CN109134641A - A kind of preparation method of chicken interferon-α - Google Patents
A kind of preparation method of chicken interferon-α Download PDFInfo
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Abstract
The present invention relates to veterinary biologics field more particularly to a kind of preparation methods of chicken interferon α.The invention discloses the preparation methods of chicken interferon α a kind of.Optimization gene sequence is utilized in preparation method of the invention, expression quantity is higher than existing gene, pass through the acquisition of chicken interferon-α gene, the building of expression vector and conversion colibacillus engineering, screening, culture, inducing expression, washing, cracking and the renaturation of bacterium and occlusion body are collected, broken to expression thallus, then purifies to obtain chicken interferon-α using chromatographic technique.Method of the invention has substantial worth to the production of chicken interferon α.
Description
Technical field
The present invention relates to veterinary biologics field more particularly to a kind of preparation methods of chicken interferon α.
Background technique
Communicable disease is to endanger the important diseases of China's aviculture sound development, especially viral disease at present, such as
Vesicular stomatitis virus, Rous sarcoma virus, newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus,
Marek's disease poison, avian influenza virus etc., these communicable diseases once break out, propagate it is very fast, caused by endanger it is very tight
Weight.Infectious bronchitis of chicken is a kind of acute highly contagious disease caused by virus, and there is hair in when annual winter-spring season
It is raw, since disease morbidity is anxious, dead fast, biggish economic loss often is caused to raiser.Bird flu is another high-incidence biography
Infectious diseases have infectivity to many animals, and highly pathogenic bird flu is the most serious, and morbidity and mortality are high, infection
Chicken group is usually all dead.
In poultry disease prevention and control, the application of various drugs plays positive work to the treatment of the generation and poultry diease that reduce poultry diease
With, such as previous treatment broiler chicken kidney type infective bronchitis often used antiviral drugs, as moroxydine, amantadine, Ribavirin,
Acyclovir etc. and its compound preparation, receive certain effect.As food-safety problem increasingly draws attention, many drugs
Remain in poultry products, the health of the mankind is generated potentially hazardous.Many state food imports use stringenter
Medicament residue examination criteria.It is announced " abrogating catalogue ", wherein sequence from October 28th, 2005, No. 560 bulletin of the Ministry of Agriculture in China
Numbers 2 drug (antiviral agent) is not used in animal epidemic prevention and treatment.Therefore the exploitation of newtype drug seems very urgent.
Interferon (Interferon, IFN) is important cell factor, it is that humans and animals cell is infected with the virus,
Or by after the effects of nucleic acid, bacterial endotoxin, cytokinin, there is high biological by one kind of recipient cell secretion
Active glycoprotein.British scientist Isaacs etc. (1957) is existing in the interference to be studied flu virus using chick chorioallantoic membrane
As when have found interferon.Lamp etc. (1963) proves that the factor is protein, and molecular mass is 20~34ka.Interferon can lure
It leads people and zooblast generates the parahormone albumen of a variety of broad-spectrum disease resistance toxalbumin, there is antiviral and antitumor and immunological regulation
Etc. biological activities and wide spectrum, it is efficient and non-specific the features such as.Since Interferon of Poultry has broad-spectrum antiviral, anti-swollen
Tumor and immunoregulation effect, to the prevention and control function well of poultry disease, in recent years by the great attention of researcher.In recent years
Come, interferon achieves good efficacy in terms of some avian viral diseases and tumor disease treatment, and interferon is in poultry body
The research of interior immunological characteristic and its clinical product has also obtained further expansion.
According to structure and the difference of receptor, interferon is usually divided into two class of I type and II type: I type interferon mainly includes
IFN-α, IFN-β, IFN- ω and IFN- τ, IFN-α are mainly generated by leucocyte, and IFN-β is mainly generated by fibroblast, it
There is similar biological activity, in conjunction with identical cell receptor, there is efficient disease-resistance cytotoxic activity.II type only includes IFN-
γ, IFN-γ are mainly generated by T cell and NK cell, mainly immunoregulation effect, are the main macrophages of mammal
Activation factor.Its physicochemical property and biological activity and I type interferon are significantly different, as I type interferon is resistant to pH 2.0
Acid processing, and II type interferon inactivates quickly under the acid condition of pH 2.0.Chicken and other birds IFN and mammal
IFN is similar, is also classified into I type IFN and II type IFN, it has now been found that and chicken I type IFN includes IFN-α, IFN-β, and II type includes IFN-γ,
IFN- ω and IFN- τ are not yet found in birds at present.
