CN104447978A - Recombinant chicken interferon alpha and preparation method thereof - Google Patents

Recombinant chicken interferon alpha and preparation method thereof Download PDF

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Publication number
CN104447978A
CN104447978A CN201410755900.7A CN201410755900A CN104447978A CN 104447978 A CN104447978 A CN 104447978A CN 201410755900 A CN201410755900 A CN 201410755900A CN 104447978 A CN104447978 A CN 104447978A
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chicken interferon
interferon alpha
recombinant
preparation
recombination
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王连民
刘珂飞
高应瑞
荆玮
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Tianjin Shengji Group Co Ltd
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Tianjin Shengji Group Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha

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Abstract

The invention provides a recombinant chicken interferon alpha and a preparation method thereof. A target gene of the chicken interferon alpha is in a sequence as shown in a sequence table 400<1>. The method comprises the following specific steps: (1) obtaining of a chicken interferon alpha gene; (2) building of recombinant plasmids; (3) building of a gene engineering strain; (4) expression of recombinant protein; (5) fermentation production of the recombinant chicken interferon alpha; and (6) biological activity determination of the recombinant chicken interferon alpha. According to the preparation method, the gene engineering strain is applied to fermentation synthesis; the obtained recombinant chicken interferon alpha is a nontoxic and harmless biological product free of residues; the recombinant chicken interferon alpha has obvious antivirus activity; escort for poultry health in China is provided; and a green guarantee is provided for food safety.

Description

Recombination chicken interferon alpha and preparation method thereof
Technical field
The present invention relates to poultry technical field of medicament, especially recombination chicken interferon alpha and preparation method thereof.
Background technology
Interferon, rabbit (Interferon, IFN) be nineteen fifty-seven, Britain virus biologist Alick Isaacs and Switzerland researchist JeanLindenmann finds when utilizing chick chorioallantoic membrane to study influenza interference phenomenon, produced by the cell of virus infection and a kind ofly can act on other cells and the factor that copies of viral interference, therefore by its called after Interferon, rabbit.This factor of the purifying such as Lampson in 1963, proves that its molecular weight is the protein of 20 ~ 34KD.The Interferon, rabbit of investigator to the mankind and mammal has carried out large quantifier elimination afterwards, achieves breakthrough progress.Research shows that Interferon, rabbit is that a class can induce people and zooblast to produce the parahormone albumen of multiple broad-spectrum disease resistance toxalbumin, has the biologic activity such as antiviral, antitumor and immunomodulatory.1980, international Interferon, rabbit NK pointed out: Interferon, rabbit is the activated protein that a class has broad-spectrum antiviral function on allogenic cell, and the performance of its activity, again by the regulation and control of cytogene, relates to the synthesis of RNA and protein.Interferon, rabbit medically definition is specifically the interferon inducers by virus and other kinds, stimulates a kind of glycoprotein that reticuloendothelial system, scavenger cell, lymphocyte and somatocyte produce.
Mammalian interferon is divided into I type and interferon type Ⅱ, and interferon type Ⅰ mainly comprises again IFN-α, IFN-β, IFN-ω and IFN-τ, and they have similar structure and enjoy same class acceptor.First three kind mainly produces the response of virus infection, and body can be induced to produce antiviral protein.Interferon type Ⅱ is also known as type II interferon, i.e. IFN-γ, is produced under the effect of inducer by the T lymphocyte activated, does not enjoy same class acceptor with interferon type Ⅰ, having regulating effect to immunity system, is the main macrophage activating factor (MAF) of Mammals.IFN-α is mainly derived from bone-marrow-derived lymphocyte, scavenger cell and NK cell; IFN-β is mainly derived from inoblast and epithelial cell, and part derives from scavenger cell; IFN-γ then produces primarily of T lymphocyte, also can be produced by NK cell under certain condition.Chicken and other Avian Interferon systems and mammalian interferon system similar, also interferon type Ⅰ and interferon type Ⅱ is divided into, have now found that chicken interferon type Ⅰ comprises IFN-α, IFN-β, II type comprises IFN-γ, not yet finds IFN-ω and IFN-τ at present in bird.
The relative molecular mass of Interferon, rabbit is little, chicken interferon molecular weight is the protein of 20 ~ 34KD, there is the protein general character, to thermally-stabilised, can preserve for a long time for 2-8 DEG C, its activity can be preserved for a long time for 20 DEG C, 56 DEG C are then destroyed, pH2 ~ 10 scope internal interference element is not destroyed, and is easily neutralized by nuclease, can by proteolytic enzyme deactivation.Natural interference element is a kind of glycoprotein, after removing sugar, does not affect the anti-disease activity of Interferon, rabbit.Interferon, rabbit effect wide spectrum, to multiple virus even bacterium all have restraining effect, but restraint performance is different; Restraining effect shows species specificity usually, and the IFN that namely a certain kind cell produces can only act on other cell of identical kind, makes its adaptive immune power, and does not have provide protection to heterogenous cell, but there is cross-reactive between indivedual kind.
