CN102628062A - Expression method of animal alpha interferon and gamma interferon - Google Patents

Abstract

The invention discloses an expression method of an animal alpha interferon and a gamma interferon. The method comprises the following step of: performing combined expression or co-expression on the alpha interferon and gamma interferon of a human being or an animal of the same type in a bioreactor. The alpha interferon gene and gamma interferon gene of a human being or an animal of the same type are subjected to combined expression or co-expression in the same insect rhabdovirus bioreactor, and an expressed recombinant alpha interferon product and recombinant gamma interferon have cooperative and synergistic actions, so that the valence and stability of a recombinant interferon product can be enhanced. The method has the advantages of high expression efficiency, high product valence, high stability and the like.

Description

The expression method of animal IFN-and IFN-

Technical field

The present invention relates to a kind of expression method of Interferon, rabbit, relate in particular to a kind of method of in the insect baculovirus bio-reactor, uniting expression or coexpression animal IFN-and IFN-, belong to the preparation field of IFN-betaser.

Background technology

(Interferon is under the effect of specific inducer IFN) to Interferon, rabbit, a kind ofly has highly bioactive gp by what cell produced, in this zooblast, has the broad-spectrum disease resistance cytotoxic activity.The known disturbances element is not a direct killing virus at present, comes the genetic transcription of viral interference or the translation of viral protein component but produce plurality of enzymes through the cell of inducing host itself.Interferon, rabbit can be divided into I type and II type IFN according to the aspects such as cell source difference, physico-chemical property and BA difference that produce; I type IFN mainly comprises IFN-α, β, ω, δ, κ, ε, ζ and τ etc., II type IFN have only one kind of IFN-γ (Cann AJ.Principles ofMolecular Virology.Beijing:Science Press, 2006:177-178).IFN-γ is mainly produced by activated T lymphocyte in the body and NK cell; T emiocytosis produces IFN-γ after antigen, PHA or ConA stimulate, and is consistent with the generation of IL-2 usually, IFN-γ acid labile; Has the virus replication of inhibition regulating effect; But its antivirus action than I type Interferon, rabbit a little less than, main expression cassette immunomodulatory effect of participating in inducing major histocompatibility antigen (MHC) is also referred to as type II interferon.The antiviral activity of I type Interferon, rabbit wants high than II type Interferon, rabbit; But because II type Interferon, rabbit has the regulating effect of inducing, so certain organism (like pig, dog, chicken etc.) is higher than the effectiveness of single any one Interferon, rabbit far away with the tiring of mixture of two types of Interferon, rabbit.

Interferon, rabbit has people such as Isaacs to find from nineteen fifty-seven after; Just cloned and in various bio-reactors, expressed always; In intestinal bacteria with in the insect cell, the exon of pig PoIFN-γ gene has been carried out the clone and (Vandenbroeck K is expressed in splicing such as Vandenbroeck etc.; Et al.Biochem Biophys Res Commun, 1991:1408-1415; Vandenbroeck K, et al.Lymphokine Cytokine Res, 1994:253-258), same more domestic investigators also successively utilize different expression systems to express PoIFN-γ.

The human IgG Tegeline can reach 19～21 days the people the intravital transformation period; The Fc fragment of IgG is used to connect and compose fusion rotein with IFN2 α; The Fc fragment increases the fusion protein molecule amount, avoid by glomerular filtration, simultaneously the Fc fragment can with newborn Fc receptors bind; Avoid fusion rotein to get in the lysosome and be degraded, thus the transformation period in the extension body.Made up IFN2 α 2b and the segmental fusion gene of human IgG immunoglobulin Fc and in pichia spp with the dimeric forms secreting, expressing, and the part glycosylation is arranged.Behind the rat skin lower injection in the blood circulation transformation period reach 65h, in the blood more than the RT 120h, prolong about 8 times than the transformation period in the body of common IFN-betaser; The blood RT prolongs 10 times; Shown its good potential applicability in clinical practice (Wang Lei, the biotechnology journal, 2008:53-62).

Same principle, α of pig, chicken, dog or the long-acting interferon of γ also can be carried out amalgamation and expression with the Fc fragment of corresponding animal IgG or HAS sequence and its α or IFN-gene, and the method for using the application can obtain corresponding long-acting interferon.

