CN101948845B

CN101948845B - Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha

Info

Publication number: CN101948845B
Application number: CN2010102705545A
Authority: CN
Inventors: 刘文军; 瞿洪仁; 李晶; 杨利敏; 贾晓娟
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2010-09-01
Filing date: 2010-09-01
Publication date: 2012-08-08
Anticipated expiration: 2030-09-01
Also published as: CN101948845A

Abstract

The invention discloses an optimization gene for coding chicken interferon alpha and application thereof in preparing the chicken interferon alpha. The optimization gene is DNA (deoxyribonucleic acid) shown as the sequence 2 in a sequence table. The expression ability of the gene is far higher than that of the traditional gene; the gene is inserted into the polyclone site of pET28b(+) to obtain a recombinant plasmid, and the recombinant plasmid is imported into escherichia coli BL21 to obtain a recombination strain. A large amount of ChIFN alpha protein can be prepared through carrying out simple induction culture on the recombination strain. The gene, the recombinant plasmid and the recombination strain has economic value for the production of the chicken interferon alpha and also has great value for broiler culture industry.

Description

The optimized gene of coding chicken alpha-interferon and the application in the preparation chicken alpha-interferon thereof

Technical field

The present invention relates to a kind of optimized gene and the application in the preparation chicken alpha-interferon thereof of the chicken alpha-interferon of encoding.

Background technology

Interferon, rabbit (Interferon; IFN) come to light during by the interference of Britain scientist Isaacs research bird flu virus at first in nineteen fifty-seven; Be a kind of active gp of broad-spectrum antiviral that has; Produce by the virus and interferon inducers stimulating endothelial cell, scavenger cell, lymphocyte and the somatocyte of other kind, have antiviral, antitumor, immunomodulatory and induce the differentiation isoreactivity.The performance of Interferon, rabbit effect is not to directly act on virus, but irritation cell produces multiple broad-spectrum disease resistance toxalbumin, can be through direct or indirect approach performance antivirus action.

According to the difference of structure and acceptor, can IFN be divided into two types: I type IFN and II type IFN.Mammals I type IFN mainly comprises IFN-α, IFN-β, IFN-ω and IFN-τ, to acid and thermally-stabilised, has the efficient disease-resistance cytotoxic activity, and wherein IFN-α is mainly produced by white corpuscle, and IFN-β is mainly produced by inoblast; II type IFN comprises IFN-γ, produces by T cell and NK cell, and to acid and thermally labile, mainly be immunoregulation effect, be the main macrophage activating factor of Mammals.Chicken and other bird IFN and Mammals IFN are similar, also are divided into I type IFN and II type IFN, have now found that chicken I type IFN comprises IFN-α, IFN-β, and the II type comprises IFN-γ, in bird, does not find IFN-ω and IFN-τ at present as yet.

Chicken IFN gene was successfully cloned from aged chick-embryo cell cDNA library by Sekellick in 1994 at first; The sequence similarity of itself and Mammals IFN is lower (to be about 20% with the amino acid identity of Mammals I type IFN; IFN is merely about 3% with the II type); But the position of its halfcystine and can be conservative relatively by the characteristic that virus induction produces, IFN is similar with Mammals I type, therefore infers that it is chicken I type IFN.Subsequently nineteen ninety-five, people such as Schultz find that this kind chicken IFN has the activity of anti-stomatitis follicularis virus, and lack the relevant biological activity of Mammals IFN-γ, thereby have confirmed this viewpoint.1996, people such as Christine Sick carried out Southern hybridization analysis and lambda phage screening to the cDNA library of preceding text, find that the genome of chicken contains 10 kinds of IFN genes at least.3 genes encoding chicken IFN-α (ChIFN-α claims ChIFN α again) of one of them gene family, the product of another independent gene is chicken IFN-β (ChIFN-β).ChIFN-α is made up of 193 amino-acid residues, and its mature peptide size is 162aa, and 31 amino acid of N-end are signal peptide.Although the amino acid identity of ChIFN-α and Mammals IFN-α is merely 24%; But the amino acid identity of high conservative region is up to 80%; The position of alpha-helix is also similar in both secondary structures; And both all can induce generation by S-26308 verivate S-28463, therefore with its called after ChIFN-α.ChIFN-β is made up of 203 amino-acid residues, and its mature peptide size is 176aa.1999, human recombinant C hIFN-α such as Plachy used rous sarcoma virus (RSV) to attack poison after handling CEF (CEF) again, found that the interference of high dosage have antineoplastic action.Simultaneously, people such as Marcus find can significantly reduce the sickness rate of NDV to the 1 Japanese instar chickling high dosage ChIFN-α that throws something and feeds.Calendar year 2001, people such as Mo C W find that ChIFN-α can suppress infectious bursal disease virus (IBDV) and form plaque, and can improve the surviving rate that infects the IBDV chicken.People such as Ellen find that ChIFN-α can suppress duplicating of IBV (IBV), thereby postpone the outbreak of disease, weaken clinical disease, show that ChIFN-α possibly be the potential immunostimulant.Calendar year 2001, people such as Jarosinski find, the MDV plaque to be formed reduce 50% with suppressing duplicating of Marek poison (MDV) behind the ChIFN-α processing CKC with the CDCC that can significantly reduce the NK cell behind the ChIFN-α processing chicken.