Chicken interferon α full genome is 582 bases, 193 amino acid of encoding, and wherein front 31 amino acid are signal peptide, and rear 162 amino acid are maturation protein, and molecular weight of albumen is about 19kDa.Sekellick equals successful clone and have expressed ChIFN2 α gene first in 1994, and carries out finishing structure analysis.The clonal expression in 2000 such as domestic scholars Wang Ming broiler chicken IFN α gene; In the same year, Xia Chun etc. have cloned Huiyang beard chicken and Silky fowl IFN α gene.
Interferon, rabbit, since nineteen fifty-seven is found, has confirmed that it has antiviral, antitumor, anti-parasitic-infectious, immunomodulatory, immunological adjuvant, affects the effects such as apoptosis successively.Interferon type Ⅰ main manifestations, for suppressing virus replication, antiproliferative effect, strengthens the ability of NK cell killing virus infected cell, strengthens the expression of mhc class i molecule and suppresses the expression of MHC class Ⅱmolecule.Interferon type Ⅱ is also known as type II interferon, and antiviral activity is low compared with I type, but the effect of its immunomodulatory and inhibition of cell proliferation is comparatively strong, and it is a kind of strong scavenger cell, NK cell, endothelial cell activation agent, and energy activating macrophage also promotes that it is active; T and bone-marrow-derived lymphocyte can be directly acted on, promote differentiation; The expression of mhc class i molecule and MHC class Ⅱmolecule can be strengthened.Marcus in 1999 etc. adopt the method for drinking water administration to carry out the research of recombinant C hIFN-α anti-new castle disease virus (NDV), show the antiviral unusual effect of IFN.Other result of study shows, recombinant C hlFN-α also has good result to virus diseases such as Marek, infectious bursal disease, infectious bronchitis, bird flus, shows wide application prospect.2004, Li Fenghua etc. reported that chicken genetically engineered IFN all has significant curative effect to virus diseases such as newcastle disease, infectious bursal disease, infectious bronchitis, Mareks.Plachy in 1999 etc. have carried out the research that chicken restructuring IFN prevents and treats RSV tumour, and the rGGIFN-α that result shows high dosage not only can Tumor suppression, can also make the RSV sarcoma completely dissolve of some chickens.1996, the research such as Weining showed, restructuring IFN is showing and mammiferous consistence by regulating and controlling in MHC class antigen presentation performance immunoloregulation function.Recombination chicken alpha-interferon (rChIFN-α) intravenous injection SPF chicken in 4 ~ 6 week age, after 24h, blood sampling is separated lymphocyte, by CD4 in Flow Cytometry Assay different time peripheral blood +and CD8 +the lymphocytic percentage of T, result shows, and rChIFN-α can significantly improve the lymphocytic percentage of CD4+T in 48 ~ 72h, proves that rChIFN-α has significant immunoregulation effect.
In clinical application, Interferon, rabbit has the irreplaceable effect of microbiotic.Current China has had commercial people to go on the market with genetic engineering interferon, but the research of the Interferon, rabbit of animal is relatively delayed, does not also have commercial launch at present.As everyone knows, China is poultry cultivation big country, and be also the consumption big country of bird product, the ups and downs of poultry cultivation industry directly affect the national economy of China.At present, high-density intensive large-scale raise in cages broiler chicken or laying hen are poultry patterns main at present, because density is excessive, cause breeding environment Quality Down, thus have manufactured opportunity to the invasion of multiple cause of disease particularly virus.The defence that virus opens chicken body is large behind the door, and the bacteriosis of various secondary occurs thereupon, causes chicken group massive mortality.In order to improve survival rate, increase culture benefit, raiser brings into use a large amount of microbiotic as fodder additives, thus reaches the object of the mortality ratio reducing chicken group.But along with antibiotic abuse is day by day serious, cause the drug residue severe overweight of bird product, various drug-resistant bacteria constantly occurs, serious harm is brought to the health of the mankind and animal and trade exports.So, find a kind of safely and effectively, " the green animal medicine " of pollution-free noresidue seem extremely urgent.
And genetically engineered chicken interferon α is biological products that are nontoxic, harmless, noresidue, there is obvious antiviral activity, therefore, be expected to be applied in poultry cultivation industry, to solving the subproblem existed in current China aquaculture, for China's avian health escorts, ensure for food safety provides green.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of recombination chicken interferon alpha.