The generation of interferon by gene control, due to interferon gene mortifier and interferon gene knot in cell DNA
It closes, inhibits dubbing system, therefore, often interfere with plain gene and be in the state of being suppressed.Under the action of inducer, inhibit to release,
Interferon operon starts to transcribe, and synthesizes mRNA, mRNA is transferred quickly to cytoplasm, before being translated into interferon on ribosomes
Body, after cutting off signal peptide, mature interferon is secreted into extracellularly.The inducement for being currently known interferon generation has: virus,
Double-stranded RNA inducer (such as PHA- phytohemagglutin phytolectin, ConA- canavalin), metabolin inducer and some pathogens.
M.J.Sekellick has cloned chicken IFN-α, IFN-γ base equal to nineteen ninety-five equal to, M.R.Digby in 1994 respectively
Cause, and its structure feature, physicochemical characteristics and biological characteristics are studied.C.Sick etc. has studied chicken IFN-β gene
Sequence, and find that the antiviral activity of IFN-α is 20 times of IFN-β.It is residual that each hypotype of IFN-α contains 165~166 amino acid
Base, structure is similar, and no glycosyl, molecular weight is about 19kD or so, and the homology between different genera is 70% or so.IFN-α point
Son contains 4 cysteines (Cys), and 2 intramolecular disulfide bonds are formed between the 99th and 199 cysteine.Domestic chicken is dry
The research for disturbing element also has greater advance, and Xia Chun etc. has cloned Huiyang beard chicken IFN-α gene, and confirms as new hypotype.Chicken
IFN gene initially in 1994 by Sekellick from the chick-embryo cell cDNA library of aging successful clone, with mammal
The sequence similarity of IFN is lower, but the position of its cysteine and the characteristic that can be generated by virus induction are relatively conservative, with lactation
Animal I type IFN is similar, therefore speculates it for chicken I type IFN.Nineteen ninety-five, Schultz et al. have found that this breeder IFN has anti-folliculus
Property Stomatovirus activity, and lack the relevant bioactivity of mammal IFN-γ, further prove that the interferon is I type.
ChIFN- α is made of 193 amino acid residues, and mature peptide size is 162aa, and 31, the end N- amino acid is signal peptide.Although
ChIFN- α and the amino acid identity of mammal IFN-α are only 24%, but the amino acid identity of high conservative region is up to
80%, the position of alpha-helix is also similar in the two secondary structure, and can both be induced and be produced by imiquimod derivative S-28463
It is raw, therefore it is named as ChIFN- α.Marcus in 1999 et al. feeds high dose ChIFN- α, Ke Yixian to 1 Japanese instar chickling
Write the disease incidence for reducing newcastle disease virus.2001, Mo C W et al. discovery ChIFN- α can inhibit infectious bursal disease virus
(IBDV) plaque is formed, and the survival rate of infection IBDV chicken can be improved.Ellen et al. discovery ChIFN- α can inhibit infectiousness branch
The duplication of bronchitis virus (IBV) weakens clinical disease to postpone the breaking-out of disease.2001, the human hairs such as Jarosinski
The cytotoxicity that NK cell can be significantly reduced after current ChIFN- α processing chicken, can inhibit Ma Li after handling CKC with ChIFN- α
The duplication of gram virus (MDV) makes MDV plaque formation reduce 50%.2009, Tao Shengli etc. was the study found that after mixed drink ChIFN- α
Broiler chicken peripheral white blood cells sum, cent lymphocytes and basophilic granulocyte percentage can be improved, it is thin to reduce neutrophil(e) granule
Born of the same parents' percentage and monocyte percentage improve broiler chicken index and spleen index and total serum protein (TP), seralbumin (ALB), blood
The concentration and ALB/GLO ratio of clear globulin (GLO)；The growth and development of broiler chicken is not influenced.Therefore, the ChIFN- of drink low dosage is mixed
The immune function of broiler chicken can be improved in α, to the growth of healthy broiler chicken without significant impact.
