back selects the clone bacterium consistent with goal gene to carry out double digestion, be connected, and correspondingly be converted into the BL-21 intestinal bacteria, cultivate respectively, identify the recombinant expressed bacterium of expression vector establishment success with expression vector pET-28a or pGEX-5X;
(3), Recombinant Protein Expression
The recombinant expressed bacterium that is connected with pET-28a or pGEX-5X expression vector of picking carries out a small amount of amplification, amplification culture respectively, adds isopropylthiogalactoside IPTG, and low temperature induction is expressed, and collects bacterium;
(4), identify recombinant protein
Use anti-alpha-interferon reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain the raw product Interferon, rabbit.
3, the preparation method of recombination porcine alpha-type interferon according to claim 1 is characterized in that more detailed steps is:
(1), makes up cloning vector
The goal gene of porcine alpha-type IFN is:
TCACTCCTTCTTCCTGAGTCTGTCTTGCAGGTTTGTGGAGGAAGAGAAGGCTCTCATGACTTCTGCCCTGACGATCTCCCAGGCACAGGGGCTGTAGCTCTTCTCTTGCAGATAGAGGGTGAGTCTGTGGAAGTATTTCCTCACAGCCAGGATGGAGTCCTCCTCCAGCAGGGGGGTCCCTTCCAGCCCGGCCTCCTGCATGACACAGGCTTCCAGGTCCCTGAGCTGCTGATCCAGTCCAGTGCAGAACTGGTGCAGGAGGCTCTCATCCCAGGCAGCAGCCGAGCCCTCTGTGCTGAAGAGCTGGAAGGTCTGCTGGAGCATCTCATGCACCAGAGCCATGGCTTGAGCCTTCTGGACCTGGTTGCCCCCCAAGGCCTCTTgGGGGAATCCAAAGTCCCTTCTGTGGTCCAGGCAGGAGAAGGGGGAGATTCTCCTCATTTGTGCCAGGAGCCTCAGGGCCCTGGTGTGAGCCAGGCTGTGGGTCTGAGGCAGGTCGCA
Specific primer sequence: upstream: GAATTC TGCGAC CTGCCT CAGACC CA
Downstream: CTCGAG TCACTC CTTCTT CCTGAG TC
Behind the goal gene with Auele Specific Primer pcr amplification porcine alpha-type IFN, with goal gene clone in pMD18-Tsimple 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.According to experimental result, PCR and the equal positive plasmid of double digestion are carried out objective gene sequencing;
(2), construction of expression vector
After identifying that the back selects the clone bacterium consistent with goal gene to carry out double digestion, be connected with expression vector pET-28a or pGEX-5X, and correspondingly be converted into the BL-21 intestinal bacteria, separate application is in the LB culture medium flat plate that contains kantlex or contain the LB culture medium flat plate overnight incubation of ammonia benzyl green grass or young crops, get the bacterium colony of growing on the aforementioned 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;
(3), Recombinant Protein Expression
The recombinant expressed bacterium that is connected with pET-28a or pGEX-5X expression vector of picking is in the LB substratum that contains kantlex 100 μ g/ml or contain amplification on a small quantity in the LB substratum of the blue or green 100 μ g/ml of ammonia benzyl respectively, after respectively the LB substratum that contains kantlex 100 μ g/ml or contain in the LB substratum of the blue or green 100 μ g/ml of ammonia benzyl amplification culture or fermentation 3h after, to bacterium arrival logarithmic phase, survey bacterium in 600nm OD value during at 0.6-0.8, add isopropylthiogalactoside IPTG, 32 ℃ of low temperature inductions are expressed, final concentration 200 μ g/ml collect bacterium;
(4), identify recombinant protein
Use anti-α type IFN reference serum and do the neutralization experiment, prove that type is errorless;
(5), purification of recombinant proteins, obtain raw product IFN.
4, according to the preparation method of one of claim 1-3 described recombination porcine alpha-type interferon, it is characterized in that identifying recombinant protein: with the recombinant plasmid pET-28a/IFN and the pGEX-5X/IFN transformed into escherichia coli BL21 of goal gene, after IPTG induces, intestinal bacteria pET-28a/IFN tropina shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compare with empty bacterium, a dense newly-increased protein band that dyes is arranged about 25KD; Intestinal bacteria pGEX-5X/IFN tropina shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compares with empty bacterium, and a dense newly-increased protein band that dyes is arranged about 45KD.Identify through Western blotting, these two kinds of tropinas all can with anti-α type IFN reference serum generation strong positive reaction.
5,, it is characterized in that purification of recombinant proteins is meant with behind the ultrasonication recombinant protein, obtains the solubility recombinant protein in the supernatant, again through AKTA according to the preparation method of one of claim 1-3 described recombination porcine alpha-type interferon
TMThe consummate albumen of explorer protein purification instrument affinity chromatography.
6, preparation method according to one of claim 1-3 described recombination porcine alpha-type interferon, it is characterized in that: purification of recombinant proteins, the concrete steps that obtain raw product IFN are: collect bacterium liquid, 4 ℃ of centrifugal 5min of 12000r/min, abandon supernatant, stay precipitation ,-20 ℃ with room temperature multigelation precipitation 3 times after will precipitate mixing in 9 times of volume PBS liquid, hatch 5min for 4 ℃, ultrasonic degradation obtains recombinant protein, power: 400W, worked 3 seconds, 3 seconds at interval, ultrasonic 6min repeats 3-4 time, can make gram's staining and observe bacterium cracking situation, the centrifugal 10min of 12000r/min, the results supernatant.
7, the preparation method of recombination porcine alpha-type interferon according to claim 6 is characterized in that: AKTA
TMThe consummate albumen of explorer protein purification instrument affinity chromatography is measured IFN and is tired and specific activity specific activity 〉=1 * 10
6International unit/mg albumen is qualified; Aseptic subpackaged ,-70 ℃ of preservations.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101845441A (en) * | 2010-03-16 | 2010-09-29 | 南京农业大学 | Composite porcine alpha-IFN gene and recombinant vector thereof |
| CN101736062B (en) * | 2009-11-27 | 2012-11-07 | 王明丽 | Method for preparing recombinant porcine alpha interferon standard substance |
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| CN107326037A (en) * | 2017-07-18 | 2017-11-07 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving Recombinant Swine interferon beta fusion protein antiviral activity |
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| CN101736062B (en) * | 2009-11-27 | 2012-11-07 | 王明丽 | Method for preparing recombinant porcine alpha interferon standard substance |
| CN101845441A (en) * | 2010-03-16 | 2010-09-29 | 南京农业大学 | Composite porcine alpha-IFN gene and recombinant vector thereof |
| CN101845441B (en) * | 2010-03-16 | 2011-10-12 | 南京农业大学 | Composite porcine alpha-IFN gene and recombinant vector thereof |
| CN104262480A (en) * | 2014-09-28 | 2015-01-07 | 重庆理工大学 | Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection |
| CN105755008A (en) * | 2014-12-17 | 2016-07-13 | 北京大北农科技集团股份有限公司动物医学研究中心 | DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon |
| CN106939042A (en) * | 2016-01-22 | 2017-07-11 | 华南农业大学 | A kind of porcine alpha-IFN and its application |
| 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 |
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