Another technical problem to be solved by this invention is the preparation method providing above-mentioned recombination chicken interferon alpha.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of recombination chicken interferon alpha, its cDNA is sequence shown in sequence table 400<1>, and the polypeptide amino acid of its protein-active is sequence shown in sequence table 400<2>.
The preparation method of above-mentioned recombination chicken interferon alpha, concrete construction step is as follows:
(1) chicken interferon α gene is obtained: with reference to the chicken interferon α mature peptide gene coded sequence delivered in Gene Bank, synthesize the cDNA sequence of chicken interferon α, this sequence is by again optimizing, replace rare codon, regulate AT content, can at high expression in e. coli bl21 (DE3), the gene order of the chicken interferon α mature peptide after optimization is sequence shown in sequence table 400<1>;
(2) structure of recombinant plasmid: cut with enzyme the method connected and respectively above-mentioned chicken interferon α gene is connected with carrier pET-21a (+), obtain recombinant expression vector pET-21a (+)/ChIFN α carrying chicken interferon α gene, and carry out double digestion checking;
(3) structure of engineering strain: by recombinant expression vector transformed host strain BL21 (DE3) correct for above-mentioned checking, obtain the engineering strain containing recombinant plasmid;
(4) expression of recombinant protein: make engineering strain synthesize chicken interferon α albumen under fermentation condition under the induction of IPTG;
(5) fermentative production of recombination chicken interferon alpha: prepare fermentation seed liquid, seed liquor is accessed in fermentor tank, make it produce chicken interferon α in a large number by adding carbon source and inductor, the recombination chicken interferon alpha of acquisition makes chicken interferon alpha production through extracting, after purifying;
(6) Determination of biological activity of recombination chicken interferon alpha: suppress method according to few cells pathology (CPE), adopts clone to be chick fibroblast as test clone, detects the antiviral activity of recombinant interferon.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, in described step (2), prokaryotic expression carrier used is coli expression carrier pET-21a, carrier construction host cell is e.colistraindh5α, and expression host cell is e. coli bl21 (DE3) bacterial strain.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, the expression of recombinant protein in described step (3): the recombinant expressed bacterium that picking is connected with pET-21a expression vector is respectively increased in containing the LB substratum of penbritin 100 μ g/ml, it is 35 ~ 37 DEG C in culture temperature, shaking speed is under 180 ~ 200r/min condition, when to be cultured to bacterium liquid OD value be about 1, take out 1mL bacterium liquid in contrast, it is that the IPTG of 0.5 ~ 2mM/mL induces that remaining part adds final concentration, induction time is 4 ~ 6 hours, induction terminates the centrifugal 2 ~ 5min of rear 12000r/min and collects thalline, and carry out SDS-PAGE electrophoresis after being boiled by the thalline before and after induction, detect the expression of recombination chicken interferon alpha albumen.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, in described step (5), the fermentative production of recombination chicken interferon alpha comprises:
A. seed activation: activated on solid LB media by the engineering strain intestinal bacteria of expressing chicken alpha-interferon, then the single bacterium colony of picking one is in 50mL LB liquid nutrient medium, and at 30 ~ 37 DEG C, 180 ~ 220rpm, cultivates 12 ~ 24 hours; Again by the seed liquor obtained by volume 1% ~ 5% inoculum size be seeded in LB substratum, at 30 ~ 37 DEG C, 180 ~ 220rpm, cultivates 6 ~ 12 hours, obtains fermentation seed liquid;
B. inoculate: in fermentor tank, fill fermention medium, then the seed liquor that above-mentioned activation is good is seeded in liquid fermentation medium by the inoculum size of 5% ~ 10% by volume;
C. ferment: the mixed gas passing into air or air/oxygen in fermentor tank, air flow is 2 ~ 4m 3/ h, leavening temperature 30 ~ 37 DEG C, agitation revolution 300 ~ 900rpm, dissolved oxygen controls to control 6.8 ~ 7.4 at 20% ~ 40%, pH, and fermentation time 11 ~ 24 hours, starts feed supplement when OD600nm=2 ~ 4, and the flow acceleration of feed supplement is 100 ~ 1000mL/h;
D. induce: start when OD600nm=4 ~ 6 to add the expression that IPTG induces chicken interferon α, induce and stop tank in 4 ~ 6 hours.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, purifying, the extraction process of the middle recombination chicken interferon alpha of described step (5) comprise:
A. inclusion body protein is extracted: 4 DEG C, 6000r pm/min, 20min collected by centrifugation coli somatic, thalline weight in wet base is 77g/L, and fragmentation in PBS damping fluid is resuspended in by ultrasonic for 10g wet thallus under power 400 watts, work 3 seconds, interval 7 seconds, 20 ~ 30 minutes working hours condition, 4 DEG C, 9000rpm/min, 15min high speed frozen centrifugation, obtain chicken alpha-interferon inclusion body protein;
B. purifying inclusion body protein: first by mass volume ratio 1: 30, inclusion body is resuspended in the first washings high speed frozen centrifugation; Then by mass volume ratio 1: 30, inclusion body is resuspended in the second washings high speed frozen centrifugation; Finally by mass volume ratio 1: 40, inclusion body is resuspended in the 3rd washings high speed frozen centrifugation, the condition of described high speed frozen centrifugation is 4 DEG C, 10000rpm/min, 15min, and wherein the component proportion of the first washings is NaCl 137mmol/L, KCl 2.7mmol/L, Na 2hPO 410mmol/L, KH 2pO 42mmol/L, 1 ~ 2% tritonX-100, pH 8 ~ 9; Second washings is 1 ~ 2mol/L urea; 3rd washings is 0.5 ~ 1.5mol/L NaCl;
C. dissolve and renaturation inclusion body protein: be 1: 35 by resuspended for previous step centrifuged pellet denaturing agent by mass volume ratio, room temperature is placed to after precipitation dissolves gradually, and 4 DEG C, 8500rpm/min, 15min high speed frozen centrifugation, collect supernatant liquor; Under 4 DEG C of conditions, supernatant liquor is made dropwise to join in the renaturation solution of above-mentioned preparation, volume ratio is 1: 80, stirs 12 ~ 16h simultaneously, and the component proportion of described denaturing agent is 1 ~ 1.5mM EDTA, 30 ~ 40mM Tris, 6 ~ 8mM DTT, pH8 ~ 9 in every 4 ~ 6M Guanidinium hydrochloride;
D. gather in the crops renaturation recombined chicken alpha interferon albumen, detect through SDS-PAGE.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, the composition of described LB substratum is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, is prepared from by ordinary method.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, consisting of of described fermention medium: glucose 5g/L, peptone 5g/L, yeast powder 5g/L, KH 2pO 42g/L, K 2hPO 44g/L, Na 2hPO 412H 2o 7g/L, (NH 4) 2sO 41.2g/L, NH 4cl 0.2g/L, MnSO 45H 2o0.001g/L, CoCl 26H 2o 0.004g/L, Na 2moO 42H 2o 0.002g/L, ZnCl 20.002g/L, CuSO 45H 2o 0.001g/L, H 3bO40.005g/L, FeSO 47H 2o 0.02g/L, CaCl2H 2o0.02g/L, MgSO 47H 2o 0.3g/L, defoamer 0.2g/L, be prepared from by ordinary method.
Preferably, the preparation method of above-mentioned recombination chicken interferon alpha, the Determination of biological activity method of recombination chicken interferon alpha in described step (6): trial-product mensuration nutrient solution is diluted to every 1ml about containing 1000IU.In 96 porocyte culture plates, do 4 times of serial dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.Aseptically operate.Make chick embryo fibroblast adherent growth in the medium.Get cultured cells and discard nutrient solution, wash 2 digestion and collecting cells afterwards with PBS, be mixed with every lml containing 2.5 × 10 with complete culture solution 5~ 3.5 × 10 5the cell suspension of individual cell, is inoculated in 96 porocyte culture plates, every hole 100ul.In 37 DEG C, cultivate 4 ~ 6 hours under 5% carbon dioxide conditions.The standard solution prepared and need testing solution are moved in the fibroblastic culture plate of inoculated into chick embryo, every hole adds 100ul.In 37 DEG C, cultivate 18 ~ 24 hours under 5% carbon dioxide conditions.Discard the supernatant liquor in Tissue Culture Plate.The vesicular stomatitis virus (VSV ,-70 DEG C of preservations) preserved is diluted to 100CCID50 with attacking malicious nutrient solution, every hole 100ul.In 37 DEG C, 5% carbonic acid gas cultivates 24 hours.
Then discard the supernatant liquor in Tissue Culture Plate, every hole adds staining fluid 50ul, and room temperature carefully washes away staining fluid with flowing water after placing 30 minutes, and blots residual moisture, and every hole adds destainer 100ul, and room temperature is placed 3 ~ 5 minutes.After mixing, be reference wavelength by microplate reader with 630nm, measure absorbancy at wavelength 570nm place, record measurement result.
Testing data adopts computer program or four parametric regression computing methods to process.And be calculated as follows test-results:
Trial-product biologic activity ( IU / ml ) = Pr &times; Ds &times; Es Dr &times; Er
In formula: Pr is standard substance biologic activity, IU/ml;
Ds is trial-product pre-dilution multiple;
Dr is standard substance pre-dilution multiple;
Es is the extension rate that trial-product is equivalent to standard substance median effective dose;
Er is that standard substance partly imitate extension rate.