2003, people such as Xia Chun cloned the IFN-α gene of having identified three kinds of Strains of Chickens of China: SH-chicken, WJ-chicken and AA-chicken.IFN-α to three kinds of chickens carries out homology analysis, and then ChIFN-α is divided into two subclass: SH-ChIFN-α and WJ-ChIFN-α.And find that both all have anti-VSV activity, and the SH-ChIFN-α of high dosage can suppress 40% chicken embryo infection H9N2, and 100% suppresses 1 age in days infects to 5 Japanese instar chicklings.2009, people such as Tao Shengli find to mix drink low dosage ChIFN-α can improve fryer PBL sum, can improve the immunologic function of fryer, and the growth of healthy fryer is not made significant difference.

Summary of the invention

The purpose of this invention is to provide a kind of optimized gene and the application in the preparation chicken alpha-interferon thereof of the chicken alpha-interferon of encoding.

The optimized gene of coding chicken alpha-interferon provided by the invention is the DNA shown in the sequence 2 in the sequence table.

The recombinant vectors, reorganization bacterium, expression cassette or the transgenic cell line that contain the DNA shown in the sequence 2 in the ordered list all belong to protection scope of the present invention.

Said recombinant vectors can be the DNA shown in the sequence in the sequence table 2 is inserted the recombinant plasmid that the MCS of pET28b (+) obtains.Said recombinant vectors specifically can be the recombinant plasmid that obtains between the NdeI of the insertion pET28b of the DNA shown in the sequence 2 of sequence table (+) and EcoRI restriction enzyme site.

Said reorganization bacterium can be the DNA shown in the sequence in the sequence table 2 is imported the reorganization bacterium that e. coli bl21 obtains; Said reorganization bacterium specifically can be the said recombinant vectors of claim 3 is imported the reorganization bacterium that e. coli bl21 obtains.Said reorganization bacterium is preferably ETEC (Escherichia coli) BL21/pET28b-ChIFN α; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 23rd, 2010 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4112.

The present invention also protects a kind of method for preparing chicken alpha-interferon; Comprise the steps: said reorganization bacterium is carried out broken thalline behind the IPTG abduction delivering; Collect inclusion body, washing inclusion body, dissolve inclusion body and, obtain containing the solution of chicken alpha-interferon its renaturation.

Said abduction delivering specifically can be in the bacterium liquid of said reorganization bacterium and adds isopropyl-(IPT6) inductor, and making its final concentration is 1mmol/L, cultivates 4h for 37 ℃ then.

Said method also can comprise the step of the said solution that contains chicken alpha-interferon being carried out protein purification.

The method of said purifying is specific as follows: with the said solution that contains chicken alpha-interferon with Superdex 75 10/300GL sieve chromatography column purification; The elutriant pH=8.0 that purifying is used is made up of Tris, NaCl and water, and the concentration of Tris is 20mM, and the concentration of NaCl is 150mM; The flow velocity of elutriant is 0.3ml/min; Collecting elution volume is the elutriant of 12.8ml-15.1ml, is chicken alpha-interferon solution.

The chicken alpha-interferon that said method prepares also belongs to protection scope of the present invention.