This bio-reactor of baculovirus expression system is that the eighties is set up.Since nineteen eighty-three utilizes baculovirus expression system to efficiently express people's alpha-interferon first (Smith, Mol.Cell Biol., 3:2156-2165,1983), existing dozens of foreign gene has obtained efficiently expressing.The baculovirus expression carrier system is one of current genetically engineered four big expression systems; For prokaryotic expression system; Baculovirus expression system is a kind of eukaryotic expression system; Have eukaryotic protein translation post-treatment, modification and delivery system, expression product aspect biological activity, antigenicity and immunogenicity near natural product.Utilize this system to produce recombinant protein and comprise that the advantage of various Interferon, rabbit is: 1. the expression efficiency of this expression system is high; Output can reach the level of milligram level/worm, thereby can reduce production costs and make the scale operation of many valuable recombinant proteins to become possibility greatly; 2. this expression system is an eukaryotic expression system, and the exogenous protein of its expression can carry out posttranslational modification, makes it similar with natural product with biological activity etc. at biochemical property, and this is that the recombinant protein that gives expression to has normal BA assurance is provided.At present, baculovirus expression system, silkworm especially wherein (Bm)-silkworm baculovirus (BmNPV) expression system are one of individual expression systems of eukaryote that has most in the world business development value.One of current genetically engineered four big expression systems (being baculovirus, intestinal bacteria, yeast, mammalian cell expression system) commonly used have been become.

The baculovirus genome is bigger, so can only earlier foreign gene be connected on the transfer vector through the way of homologous recombination, again with transfer vector and viral cotransfection cell or the reorganization of external enzymatic, thereby makes up rhabdovirus expression vector.General transfer vector all contains two homology arms of virus; One or more bacilliform virus promoters (majority is got the polyhedron promotor) are inserted into the external source goal gene after the promotor downstream, recombinate with baculovirus; Obtain recombinant virus; Again with the recombinant virus purifying, infected insect cell or polypide, foreign gene along with virus duplicate and obtain to express.What make up use when recombinant virus makes up the earliest is the cyclic wild virus, and after virus and transfer vector were recombinated, the polyhedron gene of virus was damaged; Can not form polyhedron, the cell that infects this recombinant strain forms plaque at microscopically, through repeatedly repeating screening; Can carry out purifying to recombinant virus; This screening process expends very big manpower and materials, and efficient is very low, to a great extent limit the application of baculovirus.

At present when expressing Interferon, rabbit with this bio-reactor of baculovirus expression system; All be the Interferon, rabbit of a type to be recombinated in insect host or cell, to express behind the baculovirus separately; Exist low, the expressed IFN-betaser product of the expression efficiency defectives such as low, poor stability of tiring, have much room for improvement.

Summary of the invention

Main purpose of the present invention provides a kind of expression method of new Interferon, rabbit, and this expression method has the expression efficiency height, the expressed IFN-betaser product advantages such as height, good stability of tiring.

Main purpose of the present invention realizes through following technical scheme:

A kind of expression method of Interferon, rabbit comprises: people or homozoic IFN-and IFN-are united in the insect baculovirus bio-reactor express or coexpression.

Wherein, People or homozoic alpha-IFN gene and IFN-gene united to express in the insect baculovirus bio-reactor may further comprise the steps: recombinate after with people or homozoic alpha-IFN gene and IFN-gene gang on the nonessential gene locus that duplicates or infect of baculovirus (1), the acquisition recombinant baculovirus; (2) with recombinate shape virus infection insect host or insect cell, expression alien gene obtains IFN-and IFN-associating expressed products.

Preferably, through the IRES sequence people or homozoic alpha-IFN gene and IFN-gene are connected together in the step (1), in order to promote the expression of rrna to the back interferon gene; The nucleotides sequence of said IRES sequence is classified as shown in the SEQ ID No.3.