Beneficial effect of the present invention:
Above-mentioned recombination chicken interferon alpha, there is production cost low, purity is high, active strong feature, poultry cultivation industry field can be widely used in, its preparation method is based on intestinal bacteria, genetically engineered recombinant technology is used to build engineering strain, related protein gene is obtained by gene chemical synthesis, and using microbe fermentation technique, engineering strain is acted on fermentation synthesis, the recombination chicken interferon alpha obtained is nontoxic, harmless, the biological products of noresidue, certain effect is had to suppressing viral copying and improve immunity of organisms aspect, therefore, be expected to be applied in poultry cultivation industry, to solving the subproblem existed in current China aquaculture, for China's avian health escorts, ensure for food safety provides green.
Accompanying drawing explanation
Fig. 1 is vector construction schematic diagram of the present invention;
Fig. 2 is the digestion verification electrophorogram of pET-21a (+) of the present invention/ChIFN α recombinant expression plasmid, wherein:
M is DNA marker; 1-2 is pET-21a (+)-ChIFN α/EcoR I/Sal I;
Fig. 3 is the protein expression electrophorogram of recombinant bacterium E.coli BL21 (DE3) of the present invention/pET-21a (+)-ChIFN α, wherein:
Swimming lane M is low molecular weight protein (LMWP) Marker; Swimming lane 1 is bacterium lysate complete before induction; Swimming lane 2-3 is bacterium lysate complete after induction;
Fig. 4 is the SDS-PAGE electrophorogram after recombination chicken interferon alpha protein purification of the present invention, wherein:
Swimming lane M is low molecular weight protein (LMWP) Marker; Swimming lane 1,2 is the chicken interferon α albumen after inclusion body purification.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.
Embodiment 1
A kind of preparation method of recombination chicken interferon alpha, gene synthesis technology is utilized directly to obtain the cDNA sequence of chicken interferon α, application escherichia expression system, high expression is carried out to the ChIFN α gene of chicken interferon α, build the recombination bacillus coli comprising goal gene ChIFN α, fermentation synthesis is carried out, finally by the method extracted, purifying obtains recombination chicken interferon alpha under the induction of IPTG.The present invention's goal gene used is that chicken interferon α mature peptide gene coded sequence (ACCESSIONNUMBER:DQ226094) announced with reference to NCBI synthesizes, and in the present invention, the host cell of recombinant vectors is e. coli bl21 (DE3).Constructed recombinant expression vector not only comprises the DNA sequence dna of codase, also has the controlling elements of expressing needed for this gene.
The step of concrete construction process is as follows:
1, the synthesis of recombination chicken interferon alpha gene
With reference to chicken interferon α mature peptide gene coded sequence (ACCESSION NUMBER:DQ226094), use online Optimization Software majorizing sequence again, replace rare codon, regulate AT content, make its can in the e. coli bl21 (DE3) high expression, then the gene order of this optimization is given the synthesis of Shanghai Invitrogen Corp., the sequence clone after synthesis is to pMD18-T carrier.
2, the structure of recombinant plasmid
(1) the bacterial strain activation of the ChIFN α gene containing chicken interferon α synthesis obtained, the little extraction reagent kit of plasmid is utilized to extract the plasmid of DH5 α/pMD18-T-ChIFN α, pMD18-T-ChIFN α and pET-21a (+) plasmid carries out double digestion with EcoR I and Sal I respectively, then electrophoresis, cut glue reclaim enzyme cut after plasmid and goal gene, the fragment after cutting above-mentioned enzyme with DNA purification kit (TaKaBa company) carries out purifying.Namely pET-21a (+) and the goal gene ChIFN α of the linear purifying obtained can be used to construction recombination plasmid, and vector construction schematic diagram as shown in Figure 1.
(2) enzyme cut after carrier pET-21a (+) at 16 DEG C, be connected 15h with goal gene ChIFN α, gained connects mixture and adopts DNA purification kit (TaKaRa company) to carry out purifying, and the product after purifying is used for electrotransformation transformation of E. coli DH5 α.
3, the structure of engineering strain:
(1) competent cell of bacillus coli DH 5 alpha is prepared.The connection product of above-mentioned purification process is proceeded in bacillus coli DH 5 alpha competence, the LA flat board containing ampicillin concentration being 100 μ g/mL screens transformant.The single bacterium colony of picking from flat board, being inoculated in containing ampicillin concentration is in 100 μ g/mL LB liquid medium, cultivates 14h in 37 DEG C, then extracts plasmid DNA in a small amount, carry out double digestion qualification with EcoR I and Sal I, recombinant plasmid enzyme is cut qualification result and is seen Fig. 2.