Said DNA, recombinant vectors, reorganization bacterium, expression cassette or transgenic cell line all can be used for preparing chicken alpha-interferon.

The present invention has found a kind of ChIFN α gene of optimization, and said expression of gene ability is far above existing gene, and the MCS that this gene is inserted pET28b (+) has obtained recombinant plasmid, and recombinant plasmid is imported e. coli bl21, has obtained the reorganization bacterium.This reorganization bacterium just can be prepared a large amount of ChIFN α albumen through simple inducing culture.Gene provided by the invention, recombinant plasmid and reorganization bacterium have economic worth for the production of chicken alpha-interferon, also have great value for the fryer aquaculture.

Description of drawings

Fig. 1 is the comparison of ChIFN α-1 gene and ChIFN α-2 gene.

Fig. 2 is ChIFN α-1 gene PCR amplification figure; M:marker; 1,2:ChIFN α-1 gene.

Fig. 3 is the SDS-PAGE detected result of ChIFN α-1; C: do not induce contrast; I: induce the back contrast; S: induce the back supernatant; P: induce postprecipitation; The arrow indication is the target protein of abduction delivering, about 20kD, and (21.2kD) conforms to actual size.

Fig. 4 is the western blot detected result of ChIFN α-1; C: do not induce contrast; S: induce the back supernatant; P: induce postprecipitation.

Fig. 5 is the SDS-PAGE detected result of ChIFN α-2; 1:marker; 2: do not induce contrast; 3: induce postprecipitation.

Fig. 6 is that the ChIFN α protein content of protein solution first and protein solution second compares; 2: protein solution second; 3: the protein solution first.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.PET28b (+): available from the Novagen Company products, article No. is 69865-3.Bacillus coli DH 5 alpha: available from Beijing health is the century bio tech ltd, and article No. is CW0808.E. coli bl21: available from Beijing health is the century bio tech ltd, and article No. is CW0809.DF-1 cell (CF): (ATCC clone is numbered: CRL-12203) available from Central Plains, Beijing company.VSV virus (vesicular stomatitis virus): available from China Veterinery Drug Inspection Office.All primers are synthetic to reach examining order by the completion of the living worker in Shanghai company.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

The discovery of embodiment 1, chicken ChIFN α-1 gene

1, the extraction of the total RNA of chicken

According to the DNA extraction test kit of invitrogen company, from chicken (kind is blue or green pin fiber crops chicken, available from experimentation on animals base, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Changping) whole blood, extract the genomic dna of chicken.

2, the right design of special primer

With reference to existing chicken interferon α gene order, the design primer is to as follows:

Upstream primer: 5 '-GGAATTC CATATGTGCAACCACCTTCGCC-3 ' (underscore mark NdeI restriction enzyme site);

Downstream primer: 5 '-CCG GAATTCCTAAGTGCGCGTGTTGC-3 ' (underscore mark EcoRI restriction enzyme site).

3, the amplification of ChIFN α gene

Genomic dna with step 1 is a template, to carrying out pcr amplification, obtains pcr amplification product with the special primer of step 2 design.

Pcr amplification product is checked order, and sequencing result is shown in the sequence 2 of sequence table.

Disclosed chicken ChIFN α gene (GENBANK ACCESSION NO.DQ226094) shown in the sequence 3 of sequence table.Fig. 1 is seen in the comparison of gene shown in gene shown in the sequence 2 and the sequence 3, and there are 5 different IPs thuja acids in both, all the ChIFN α albumen shown in the sequence 1 of code sequence tabulation.With DNA called after ChIFN α-1 gene shown in the sequence 2 of sequence table, with DNA called after ChIFN α-2 gene shown in the sequence 3 of sequence table (existing gene).

Embodiment 2, ChIFN α-1 gene and ChIFN α-2 expression of gene

One, the preparation of ChIFN α-1 gene and ChIFN α-2 gene

1, the preparation of ChIFN α-1 gene

DNA shown in the sequence 2 of preparation sequence table as template, carries out pcr amplification with it, obtains pcr amplification product (ChIFN α-1 gene).

The pcr amplification primer is to as follows:

The PCR condition is: 95 ℃ of 5min; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 50s, 30 circulations; 72 ℃ of 10min.