In recent years, the test result of humans and animals (mouse, dog, cat, horse, pig, ox etc.) and clinical application show that IFN-α passes through
Mucous membrane of mouth administration can also play anti-infective, antitumor and immunoregulation effect etc. (Cumins etc., 2005), this is interferon
It is convenient to use to open good prospect.The demand of the oral mixed drink of interferon is very big, and 2010, Liu Wenjun etc. disclosed one
It plants the optimization gene of coding chicken interferon alpha and its is preparing the application in chicken alpha interferon.2012, it is dry that Yi Lin etc. has studied chicken α
Disturb the prokaryotic expression and anti-new castle disease virus effect observation of element.2010, Ma Fenglong etc. disclosed the big of expression chicken alpha-interferon
Enterobacteria and its application.2013, Wang Mingli etc. disclosed a kind of recombination chicken interferon-' alpha ' preparation method.What above method obtained
The boot sequence on expression vector is all utilized in recombination chicken IFN-α to some extent, and expression product chicken IFN-α is all with fusion protein
Form be used for antivirus test, and interferon category peptide matters, if to pass through mucous membrane of mouth administration obtains good absorption
Effect, molecule itself want the smaller the better, therefore are highly desirable to develop a kind of high efficient expression recombination chicken gizzard interferon-' alpha ' native peptides
Method with meet herding medicine the needs of.
Summary of the invention
The object of the present invention is to provide the preparation methods of chicken interferon-α a kind of, can efficiently prepare chicken interferon-α, be
It achieves the object of the present invention, the invention adopts the following technical scheme:
A kind of preparation method of chicken interferon-α, comprising: (1) acquisition of chicken interferon-α gene；(2) structure of expression vector
It builds and converts colibacillus engineering: chicken interferon-α gene order being inserted into the restriction enzyme site of expression vector plasmid, then
Convert e. coli host bacteria；(3) it is enterprising in culture medium the screening, culture of positive colony, inducing expression: to filter out positive colony
Row culture expresses target protein through induction；(4) separation, purifying of chicken interferon-α: first collect coli somatic,
Broken bacterium, is collected by centrifugation occlusion body, then carries out washing, cracking and the renaturation of occlusion body, then purify to obtain using chromatographic technique
Chicken interferon-α.
A kind of expressing gene that the preparation method of chicken interferon-α uses of the present invention is the chicken interferon-α DNA sequence of SEQ.1
Column.
A kind of preparation method of chicken interferon-α of the present invention, the expression vector of use is pET serial carrier plasmid.
A kind of preparation method of chicken interferon-α of the present invention, in step (3) the temperature control of recombinant 25 DEG C~
40 DEG C, preferably at 30 DEG C~37 DEG C.
A kind of preparation method of chicken interferon-α of the present invention, the inducer in step (3) is isopropyl-beta D-thio gala
Glucosides makes its final concentration of 0.1~5mmol/L, preferably 1mmol/L.
A kind of preparation method of chicken interferon-α of the present invention, step (4) are split with urea, guanidine hydrochloride, guanidinium isothiocyanate, hydrochloric acid
Solve occlusion body.
A kind of preparation method of chicken interferon-α of the present invention, the urea concentration that step (4) cracks occlusion body is 8mol/L.
A kind of preparation method of chicken interferon-α of the present invention, step (4) crack the concentration of guanidine hydrochloride of occlusion body be 5.5~
7mol/L, preferably 6mol/L.
A kind of preparation method of chicken interferon-α of the present invention, the renaturing inclusion bodies in step (4) are using Urea Gradient dialysis
Method and dilution method.
A kind of preparation method of chicken interferon-α of the present invention, using affinity chromatography technology, ion-exchange chromatography, molecular sieve layer
Analysis, the one or more methods of hydrophobic chromatography are purified.
A kind of preparation method of chicken interferon-α of the present invention, expressional scheme is simple, and expression quantity is high, reaches 30%, purifying
Technique, high income.For the chicken interferon-α that the present invention obtains without there is non-natural amino acid sequence, component amount is small, is conducive to
Oral mucosal absorption improves the effect of the mixed drink interferon of poultry, improves the benefit of husbandry sector.
Detailed description of the invention
The plasmid-encoded chicken interferon of attached drawing 1, pET-32a-chIFN- α-alpha expression whole bacterial protein SDS-PAGE analysis
M: standard protein molecular weight (97.4,66.2,43,31,20,14.4kd from top to bottom)；
1: not inducing bacterial protein；
2 and 3: induction bacterial protein.