Preferably; In the step (1) in the following manner with the recombination of gang to the nonessential gene locus that duplicates or infect of baculovirus: the alpha-IFN gene of gang and IFN-gene clone to baculovirus transfer vector, are obtained the recombinant baculovirus transfer vector; Recombinant baculovirus transfer vector and baculovirus are recombinated in insect cell, with the alpha-IFN gene of gang and IFN-recombinate duplicating of baculovirus or the nonessential gene locus that infects on; Wherein, described baculovirus transfer vector is preferably the pVL1393 carrier; The nonessential gene locus that duplicates or infect of described baculovirus comprises sites such as polyhedrosis gene site, p10, egt, p74, p26, is preferably the polyhedrosis gene site.

Mode of infection described in the step (2) is preferably: with recombinant virus infection insect cell or percutaneous puncture-inoculation insect larvae or pupa; After infecting 3-6 days, collect and contain the insect cell of expression product or the body fluid or the tissue homogenate of larva or pupa.

Describedly people or homozoic alpha-IFN gene and IFN-gene carried out coexpression may further comprise the steps in the insect baculovirus bio-reactor: (a) people or homozoic alpha-IFN gene and IFN-gene are recombinated duplicating of baculovirus respectively or two nonessential gene locuss infecting on, obtain recombinant baculovirus; (b) with recombinate shape virus infection insect host or insect cell, expression alien gene obtains the product of IFN-and IFN-coexpression;

Preferably; On two nonessential gene locuss of in such a way people or homozoic alpha-IFN gene and IFN-gene being recombinated duplicating of baculovirus respectively in the step (a) or infecting; Obtain recombinant baculovirus: the same EGFP of IFN-gene (enhanced green fluorescence protein) labelled protein gene is connected; On the p10 gene locus of method with its ReBm-dcc-d1629-d10 baculovirus shuttle vectors of recombinating (silkworm baculovirus shuttle vectors ReBm-dcc-d1629-d10 carries out the external shuttle vectors that acquisition can't normally get into viral life cycle that knocks out through the ORF1629 gene locus to BmNPV) that in intestinal bacteria, carries out vitro recombination, the phenotypic screen through EGFP goes out recombinant vectors; The EGFP marker gene is knocked out defective type (ReBm-dcc-d1629-d10) the baculovirus shuttle vectors of the IFN-that obtains to have recombinated; Alpha-IFN gene recombinated to contain in the baculovirus transfer vector of ORF1629 homology arm, obtain the recombinant baculovirus transfer vector; With the defective type baculovirus shuttle vectors and the recombinant baculovirus transfer vector cotransfection bombyx mori cell of the IFN-of having recombinated, the method for saving through inactivation obtains the recombinant baculovirus that the ORF1629 gene is repaired; Thereby in silkworm biological reactor, express the coexpression product that obtains two types of Interferon, rabbit.Wherein, when coexpression IFN-and IFN-, the present invention preferably recombinates IFN-on the polyhedrosis gene site of baculovirus, and IFN-is recombinated on the p10 gene locus of baculovirus.

In addition; Low antiviral vitality or unvital situation that the various foreign interferon of expressing for fear of this bio-reactor of usefulness cause owing to expression environment difference; The present invention is optimized through the gene to the various Interferon, rabbit that obtained; It mainly is the transformation of codon preference type and kozak sequence; Make it can be suitable in applied bio-reactor, giving expression to activated composition as far as possible,, improved the expression efficiency of Interferon, rabbit and tiring of expression product through optimization to interferon gene.

The present invention optimizes porcine alpha-IFN gene and IFN-gene, and the nucleotides sequence of the porcine alpha-IFN gene after the optimization is classified as shown in the SEQ ID No.1, and the nucleotides sequence of the pig gamma interferon gene after the optimization is classified as shown in the SEQID No.2; In addition, the present invention suddenlys change to the sequence of porcine alpha-IFN, it is obtained than higher protease resistant guaranteeing under its active situation it is transformed; Help the time that Interferon, rabbit exists in vivo; Through a large amount of point mutation experiment, the present invention finds, the effect that the 64th L-glutamic acid of porcine alpha-IFN is sported Stimulina is the best; Effectively reduce susceptibility to proteolytic ferment in blood and the tissue; Obtained long-acting porcine alpha-IFN two mutants, the nucleotides sequence of this two mutants of encoding is classified as shown in the SEQ ID No.9, and its aminoacid sequence is shown in the SEQID No.10.