The pcr amplification product of step 2 is carried out electrophoresis, and electrophoresis result is seen Fig. 2.Among Fig. 2, the arrow indication is a goal gene, about 500bp, and (492bp) conforms to actual size.

2, the preparation of ChIFN α-2 gene

DNA shown in the sequence 3 of preparation sequence table as template, carries out pcr amplification with it, obtains pcr amplification product (ChIFN α-2 gene).The pcr amplification primer to the PCR condition with step 1, pcr amplification product is carried out electrophoresis.

Two, construction of recombinant plasmid

1, the structure of recombinant plasmid pET28b-ChIFN α-1

1. cut ChIFN α-1 gene of glue recovering step one, behind restriction enzyme NdeI and EcoRI double digestion, reclaim enzyme and cut product.

2. use restriction enzyme NdeI and EcoRI double digestion pET28b (+), reclaim carrier framework.

3. with the T4DNA ligase enzyme step enzyme is 1. cut product and be connected, obtain connecting product with step carrier framework 2..

4. with step connection product transformed into escherichia coli DH5 α competent cell 3., choose mono-clonal and carry out PCR and identify, the evaluation of checking order of positive colony upgrading grain, sequencing result shows, has obtained recombinant plasmid pET28b-ChIFN α-1.Among the pET28b-ChIFN α-1, between the NdeI of pET28b (+) and EcoRI restriction enzyme site, inserted ChIFN α-1 gene shown in the sequence 2 of sequence table.

2, the structure of recombinant plasmid pET28b-ChIFN α-2

1. cut ChIFN α-2 gene of glue recovering step one, behind restriction enzyme NdeI and EcoRI double digestion, reclaim enzyme and cut product.

4. with step connection product transformed into escherichia coli DH5 α competent cell 3., choose mono-clonal and carry out PCR and identify, the evaluation of checking order of positive colony upgrading grain, sequencing result shows, has obtained recombinant plasmid pET28b-ChIFN α-2.Among the pET28b-ChIFN α-2, between the NdeI of pET28b (+) and EcoRI restriction enzyme site, inserted ChIFN α-2 gene shown in the sequence 3 of sequence table.

Three, the preparation of reorganization bacterium

With pET28b-ChIFN α-1 transformed into escherichia coli BL-21 (expressing bacterium); Containing the single reorganization bacterium bacterium colony that picking on the LB agar plate of kantlex contains recombinant plasmid; With one of them reorganization bacterium called after ETEC (Escherichia coli) BL21/pET28b-ChIFN α; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 23rd, 2010 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4112.

Adopt identical method with pET28b-ChIFN α-2 transformed into escherichia coli BL-21, obtain contrasting bacterium.

Four, ChIFN α-1 gene and ChIFN α-2 expression of gene

1, ChIFN α-1 expression of gene

1. the bacterium E.coli BL21/pET28b-ChIFN α that will recombinate is inoculated in 3ml and contains in the LB liquid nutrient medium of kantlex 37 ℃ of overnight cultures activation.

2. by 1: 100 volume ratio the activation bacterium is inoculated in 5ml and contains in the LB substratum of kantlex, cultivate about 2h to logarithmic phase (D for 37 ℃ _600nmValue reaches 0.5～0.8), take out a small amount of contrast of not inducing, remainder adds IPTG inductor (final concentration is 1mmol/L), 37 ℃ of abduction delivering 4h.

3. use 500 μ l PBS (8g NaCl, 0.2g KCl, 1.38g Na ₂HPO ₄, 0.2g KH ₂PO ₄Be dissolved in 1l ddH ₂O transfers pH to 7.4) resuspended bacterium, small size probe ultrasonic degradation (ultrasonic 2s stops 4s, 20 times) takes out a small amount of as inducing the back contrast; Then 12, the centrifugal 10min of 000rpm; Get supernatant (inducing the back supernatant), with 500 μ l PBS (8gNaCl, 0.2g KCl, 1.38g Na ₂HPO ₄, 0.2g KH ₂PO ₄Be dissolved in 1L ddH ₂O transfers pH to 7.4) resuspended deposition (inducing postprecipitation).

To not induce contrast respectively, contrast after inducing, induce the back to go up the cleer and peaceful postprecipitation of inducing to carry out 12%SDS-PAGE, the result sees Fig. 3.