Attached drawing 2, ion exchange DEAE column tomographic results map
Attached drawing 3, hydrophobic chromatography result map
Attached drawing 4, purifying chicken interferon-α (pET-32a-chIFN- α is plasmid-encoded) SDS-PAGE analysis
M: standard protein molecular weight (97.4,66.2,43,31,20,14.4kd from top to bottom)；
1 and 2: chicken interferon-α each 5 μ g.
Specific embodiment
The further explanation present invention combined with specific embodiments below, but and the non-limiting present invention.
The acquisition of one chicken interferon-α gene of embodiment
Jungle fowl (Gallus gallus) gene order of (GenBank:U07868.1) according to the literature carries out gene
Expressed sequence optimization, sequence is shown in sequence table 1 after optimization, synthesizes 15 sections of complementary oligonucleotides, by the conventional method of molecular cloning,
37 DEG C of processing 30min of T4 bacteriophage polynucleotide kinase are used first, each oligonucleotide fragment of phosphorylation is mixed with equimolar, and 94
DEG C denaturation 5min, immediately 65 DEG C of annealing 10min, are then added T4 ligase, 16 DEG C of connections overnight, acquisition target gene template piece
Section.The microcentrifugal tube of 4 sterilizings is taken, is added:
Said mixture gently shakes of short duration centrifugation again after mixing, is subsequently placed in heat preservation connection in 15 DEG C of water-baths and stays overnight (12
~16h).5 μ l connection products are taken to be added in the Escherichia coli TOP10 competent cell that 50 μ l thaw on ice, light rotation mixes several times
It is even, it is put into the water-bath for being preheated to 42 DEG C after placing 30 minutes on ice, heat shock 90 seconds.Then it is immediately placed on ice, keeps cell cold
But 10 minutes.Every 800 μ l LB culture medium of Guan Zhongjia (is free of antibiotic), 37 DEG C shaken cultivation 1 hour.Room temperature 3,500rpm from
The heart 3 minutes, after discarding 800 μ l supernatants, cell is resuspended with remaining 50 μ l culture mediums and is applied to the LB agar plate table containing Amp
Face, then 40 μ l 20mg/ml X-gal, 7 μ l200mg/ml IPTG are added dropwise on plate.Plate is placed in room temperature until liquid quilt
It absorbs.It is inverted plate, is cultivated in 37 DEG C, may occur in which that bacterium colony, white colony are to obtain positive bacterium colony after 12-16 hours.
The expression of two chicken interferon-α gene of embodiment
Pair of primers is designed, primer 1 and 2 is shown in sequence 2 and sequence 3 respectively, and ' end primer has Nde I enzyme to gene 5
Enzyme site, 3 ' end primers have EcoR I restriction enzyme site, 25 μ l reaction systems are prepared in 0.2ml PCR microcentrifugal tube:
94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 2min are arranged in 94 DEG C of initial denaturation 5min, totally 35 circulations, and last 72 DEG C
15min.10 μ l products are taken to carry out agarose gel electrophoresis, the size about 500bp segment of clip size and design after PCR
It is in the same size, see sequence 1, the amino acid sequence of coding is shown in sequence 4.
PET-32a plasmid Nde I, EcorR I double digestion recycle large fragment, connect with the chicken interferon genetic fragment of PCR
It connects, the ratio of 20 μ L reaction system genetic fragments and carrier large fragment is 10: 1, adds 300 unit of T4 DNA ligase, 16 DEG C of companies
Night is taken over, takes 5 μ L connection products directly to convert e. coli host bacteria BL21 (DE3) competent cell, is coated on ammonia benzyl mould
Plain resistant panel, 37 DEG C of overnight incubations obtain engineering bacteria and are further screened.
Ampicillin does resistance screening, obtains positive colony pET-32a-chIFN- α.Plasmid is extracted, with restricted interior
Enzyme cutting is identified.Positive transformant carries out sequence analysis with universal primer, and as a result cloned sequence and implementation sequence are completely the same.
Inoculation positive colony is cultivated, and IPTG through 0.5mmol/L induction, expression of results is shown in attached drawing 1, and to photograph
Than the expression quantity of chicken interferon-α reaches 30% or more.