The present invention optimizes dog alpha interferon gene and IFN-gene, and the nucleotides sequence of the dog alpha interferon gene after the optimization is classified as shown in the SEQ ID No.5, and the nucleotides sequence of the dog IFN-gene after the optimization is classified as shown in the SEQID No.6; In addition, the present invention also optimizes chicken alpha interferon gene and IFN-gene, and the nucleotides sequence of the chicken alpha interferon gene after the optimization is classified as shown in the SEQ ID No.7, and the nucleotides sequence of the chicken IFN-gene after the optimization is classified as shown in the SEQ ID No.8.

Insect baculovirus described in the present invention comprises BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV, TnNPV etc.; Described insect host comprises silkworm (Bombyx mori); Wild silkworm (Bombyx mandarina); Semen Ricini silkworm (Philosamia cynthia ricim); Wild silkworm (Dictyoploca japanica); Philosamia cynthia (Philosamia cynthiapryeri); Tussah (Antheraea pernyi); Yamama (Antheraea yamamai); Wild giant silkworm (Antheraea polyphymus); Autographa california (Atographa califorica); Tea geometrid (Ectropis obliqua); Cabbage army worm (Mamestra brassicae); Prodenia litura (Spodoptera littoralis); Autumn mythimna separata (Spodoptera frugiperda); Trichoplusia ni (Trichoplusia ni); Armyworm (Thaumetopoea wilkinsoni); Bollworm (Heliothis armigera); U.S. bollworm (Heliothis zea); Oriental tobacco budworm (Heliothis assulta); Cigarette beetle (Heliothis virescens); Oriental armyworm (Pseudaletia separata); Gypsymoth (Lymantria dispar) etc.

According to expression method of the present invention can baculovirus duplicate or sites such as the nonessential gene locus that infects such as polyhedrosis gene, p10, egt, p74, p26 through in the baculovirus shuttle plasmid, importing the interferon gene expression cassette successively, realize with a recombinant virus while at host insect or a plurality of Interferon, rabbit of insect cell inner expression.

The inventive method is united people or homozoic alpha-IFN gene and IFN-gene and is expressed or coexpression in same insect baculovirus bio-reactor; Has synergistic function between the expressed IFN-betaser product; Can improve tiring and stability of IFN-betaser product; The inventive method has the expression efficiency height, the expression product advantages such as height, good stability of tiring.

Description of drawings

Fig. 1 contains the pVL1393 carrier collection of illustrative plates of ORF1629 homology arm.

PCR detected peaks figure behind Fig. 2 SIFNG reorganization BmBacmid.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.

1, bacterial strain, virus strain and carrier e. coli strains: BmNPV, delivery carrier pVL1393, pBm035, bombyx mori cell BmN, Bm-5, Sf-21 cell, (Top10 DH10B) waits available from Invitrogen company or Japanese Riken BRC intestinal bacteria; PGL-3 carrier, luciferase test kit Luciferase Assay System are available from Promega; PMD-18T carrier and pMD-18T-simple carrier are available from TaKaRa company; The BW25113/pKD46 that contains recombinase, DH5alpha/pCP20 is available from Depart of MCD biology 830KBT, Yale University.

2, enzyme and reagent: restriction enzyme, ligase enzyme are the Promega Company products.

3, biochemical reagents: IPTG, X-Gal, SDS are the Sigma Company products.Lipofectin, low melting point agarose LMP, PCR test kit, T4DNA ligase enzyme, RNA enzyme, Proteinase K, cell culture medium TC-100, foetal calf serum and other reagent are purchased the company in Invitrogen.

4, substratum: the intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0); The insect cell substratum is TC-100.

5, the primer:

Used primer sequence in table 1 experiment

Embodiment 1 porcine alpha-IFN and IFN-unite expression

1 experimental technique

At first to obtain the sequence of porcine alpha-IFN and IFN-; Through transforming the codon that the codon in the pig interferon sequence of wild-type is replaced with preference in the silkworm baculovirus; Synthetic rear clone also is connected on the pVL1393 carrier; Link to each other through the IRES sequences in the middle of two Interferon, rabbit, method that then can be through homologous recombination with the process of BmNPV DNA cotransfection in obtain being used to express the recombinant shuttle plasmid of target protein.