Because have the His label on the recombinant protein, carry out Western Blot checking with the antibody of Anti-His, the result sees Fig. 4.Fig. 4 shows that expressed is target protein really, with the formal representation of inclusion body.

2, ChIFN α-2 expression of gene

To contrast bacterium and replace reorganization bacterium E.coli BL21/pET28b-ChIFN α, other is fully with step 1.

To not induce contrast respectively and induce postprecipitation to carry out 12%SDS-PAGE, the result sees Fig. 5.

Because have the His label on the recombinant protein, carry out Western Blot checking with the antibody of Anti-His, the result shows that expressed is target protein really, with the formal representation of inclusion body.

Five, proteic purifying

2 bacterium liquid 2. with 1 bacterium liquid 2. of step 4 and step 4 extracts purifying protein respectively, and step is following:

1. the bacterium liquid after getting 2L and inducing, centrifugal collection thalline, ultrasonication (ultrasonic 6s, 12s at interval, 99 times, 300W), centrifugal collecting precipitation (inclusion body).

2. use washing buffer (0.5%Triton-100,50mM Tris pH=8.0,300mM NaCl successively; 10mM EDTA; 10mM DTT), resuspension buffer (50mM Tris pH=8.0,100mM NaCl, 10mMEDTA; 10mM DTT) washing inclusion body, centrifugal collecting precipitation.

3. use dissolution buffer (6M Gua-HCl, 10% glycerine, 50mM Tris pH=8.0,100mM NaCl, 10mM EDTA, 10mM DTT) to dissolve inclusion body, the centrifugal deposition of abandoning fully.

4. dilution method is carried out renaturation: supernatant adds in the 5mL syringe, slowly splashes into refolding buffer (100mMTris pH=8.0,400mM L-Arg HCl, 2mM EDTA, 5mM GSH, 0.5mM GSSG), 4 ℃ of stirring at low speed 12-24h.

5. step solution is 4. concentrated in 4 ℃ with concentrating cup; Add molecular sieve damping fluid (20mM Tris pH=8.0 when being concentrated into the 20ml left and right sides; 150mM NaCl) continue to concentrate to 100ml, move to evaporating pipe when repeating to be concentrated into the 20ml left and right sides once more 2 times and be concentrated into 4ml.

6. get the above-mentioned liquid concentrator 12000rpm of 1ml, 10min is centrifugal, and (column volume is 24ml with Superdex 7510/300GL sieve chromatography column purification; Weighting material is a polydextran gel; Elutriant: 20mM Tris pH=8.0,150mMNaCl; Flow velocity: 0.3ml/min), collecting elution volume is the elutriant of 12.8ml-15.1ml, is target protein solution.

The protein solution that obtains with 1 bacterium liquid purifying 2. of step 4 is as protein solution first (batch 2009001).The protein solution that obtains with 2 bacterium liquid purifying 2. of step 4 is as protein solution second (batch 2009001).

Six, the ChIFN α protein content of protein solution first and protein solution second relatively

The protein solution first and the protein solution second that respectively step 5 are obtained are carried out Western Blot, and one anti-(available from Santa Cruz company, article No. is: SC-8036), the result sees Fig. 6 for Anti-His antibody.The result shows that the proteic content of ChIFN α is far above the proteic content of ChIFN α in the protein solution second in the protein solution first.

Embodiment 3, ChIFN α-1 gene and chiIFN α-2 expression of gene

One, the preparation of reorganization bacterium and contrast bacterium

PET28b-ChIFN α-1 transformed into escherichia coli BL-21 with the preparation of the step 2 of embodiment 2 obtains the bacterium of recombinating.PET28b-ChIFN α-2 transformed into escherichia coli BL-21 with the preparation of the step 2 of embodiment 2 obtains contrasting bacterium.

Two, ChIFN α-1 gene and ChIFN α-2 expression of gene

1, ChIFN α-1 expression of gene

1. the bacterium of will recombinating is inoculated in 3ml and contains in the LB liquid nutrient medium of kantlex 37 ℃ of overnight cultures activation.

2, ChIFN α-2 expression of gene

1. will contrast bacterium is inoculated in 3ml and contains in the LB liquid nutrient medium of kantlex 37 ℃ of overnight cultures activation.