Three chicken interferon-α's of embodiment isolates and purifies
1, thallus culture
It prepares 1 liter of LB culture medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.0), cultivates
After basigamy is good at a temperature of 121 DEG C damp and hot sterilization 40min.In super-clean bench when culture medium cooling non-scald on hand after sterilizing
In plus Amp make its final concentration of 50 μ g/mL, it is spare in cooling 4 DEG C of refrigerators of postposition.
Picking pET-32a-chIFN- α single bacterium falls within 100ml LB culture medium and (contains in plate under aseptic technique
Aamp it in), is subsequently placed at 37 DEG C, 250rpm shaking table culture is stayed overnight.From the inoculum of overnight incubation by 0.5% inoculation
Amount switching is cultivated 4 hours, the OD600 of culture solution is about 0.6~0.8, is then added in LB culture medium in 37 DEG C, 250rpm
Inducer IPTG to final concentration 0.5mmol/L.Continue culture 5 hours, thalline were collected by centrifugation (5,000rpm × 10min).Thallus
It is washed 2-3 times with cleaning solution 20mM PB, pH7.0,0.15M NaCl.
2, bacterial cell disruption
The 10g wet thallus for having used the IPTG inducing expression 5h of 0.5mmol/L is taken, adds 10ml to break bacterium buffer by 1g wet thallus
(50mM Tris-HCl buffer, 0.1M NaCl, 1mM EDTA, pH 8.5) suspension thalline, i.e., break bacterium buffer with 100mL
Suspension thalline, then ice-bath ultrasonic broken (ultrasonic power 5000W, each ultrasound 5s, interval 5s, i.e. duty ratio 50%, altogether progress
30 circulations), collect bacteria break supernatant: 4 DEG C, 10,000rpm centrifugation 15min collect precipitating occlusion body.
3, the cracking of occlusion body
Sediment through inclusion body cleaning solution (2mol/L urea, 0.5% Triton X-100,20mmol/L Tris.Cl,
1mmol/ LEDTA, pH 8.5) after supersound washing 10min, it is centrifuged 20min in 19 000rpm, precipitating is taken to obtain purer chicken
Interferon-' alpha ' occlusion body.
Institute is resuspended with protein denaturation liquid (8mol/L urea, 100mmol/LBME, 50mmol/L Tris.Cl, pH 8.0)
Obtained chicken interferon-α occlusion body.In oscillation cracking 1 hour on 37 DEG C, the shaking table of 250rpm after sufficiently suspending.Then in 10
000rpm, 4 DEG C of centrifugation 15min, taking centrifuged supernatant to survey protein concentration is about 28mg/mL.
4, renaturation
Chicken interferon-α occlusion body lysate is diluted in 4mol/L, 50mM Tris, 20mM NaCl, 1mM 2-ME, 1mM
In EDTA, pH8.5 solution, make the final concentration of protein 0.2mg/ml of chicken interferon-α.Renaturation solution is loaded on and uses 5%NaHCO3,
5mM EDTA boils in the bag filter that processed molecular interception amount is 8000Da, is placed in 20mM Tris with 1: 10 ratio,
In 20mM NaCl, 1mM EDTA, pH8.5 solution, 4 DEG C are dialysed 12~16 hours, replace 3 outer transparent liquid.
5, it isolates and purifies
Chicken interferon-α renaturation solution is subjected to ion exchange chromatography purifying, process is the 20mmol/ for being 8.0 with pH value
The dilution of L Tris.CL buffer, makes its conductivity be less than 5mS/cm.DEAE column is balanced with same buffer, is used after loading flat
Weighing apparatus buffer is washed till baseline, with 0.01~0.5mol/LD NaCL gradient elution, collects target protein peak, chromatography map is shown in Fig. 2.
Target protein adds (NH4)2SO4Make final concentration of 1.0-1.5mol/L, 1.0mol/L (NH4) on sample2SO4, 20mmol/L
The Phenyl-Sepharose Fast Flow column of the buffer balance of PB, pH7.4, is washed by reduction salt concentration gradient and is dragged, received
Ji Mubiaodanbaifeng is shown in the hydrophobic chromatography map of Fig. 3.
Through Sephadex G-25 desalination, equilibrium liquid is 20mmol/L PB, pH7.4 at obtained hydrophobic chromatography peak.Sample is de-
It is polishing purification after salt by chicken interferon-α, as a result 15%SDS-PAGE electrophoretic analysis sample purity is shown in that Fig. 4, sample are pure
Degree reaches 99% or more.The yield of chicken interferon-α can achieve 300~500mg with every liter of culture solution.