1.1. about solution and culture medium preparation referring to relevant document (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998)

1.2 the acquisition of porcine alpha-IFN, IFN-sequence and IRES sequence and the transformation of two Interferon, rabbit sequences and synthetic

According to delivering document; And the data among the combination GenBank; Obtain several kinds of porcine alpha-IFN sequences and be respectively Sus scrofa interferon alpha-1gene (GenBank:AY526089.1), Sus scrofa interferon-alpha-6 (GenBank:GQ415060.1), Sus scrofa interferon-alpha-4 (NCBI Reference Sequence:NM_001166319.1), Sus scrofa interferon-alpha-12 gene (GenBank:GQ415066.1), Sus scrofa interferon alpha-14 (NCBI Reference Sequence:NM_001130230.1); Analyze its sequence characteristic so that reasonable reconstruction, obtain the sequence Sus scrofa interferon gamma mRNA (GenBank:GU433229.1) of pig gamma interferon.

According to the gene order that obtains and as the silkworm of bio-reactor and the characteristics of Bombyx mori nuclear polyhydrosis virus two gene orders are carried out GC content transformation (GC Content Adjustment) in the sub-preference property transformation of translation cipher (Codon usage bias adjustment), the sequence, used the removal of restriction endonuclease sites etc. always; Obtain the improved sequence of two Interferon, rabbit; In addition according to will with carrier pVL1393 sequence on the sequence that obtains behind two gene optimizations of polyclone restriction enzyme site design be respectively SIFNA (SEQ ID No.1) and SIFNG (SEQ ID No.2); SIFNA upstream and downstream restriction enzyme site is BamH I and Xba I, and SIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.The IRES sequence is seen SEQIDNo.3, and the upstream and downstream restriction enzyme site is Xba I and EcoR I, these three sections sequences is synthesized respectively be used for subsequent operations; Be connected to above the pUC57 carrier after above-mentioned several gene is all synthetic.

1.3IRES sequence is connected on the pVL1393 carrier

The pVL1393 carrier synoptic diagram that contains the ORF1629 homology arm is seen Fig. 1.Its main element is following: upper reaches homology arm is Recombination sequence (ORF603+): bases 1-3997; The polyhedron upstream promoter is Polyhedrin promoter:bases 3998-4092; Polyhedrosis gene is Polyhedrin gene:bases 4093-4738; MCS is Multiple cloning site:bases 4128-4179; The downstream homology arm is Recombinationsequence (ORF1629+): bases 4738-7002; The intestinal bacteria replication origin is ColE1 origin:bases8029-7356, and the resistance screening gene is ammonia benzyl mycin resistant gene Ampicillin resistance gene:bases8965-8177.

1.3.1IRES the sequence enzyme is cut

The enzyme system of cutting is seen table 2

Table 2 enzyme is cut system

10 * damping fluid H, 5 μ L contain IRES plasmid 8.0 μ L

Enzyme (Xba I) 1 μ L enzyme (EcoR I) 1 μ L

BSA 0.5μL ddH ₂O 34.5μL

37 ℃ were reacted 2.5 hours.

1.3.2 enzyme is cut the glass milk method of product and is reclaimed (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998)

1.3.3pVL1393 the carrier enzyme is cut

The enzyme system of cutting is seen table 3.

Table 3 enzyme is cut system

10 * damping fluid H, 5 μ L contain IRES plasmid 5.0 μ L

Enzyme (Xba I) 1 μ L ddH ₂O 38.5 μ L

BSA 0.5μL

37 ℃ of endonuclease reactions took a morsel and carry out agarose gel electrophoresis after 1.5 hours, added 1 μ L EcoR I reaction 2 hours again after the check enzyme cuts entirely, and 65 ℃ of temperature are bathed 15 minutes with enzyme-deactivating then, obtain the carrier that enzyme cuts.

1.3.4IRES endonuclease bamhi connects the pVL1393 carrier

Linked system is seen table 4.