Three, proteic purifying

1, the preparation of protein solution first

1 the bacterium liquid that 2. obtains of step 2 is carried out purifying, and method is with the step 5 of embodiment 2, obtains protein solution first (batch 2009002).

2 the bacterium liquid that 2. obtains of step 2 is carried out purifying, and method is with the step 5 of embodiment 2, obtains protein solution second (batch 2009002).

Embodiment 4, ChIFN α-1 gene and ChIFN α-2 expression of gene

One, the preparation of reorganization bacterium and contrast bacterium

Two, ChIFN α-1 gene and ChIFN α-2 expression of gene

1, ChIFN α-1 expression of gene

2, ChIFN α-2 expression of gene

Three, proteic purifying

1, the preparation of protein solution first

1 the bacterium liquid that 2. obtains of step 2 is carried out purifying, and method is with the step 5 of embodiment 2, obtains protein solution first (batch 2009003).

2 the bacterium liquid that 2. obtains of step 2 is carried out purifying, and method is with the step 5 of embodiment 2, obtains protein solution second (batch 2009003).

Embodiment 5, ChIFN α-1 gene and ChIFN α-2 expression of gene ability are relatively

Three batches protein solution first of comparing embodiment 2 to embodiment 4 preparations and the protein content difference in the protein solution second, to compare ChIFN α-1 gene and ChIFN α-2 expression of gene ability, method is following:

By 50: 1 volume ratio get BCA protein quantification test kit (available from Beijing health is the century bio tech ltd, article No.: solution A and solution B CW0014) are mixed into working fluid, and with BSA standard substance gradient dilution to 500mg/ml; 400mg/ml, 300mg/ml, 200mg/ml; 100mg/ml, 50mg/ml, 25mg/ml; Get the standard substance or the testing protein sample 20 μ l of each concentration; The working fluid that adds 200 μ l mixes, and seals 37 ℃ with preservative film and hatches 30min, reads OD with ELIASA after returning to room temperature ₅₆₅Light absorption value.According to standard substance concentration and light absorption value drawing standard curve, calculate the protein concentration in the testing protein sample according to typical curve again.

The protein concentration of protein solution first (batch 2009001) is 3.6mg/ml, and the protein concentration of protein solution second (batch 2009001) is 2.3mg/ml.The protein concentration of protein solution first (batch 2009002) is 3.7mg/ml, and the protein concentration of protein solution second (batch 2009002) is 2.2mg/ml.The protein concentration of protein solution first (batch 2009003) is 3.55mg/ml, and the protein concentration of protein solution second (batch 2009003) is 2.35mg/ml.

The result shows that though only differ 5 Nucleotide, ChIFN α-1 expression of gene ability provided by the invention is higher than existing C hIFN α-2 gene far away.

Embodiment 6, with the proteic interferon activity of ChIFN α-1 of method of the present invention preparation

Three batches protein solution first of embodiment 2 to embodiment 4 preparations is divided into two groups respectively: first group: 4 ℃ of-8 ℃ of preservations, respectively at 6 months, 12 months, 18 months and 24 months sampling detection interferon activities; Second group: 25 ℃ of preservations, respectively at 1 month, 3 months and 6 months sampling detection interferon activities.

The interferon activity detection method is following:

1, cell preparation

Get well-grown DF-1 cell, the digestion back prepares cell suspension with the DMEM that contains 10%FBS; Pair cell is counted, and the adjustment cell concn is 5 * 10 ⁵The suspension of individual/mL joins in (100 μ l/ hole) on the 96 porocyte culture plates 37 ℃, 5%CO then ₂Cultivating 8-10h makes it become monolayer.

2, add the interferon-treated cell

Use the DMEM that contains 10%FBS to be diluted to final concentration in advance the protein solution first and be 0.001mg/ml,, again mother liquor is carried out 4 times of gradient dilutions, dilute 6 gradients, obtain various diluents as mother liquor.

3, the nutrient solution sucking-off in the every hole of the Tissue Culture Plate of step 1; 6 holes in the Tissue Culture Plate (3 positive control holes, 3 negative control holes) add the cell culture fluid in 100 μ l/ holes, and all the other holes add the diluent (100 μ l/ holes, three repetitions of each gradient) of step 2 respectively; 37 ℃, 5%CO ₂Cultivate 12-15h.