(2) competent cell of e. coli bl21 is prepared.Recombinant plasmid pET-21a (+)-ChIFN αization correct for digestion verification is proceeded to e. coli bl21, obtains recombinant bacterial strain E.coliBL21 (DE3)/pET21a (+)-ChIFN α containing chicken interferon α.
4, the abduction delivering of genetic engineering bacterium:
(1) verify correct single bacterium colony from picking flat board, be inoculated in penbritin (100 μ g/mL) LB liquid medium, cultivate 14h in 37 DEG C.
(2) overnight culture is connect in 50mL containing in the LB substratum of 100 μ g/mL penbritins by the inoculum size of 1%.
(3) 37 DEG C of shaking culture, work as OD 600add IPTG when being about 1, final concentration is to 0.5mM/mL.
(4) at 37 DEG C, 4h is induced.Induction terminates the centrifugal 2min of rear 12000r/min and collects thalline, and carries out the expression of SDS-PAGE electrophoresis detection albumen after being boiled by the thalline before and after induction.
(5) SDS-PAGE expression analysis
1. gel preparation, sees the following form 1.
Table 1
2. sample preparation
A) 1mL nutrient solution is in small-sized centrifuge tube, the centrifugal 3min of 12000r/min at 4 DEG C.
B) except supernatant, the thalline of precipitation " drying " is as far as possible made.
C) resuspended thalline is in 100 μ L 1 × SDS-PAGE electrophoresis sample-loading buffers, and fully mixes.
D) boil 5min, centrifuging and taking supernatant at 100 DEG C, sample retention is in-20 DEG C until carry out protein electrophoresis analysis.Electrophoresis result is shown in Fig. 3.
5, the zymotechnique of recombination chicken interferon alpha:
(1) seed activation: activated on solid LB media by the engineering strain intestinal bacteria of expressing chicken alpha-interferon, then the single bacterium colony of picking one is in 50mL LB liquid nutrient medium, and at 37 DEG C, 180rpm, cultivates 14 hours; Again by the seed liquor obtained by volume 2% inoculum size be seeded in LB substratum, at 37 DEG C, 180rpm, cultivates 6 hours, obtains fermentation seed liquid;
(2) inoculate: in fermentor tank, fill fermention medium, then the seed liquor that above-mentioned activation is good is seeded in liquid fermentation medium by the inoculum size of 10% by volume;
(3) ferment: the mixed gas passing into air or air/oxygen in fermentor tank, air flow is 4m 3/ h, leavening temperature 37 DEG C, agitation revolution 500rpm, dissolved oxygen controls to control 6.8 at 20%, pH, and fermentation time 18 hours, starts feed supplement as OD600nm=2, and the flow acceleration of feed supplement is 600mL/h;
(4) induce: start as OD600nm=4 to add the expression that IPTG induces chicken interferon α, induce and stop tank in 6 hours.
6, purifying, the extraction process of recombination chicken interferon alpha:
(1) inclusion body protein is extracted: 4 DEG C, 6000rpm/min, 20min collected by centrifugation coli somatic, thalline weight in wet base is 77g/L, and fragmentation in PBS damping fluid is resuspended in by ultrasonic for 10g wet thallus under power 400 watts, work 3 seconds, interval 7 seconds, 30 minutes working hours condition, 4 DEG C, 9000rpm/min, 15min high speed frozen centrifugation, obtain chicken alpha-interferon inclusion body protein;
(2) purifying inclusion body protein: first by mass volume ratio 1: 30, inclusion body is resuspended in the first washings high speed frozen centrifugation; Then by mass volume ratio 1: 30, inclusion body is resuspended in the second washings high speed frozen centrifugation; Finally by mass volume ratio 1: 40, inclusion body is resuspended in the 3rd washings high speed frozen centrifugation, the condition of described high speed frozen centrifugation is 4 DEG C, 10000r pm/min, 15min, and wherein the component proportion of the first washings is NaCl 137mmol/L, KCl2.7mmol/L, Na 2hPO 410mmol/L, KH 2pO 42mmol/L, 1.2% tritonX-100, pH 8.2; Second washings is 2mol/L urea; 3rd washings is 1.5mol/L NaCl;
(3) dissolve and renaturation inclusion body protein: be 1: 35 by resuspended for step (2) centrifuged pellet denaturing agent by mass volume ratio, room temperature is placed to after precipitation dissolves gradually, 4 DEG C, 8500rpm/min, 15min high speed frozen centrifugation, collect supernatant liquor; Under 4 DEG C of conditions, make supernatant liquor dropwise join in the renaturation solution of above-mentioned preparation, volume ratio is 1: 80, stirs 16h simultaneously, and the component proportion of described denaturing agent is 1.5mM EDTA, 30mM Tris, 8mM DTT, pH8.5 in every 6M Guanidinium hydrochloride;
(4) gather in the crops renaturation recombined chicken alpha interferon albumen, as shown in Figure 4, prove that electrophoresis is single band through SDS-PAGE result, purity can reach more than 95%, its molecular weight of albumen is approximately 18KD, and westernblot confirms, has the attribute of natural chicken interferon α.