Table 4 linked system

16 ℃ of reactions are spent the night.

Transform the TOP10 competent cell 1.3.5 connect product

1.3.5.1 competent preparation (Joseph et al., the molecular cloning experiment guide third edition, 2002)

Transform (Joseph et al., the molecular cloning experiment guide third edition, 2002 1.3.5.2 connect product; Ao Sibai et al., fine works molecular biology guide, 1998)

(1) DNA 20～100ng or connect mixture 3 μ L and be added in the competent cell of the above-mentioned preparation of 100 μ L, mixing gently, ice bath 30min;

(2) carry out heat-shocked (42 ℃ of insulation 90s), put 1～2min on ice rapidly, add 1mL and be incubated to 37 ℃ LB substratum, 37 ℃ of shaking culture 1h,

(3) centrifugal slightly, go resuspended deposition behind the part supernatant, coat several and contain on the corresponding antibiotic LB flat board.Be inverted overnight cultures for 37 ℃.

1.3.6 the evaluation of recombinant plasmid

Cytoclasis method Rapid identification

When having certain difference, plasmid after the reorganization and original plasmid molecule amount can detect recon (Beuken, et al., Biotechniques, 72:3827-3836,1998) with this method.

(a) a plurality of single colony transformation of picking are inoculated in 4mL and contain in the LB substratum of 80 μ g/mLAmp respectively, and 37 ℃ of shaking culture are spent the night;

(b) get 300～500 μ L bacterium liquid in the Eppendorf pipe, 12, the centrifugal 10sec of 000g abandons supernatant, adds 30 μ L damping fluids (6% sucrose, 0.1% tetrabromophenol sulfonphthalein), adds 20 μ L phenol/chloroforms (1: 1) again, and fully thalline is upspring in vibration;

(c) 12, the centrifugal 5min of 000g gets appearance electrophoresis on the supernatant, observations.

The further enzyme of recombinant plasmid is cut evaluation (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998);

(1) extraction of common DNA

(2) extraction of Bacmid DNA

(3) enzyme of recombinant plasmid is cut evaluation

Choose each restriction enzyme site of connection site two ends and carry out double digestion, can identify.

Identification system is seen table 5.

Table 5 identification system

10 * damping fluid H, 1.5 μ L recombinant plasmids, 2.0 μ L

Enzyme 0.5 μ L ddH ₂O 11 μ L

37 ℃ of enzymes are cut 1～2h, add Marker standard as a reference, detect enzyme with agarose gel electrophoresis and cut the result.

If the buffer difference of two kinds of enzymes is then used single endonuclease digestion 1～2h earlier, add the 3M sodium-acetate of 1/10 volume and the absolute ethyl alcohol deposit D NA of 2 times of volumes, remove supernatant and carry out second enzyme again and cut.Obtain being connected with the pVL1393-IRES vector plasmid of IRES sequence after identifying correctly.

1.4 porcine alpha-IFN (SIFNA) is connected on the pVL1393-IRES carrier

1.4.1SIFNA receive with the enzyme switchback of pVL1393-IRES

To SIFNA with pVL1393-IRES carries out BamH I (Buffer E) respectively and Xba I (Buffer E) enzyme is cut, method is seen 1.3.1, and glass milk method reclaims the endonuclease bamhi that obtains SIFNA and pVL1393-IRES.

See 1.3.4 1.4.2SIFNA be connected to last also transformed into escherichia coli TOP10 competent cell linked system of pVL1393-IRES and method, will connect product and be transformed in the TOP10 competent cell.

1.4.3 the detection of recombinant plasmid

Picking transforms the positive bacterium colony of back antibiotic-screening, slightly carries picking out recombinant clone and extracting plasmid and carry out enzyme and cut evaluation, and method is seen 1.3.6.Obtain plasmid pVL1393-SA-IRES after detecting correctly.

1.5 pig gamma interferon (SIFNG) is connected on the pVL1393-SA-IRES carrier

The plasmid and the pVL1393-SA-IRES carrier that will contain SIFNG carry out EcoR I (BufferH) and Bgl II (BufferD) double digestion; Because endonuclease reaction Buffer is different; So carry out an endonuclease reaction earlier; Detect enzyme after 1-2 hour and cut full postprecipitation plasmid, carry out second kind of enzyme and cut, method is seen 1.3.6.