The dilution refolding of example IV chicken interferon-α
Chicken interferon-α occlusion body lysate is diluted in 50mM Tris, 1mM GSH, 0.1mmol/L GSSG, 1mM
In EDTA, 0.5M L-Arg, 1M Urea, pH8.5 solution, answering in pre-cooling is added dropwise in albuminate lysate under ice bath environment
In property liquid, make the final concentration of protein 0.5mg/ml of chicken interferon-α, in 20mM Tris, 1mM after 4 DEG C of standing renaturation 48hr
Dialysis removes L-Arg in the outer transparent liquid of the dialysis of EDTA, pH8.0, and the outer transparent liquid of every 12hr replacement is primary, replaces 4 times altogether, the total time
For 96hr.It is centrifuged or filters after dialysis, remove a small amount of suspended matter, supernatant is further isolated and purified.
The analysis of biological activity of five chicken interferon-α of embodiment
Aseptically, after taking the national standard of human interferon Determination of biological activity, by specification to redissolve, with survey
Determine culture solution and is diluted to every 1ml containing 1000IU.In 96 porocyte culture plates, does 4 times and be serially diluted, totally 8 dilutions, each
Dilution does 2 holes.Chicken interferon-α is diluted to every 1ml containing about 1000IU with measurement culture solution.In 96 porocyte culture plates, do
4 times are serially diluted, totally 8 dilutions, and each dilution does 2 holes.
Make WISH cell adherent growth in the medium.By 1: 3 passage, 2 times a week, grown in complete culture solution.It takes
The cell of culture discards culture solution, and cell is digested and collected after washing 2 times with PBS, is configured to every 1ml containing 2.5 with complete culture solution
×105~3.5 × 105The cell suspension of a cell is inoculated in 96 porocyte culture plates, every 100 μ l of hole.In 37 DEG C, 5%CO2
Under the conditions of cultivate 4~6 hours.The standard solution completed will be prepared and test solution moves into the culture plate of inoculation WISH cell
In, 100 μ l are added in every hole.In 37 DEG C, 5%CO2Under the conditions of cultivate 18~24 hours.Discard the supernatant in tissue culture plate.
The vesicular stomatitis virus of preservation is diluted to about 100CCID50, every 100 μ l of hole with malicious culture solution is attacked.In 37 DEG C, 5%CO2Training
It supports 24 hours (50% lesion point of microscopic criteria product solution is in 1IU/ml).Then the supernatant in tissue culture plate is discarded, often
50 μ l of dyeing liquor is added in hole, after being placed at room temperature for 30 minutes, carefully washes away dyeing liquor with flowing water, and blot residual moisture, every hole adds
Enter 100 μ l of destainer, is placed at room temperature for 5 minutes.After mixing, with microplate reader using 630nm as reference wavelength, surveyed at wavelength 570nm
Determine absorbance, record measurement result is shown in Table 1.
1. chicken interferon-α Determination of biological activity result of table
Test data is handled using computer program, verification result: chicken interferon-α bioactivity is 1.17 ×
106IU/ml, specific activity are 1.17 × 107IU/mg
The antivirus test of six chicken interferon-α of embodiment
Conventional H 9N2 subtype avian influenza virus is inoculated with 10 3 pieces of embryo age SPF chicken embryos, 0.1ml/ according to 2 unit quantity of HA potency
Embryo, after inoculation for 24 hours within dead embryo be invalid embryo, for 24 hours after dead embryo takes out is put in 4 DEG C of refrigerators preservations at any time, 96h is not dead
Chicken embryo, which is all taken out, puts 4 DEG C of refrigerator overnights.It takes out all chicken embryos and collects allantoic fluid, do HA potency and bacteriologic test, it is invalid to discard
Valence and cyst fluid containing bacteruria, remaining allantoic fluid are sufficiently mixed, and multitube, -20 DEG C of freezen protectives are packed as
The EID50 method that virus is demarcated after taking wherein 1 pipe to melt is as follows: 10 times are serially diluted, and respectively take 10-5、10-6、10-7、
10-8、10-9Each dilution is inoculated with 6 pieces of SPF chicken embryo of 10 embryo ages by 5 dilutions respectively at allantoic cavity, and every embryo 0.1ml is set
Continue to hatch in hatch machine.After inoculation for 24 hours within dead embryo be invalid embryo, for 24 hours after dead embryo take out is put in 4 DEG C of refrigerators at any time
It saves, the not dead chicken embryo of 96h, which is all taken out, puts 4 DEG C of refrigerator overnights.It takes out all chicken embryos and collects allantoic fluid detection HA potency, HA
The positive is to infect.