7, the Determination of biological activity of recombination chicken interferon alpha
Detect the antiviral activity of recombinant interferon, clone used is chick embryo fibroblast (DF-1), viral VSV (vesicular stomatitis virus).Suppression micromethod based on measuring method suppresses in order to cytopathy (CPE).Half cell (50%) still can be protected to be Interferon, rabbit unit from the dilution inverse of virus attack with the most high dilution of every milliliter of Interferon, rabbit inspection product.
Trial-product mensuration nutrient solution is diluted to every 1ml about containing 1000IU.In 96 porocyte culture plates, do 4 times of serial dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.Aseptically operate.Make chick embryo fibroblast adherent growth in the medium.Get cultured cells and discard nutrient solution, wash 2 digestion and collecting cells afterwards with PBS, be mixed with every lml containing 2.5 × 10 with complete culture solution 5~ 3.5 × 10 5the cell suspension of individual cell, is inoculated in 96 porocyte culture plates, every hole 100ul.In 37 DEG C, cultivate 4 hours under 5% carbon dioxide conditions.The standard solution prepared and need testing solution are moved in the fibroblastic culture plate of inoculated into chick embryo, every hole adds 100ul.In 37 DEG C, cultivate 18 hours under 5% carbon dioxide conditions.Discard the supernatant liquor in Tissue Culture Plate.The vesicular stomatitis virus (VSV ,-70 DEG C of preservations) preserved is diluted to 100CCID50 with attacking malicious nutrient solution, every hole 100ul.In 37 DEG C, 5% carbonic acid gas cultivates 24 hours.
Then discard the supernatant liquor in Tissue Culture Plate, every hole adds staining fluid 50ul, and room temperature carefully washes away staining fluid with flowing water after placing 30 minutes, and blots residual moisture, and every hole adds destainer 100ul, and room temperature places 5 minutes.After mixing, be reference wavelength by microplate reader with 630nm, measure absorbancy at wavelength 570nm place, record measurement result.
Testing data adopts computer program or four parametric regression computing methods to process.And be calculated as follows test-results:
Trial-product biologic activity ( IU / ml ) = Pr &times; Ds &times; Es Dr &times; Er
In formula: Pr is standard substance biologic activity, IU/ml;
Ds is trial-product pre-dilution multiple;
Dr is standard substance pre-dilution multiple;
Es is the extension rate that trial-product is equivalent to standard substance median effective dose;
Er is that standard substance partly imitate extension rate.
Result proves, when clone is chick fibroblast (DF-1), viral VSV (vesicular stomatitis virus), the antiviral activity of recombination chicken interferon alpha is about 2 × 10 8u/mg.
Above-mentioned detailed description of this recombination chicken interferon alpha and preparation method thereof being carried out with reference to embodiment; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.

Claims (7)

1. a recombination chicken interferon alpha, it is characterized in that: the cDNA of described chicken interferon α goal gene is sequence shown in sequence table 400<1>, the polypeptide amino acid of its protein-active is sequence shown in sequence table 400<2>.
2. the preparation method of recombination chicken interferon alpha according to claim 1, is characterized in that: concrete construction step is as follows:
(1) chicken interferon α gene is obtained: with reference to chicken interferon α mature peptide gene coded sequence, the cDNA sequence of synthesis chicken interferon α, this sequence is by again optimizing, replace rare codon, regulate AT content, can at high expression in e. coli bl21 (DE3), the gene order of the chicken interferon α mature peptide after optimization is sequence shown in sequence table 400<1>;
(2) structure of recombinant plasmid: cut with enzyme the method connected and respectively above-mentioned chicken interferon α gene is connected with carrier pET-21a (+), obtain recombinant expression vector pET-21a (+)/ChIFN α carrying chicken interferon α gene, and carry out double digestion checking;
(3) structure of engineering strain: by recombinant expression vector transformed host strain BL21 (DE3) correct for above-mentioned checking, obtain the engineering strain containing recombinant plasmid;
(4) expression of recombinant protein: make engineering strain synthesize chicken interferon α albumen under fermentation condition under the induction of IPTG;
(5) fermentative production of recombination chicken interferon alpha: prepare fermentation seed liquid, seed liquor is accessed in fermentor tank, make it produce chicken interferon α in a large number by adding carbon source and inductor, the recombination chicken interferon alpha of acquisition makes chicken interferon alpha production through extracting, after purifying.