4, virus infection

Get VSV virus, use serum-free DMEM to be diluted to final concentration and be 1000TCID ₅₀The viral dilution liquid of/ml; Every hole, positive control hole adds 100 μ l viral dilution liquid, and the every hole of negative control hole adds 100 μ l serum-free DMEM nutrient solutions, and all the other every holes, each hole add 100 μ l viral dilution liquid; Cultivate 24h.

5, violet staining

Abandon cell culture fluid, in every hole, add violet staining liquid 100 μ l, room temperature is placed 30min.

6, decolouring

Abandon dyestuff, and wash not illuminating colour with tap water or distilled water, in every hole, add destainer 100 μ l then, room temperature is placed 10min.

7, utilize ELIASA to measure OD ₅₇₀Value and record.

8, data processing

Be defined as an activity unit can suppress 50% cytopathic Interferon, rabbit content, utilization Reed-Muench method is calculated Interferon, rabbit and is tired, and can suppress 50% cytopathic extension rate, and the result sees table 1.

Table 1 utilization Reed-Muench method is calculated Interferon, rabbit and is tired

X: the OD of negative control (virus-free contrast) ₅₇₀MV;

Y: the OD of positive control (virus control) ₅₇₀MV.

Calculate each item value according to last table, calculate Interferon, rabbit tire (suppose in the aforementioned calculation G4 item calculated value greater than 0.5, and G5 item calculated value being less than 0.5) according to the G item according to following formula at last:

Interferon, rabbit to be detected (U/0.001mg)=in advance extension rate * 4 of tiring ^{(4+ (G4-0.5)/(G4-G5))}

The Interferon, rabbit detected result of tiring is seen table 2.

The table 2 Interferon, rabbit detected result of tiring

The result shows: adopt the protein solution first of method preparation of the present invention to deposit 24 months at 4 ℃-8 ℃, deposited 3 months for 25 ℃, biological activity does not have considerable change.

Claims

1. the DNA shown in the sequence 2 of sequence table.

2. the recombinant vectors that contains the DNA shown in the sequence 2 of ordered list.

3. the reorganization bacterium that contains the DNA shown in the sequence 2 of ordered list.

4. the expression cassette that contains the DNA shown in the sequence 2 of ordered list.

5. the transgenic cell line that contains the DNA shown in the sequence 2 of ordered list.

6. recombinant vectors as claimed in claim 2 is characterized in that: said recombinant vectors is the recombinant plasmid that obtains between the NdeI of the insertion pET28b of the DNA shown in the sequence 2 of sequence table (+) and EcoRI site.

7. reorganization bacterium as claimed in claim 3 is characterized in that: said reorganization bacterium is ETEC (Escherichia coli) BL21/pET28b-ChIFN α, CGMCC No.4112.

8. method for preparing chicken alpha-interferon; Comprise the steps: the said reorganization of claim 7 bacterium is carried out broken thalline behind the IPTG abduction delivering; Collect inclusion body, washing inclusion body, dissolve inclusion body and, obtain containing the solution of chicken alpha-interferon its renaturation.

9. method as claimed in claim 8 is characterized in that: said abduction delivering is in the bacterium liquid of said reorganization bacterium, to add isopropyl-, and making its final concentration is 1mmol/L, cultivates 4h for 37 ℃ then.

10. like claim 8 or 9 described methods, it is characterized in that: said method also comprises the step of the said solution that contains chicken alpha-interferon being carried out protein purification.

11. method as claimed in claim 10 is characterized in that: the method for said purifying is following: with the said solution that contains chicken alpha-interferon with Superdex 7510/300GL sieve chromatography column purification; The used elutriant pH of purifying is 8.0, is made up of Tris, NaCl and water, and the concentration of Tris is 20mM, and the concentration of NaCl is 150mM; The flow velocity of elutriant is 0.3ml/min; Collecting elution volume is the elutriant of 12.8ml-15.1ml, is chicken alpha-interferon solution.

12. said DNA of claim 1 or claim 2 or 6 described recombinant vectorss, claim 3 or 7 said reorganization bacterium, the said expression cassette of claim 4 or the said transgenic cell of claim 5 tie up to the application in the preparation chicken alpha-interferon.