Chicken embryo median infective dose (EID50) is calculated referring to Reed-Muench method, calculation formula is as follows:
IgEID50=is higher than logarithm+distance proportion value × dilution times of the viral dilution of 50% death rate (infection rate)
Several logarithms
Distance proportion value=(being higher than 50% death rate -50%)/(higher than the death rate that 50% death rate-is lower than 50%)
The 9 embryo age SPF chicken embryos through being incubated for are divided into 4 groups of A, B, C, D, and A group is 2 pieces of blank control group, other every group 20
Piece.It is inoculated with through allantoic cavity, C group is inoculated with chicken interferon α 0.1ml (1.6 ten thousand IU/ being sterile filtered after sterilized normal saline dilution
Piece), D group is inoculated with the chicken interferon 0.1ml (0.8IU/ pieces) being sterile filtered after sterilized normal saline dilution, and 37 DEG C of incubations 24 are small
When.Then the bird flu H9 virus 0.1ml/ embryo of 100 × EID50 of the every embryonic breeding kind of B, C, D group continues to be incubated for, after inoculation operation
Dead chicken embryo is that invalid chicken embryo discards in for 24 hours, B, C, D after attacking poison for 24 hours, 36h, 48h, 60h, 72h take out every group and take 3
Piece chicken embryo, also 72h takes out 2 pieces of chicken embryos of A group after other groups attack poison, collects all chick embryo allantoic liquids respectively according to label and carries out HA
Bioactivity the results are shown in Table 2.
Table 2, experiment each group different time points chicken embryo death situation
| Experimental group | The normal incubation group of A | B attacks malicious control group | C high dose IFN group | D low dosage IFN group |
| 24 hours after virus attack | / | 2 | 0 | 0 |
| 48 hours after virus attack | / | 7 | 3 | 2 |
| 72 hours after virus attack | / | 18 | 8 | 10 |
Claims (10)
1. a kind of preparation method of chicken interferon-α, it is characterised in that: (1) acquisition of chicken interferon-α gene；(2) expression vector
Building and conversion colibacillus engineering: chicken interferon-α gene order is inserted into the restriction enzyme site of expression vector plasmid,
Then e. coli host bacteria is converted；(3) screening, culture of positive colony, inducing expression: positive colony is filtered out in culture medium
On cultivated, through induction express target protein；(4) separation, purifying of chicken interferon-α: coli somatic is received first
Collection, broken bacterium, are collected by centrifugation occlusion body, then carry out washing, cracking and the renaturation of occlusion body, then purify using chromatographic technique
To chicken interferon-α.
2. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: expressing gene is SEQ.1
Chicken interferon-α DNA sequence dna.
3. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: expression vector is pET system
Column vector plasmid.
4. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: cultivated in step (3)
Temperature is controlled at 25 DEG C~40 DEG C, preferably at 30 DEG C~37 DEG C.
5. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: the induction in step (3)
Agent is isopropyl-β-D-thiogalactoside, makes its final concentration of 0.1~5mmol/L, preferably 1mmol/L.
6. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: step (4) urea, salt
Sour guanidine, guanidinium isothiocyanate, salt acid cleavage occlusion body.
7. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: step (4), which cracks, includes
The urea concentration of body is 8mol/L.
8. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: step (4), which cracks, includes
The concentration of guanidine hydrochloride of body is 5.5~7mol/L, preferably 6mol/L.
9. the preparation method of chicken interferon-α according to claim 1 a kind of, it is characterised in that: forgiving in step (4)
Body renaturation uses Urea Gradient dialysis and dilution method.
10. the preparation method of chicken interferon-α according to claim 7 a kind of, it is characterised in that: use affinity chromatography skill
The one or more methods of art, ion-exchange chromatography, sieve chromatography, hydrophobic chromatography are purified.
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