3. the preparation method of recombination chicken interferon alpha according to claim 2, it is characterized in that: in described step (2), prokaryotic expression carrier used is coli expression carrier pET-21a, carrier construction host cell is e.colistraindh5α, and expression host cell is e. coli bl21 (DE3) bacterial strain.
4. the preparation method of recombination chicken interferon alpha according to claim 2, it is characterized in that: the expression of recombinant protein in described step (3): the recombinant expressed bacterium that picking is connected with pET-21a expression vector is respectively increased in containing the LB substratum of penbritin 100 μ g/ml, it is 35 ~ 37 DEG C in culture temperature, shaking speed is under 180 ~ 200r/min condition, when to be cultured to bacterium liquid OD value be about 1, take out 1mL bacterium liquid in contrast, it is that the IPTG of 0.5 ~ 2mM/mL induces that remaining part adds final concentration, induction time is 4 ~ 6 hours, induction terminates the centrifugal 2 ~ 5min of rear 12000r/min and collects thalline, and carry out SDS-PAGE electrophoresis after being boiled by the thalline before and after induction, detect the expression of recombination chicken interferon alpha albumen.
5. the preparation method of recombination chicken interferon alpha according to claim 2, is characterized in that: in described step (5), the fermentative production of recombination chicken interferon alpha comprises: the preparation of a. recombinant expressed engineering bacteria E.coliBL21 (DE3)/pET21a (+)/ChIFN α fermentation seed liquid; B. the cultivation stage of fermentation thalli; C. carbon source feeding period; D. the abduction delivering stage; E. the extraction of recombination chicken interferon alpha, purifying.
6. the preparation method of recombination chicken interferon alpha according to claim 4, is characterized in that: the composition of described LB substratum is peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L.
7. the preparation method of recombination chicken interferon alpha according to claim 5, is characterized in that: consisting of of described fermention medium: glucose 5g/L, peptone 5g/L, yeast powder 5g/L, KH 2pO 42g/L, K 2hPO 44g/L, Na 2hPO 412H 2o 7g/L, (NH 4) 2sO 41.2g/L, NH 4cl 0.2g/L, MnSO 45H 2o 0.001g/L, CoCl 26H 2o 0.004g/L, Na 2moO 42H 2o 0.002g/L, ZnCl 20.002g/L, CuSO 45H 2o 0.001g/L, H 3bO40.005g/L, FeSO 47H 2o 0.02g/L, CaCl2H 2o 0.02g/L, MgSO 47H 2o 0.3g/L, defoamer 0.2g/L.
CN201410755900.7A 2014-12-10 2014-12-10 Recombinant chicken interferon alpha and preparation method thereof Pending CN104447978A (en)

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CN107177613A (en) * 2017-07-18 2017-09-19 哈尔滨紫霞生物科技有限公司 A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity
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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
CN117343942A (en) * 2023-12-06 2024-01-05 南京市食品药品监督检验院 PagA recombinant protein and preparation method thereof

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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
CN106399321A (en) * 2016-08-27 2017-02-15 华南农业大学 Production process of fusion expression recombinant chicken interferon alpha
CN107141347A (en) * 2016-12-26 2017-09-08 河南后羿生物工程股份有限公司 Escherichia coli recombined chicken alpha interferon, recombinant expression carrier, recombination expression engineering bacteria and its preparation method and application
CN107141347B (en) * 2016-12-26 2020-05-05 河南后羿生物工程股份有限公司 Escherichia coli recombinant chicken α interferon, recombinant expression vector, recombinant expression engineering bacterium, preparation method and application thereof
CN109125713A (en) * 2017-06-19 2019-01-04 杭州俊丰生物工程有限公司 A kind of chicken interferon-α Pharmaceutical composition
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
CN107217068A (en) * 2017-07-18 2017-09-29 哈尔滨紫霞生物科技有限公司 A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity
CN107177613A (en) * 2017-07-18 2017-09-19 哈尔滨紫霞生物科技有限公司 A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity
CN107177614A (en) * 2017-07-18 2017-09-19 哈尔滨紫霞生物科技有限公司 A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity
CN108165541A (en) * 2018-01-09 2018-06-15 中国海洋大学 A kind of zymoprotein and its application with betagalactosidase activity
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
CN111455006B (en) * 2020-04-10 2021-09-07 天津生机集团股份有限公司 Recombinant chicken interferon alpha product expressed by escherichia coli and preparation method and application thereof
CN117343942A (en) * 2023-12-06 2024-01-05 南京市食品药品监督检验院 PagA recombinant protein and preparation method thereof
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Application publication date: 20150325