Enzyme is cut evaluation after filtering out positive colony, and the sequence of three fragment genes that add in the pVL1393 carrier polyclone restriction enzyme site is measured in the plasmid that the obtains detection of checking order, and obtains pVL1393-SAIG after correct.

1.6 interferon gene and BmNPV DNA cotransfection bombyx mori cell carry out the virus reorganization

With the pVL1393-SAIG plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell because the pVL1393 plasmid that BmNPV DNA carries out homology arm recombinates, be the baculovirus after the reorganization in the no polyhedron plaque of the cell culture that obtains.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-SAIG DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%; Seal; Cultivate 4～5d for 27 ℃, after floating to cell, collect screening and expression that supernatant is used for recombinant virus.

1.7 the screening of recombinant virus Bm-SAIG, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths; Be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, add X-gal and make its final concentration reach 150 μ g/mL, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ L TC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 33 spots all is the recombinant virus Bm-SAIG that contains porcine alpha-IFN gene and pig gamma interferon gene, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations were in order to injection.

1.8 recombinant virus Bm-SAIG uniting of α and IFN-in silkworm expresses and detects

1.8.1 the expression of recombinant virus Bm-SAIG

Above-mentioned 33 recombinant virus Bm-SAIG nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain porcine alpha-IFN and the IFN-associating expression product in silkworm.

1.8.2ELISA method detects

1.8.2.1 the ELISA of porcine alpha-IFN detects in the associating expression product

Use DZ011 porcine alpha-IFN (IFN-α) the ELISA test kit of Beijing winter song bio tech ltd to detect the associating expression product, the ELISA operation steps is following:

A: the dilution of standard substance and application of sample: encapsulate at enzyme mark and to establish 10 holes, standard substance hole on the plate, in first, second hole, add standard substance 100 μ l respectively, in first, second hole, add standard substance diluent 50 μ l then, mixing; From first hole, second hole, respectively get 100 μ l then and be added to the 3rd hole and the 4th hole respectively, add standard substance diluent 50 μ l, mixing again in the 3rd, the 4th hole respectively; In the 3rd hole and the 4th hole, respectively get 50 μ l earlier then and discard, respectively get 50 μ l again and be added to respectively in the 5th, the 6th hole, in the 5th, the 6th hole, add standard substance diluent 50ul, mixing more respectively; Get respectively from the 5th, the 6th hole behind the mixing that 50 μ l are added to the 7th respectively, in the octal; Again the 7th, add standard substance diluent 50 μ l respectively in the octal; Behind the mixing from the 7th, get 50 μ l respectively the octal and be added in the 9th, the tenth hole; Add standard substance diluent 50 μ l again in the 90 hole respectively, from the 90 hole, respectively get 50 μ l behind the mixing and discard.(each hole application of sample amount of dilution back all is 50 μ l, and concentration is respectively 900pg/ml, 600pg/ml, and 300pg/ml, 150pg/ml, 75pg/ml).

B: application of sample: establish blank well (the blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole respectively.Encapsulate at enzyme mark and to add sample diluent 40 μ l on the plate in the testing sample hole earlier, and then add testing sample 10 μ l (the final extent of dilution of sample is 5 times), application of sample is added on bottom, enzyme plate hole with sample, does not touch hole wall as far as possible, rocks mixing gently.

C: incubation: with rearmounted 37 ℃ of incubations of shrouding film shrouding 30 minutes.

D: dosing: doubly the dilution back is subsequent use with zero(ppm) water 30 (48T 20 times) for (48T 20 times) times concentrated cleaning solution with 30.

E: washing: carefully take the shrouding film off, discard liquid, dry, washings is filled it up with in every hole, leaves standstill after 30 seconds to discard, and so repeats 5 times, claps and does.

F: enzyme-added: every hole adds enzyme marking reagent 50 μ l, except the blank well.

G: incubation: operate same C.

H: washing: operate same E.

I: colour developing: every hole adds developer A50 μ l earlier, adds developer B50 μ l again, the light shaking mixing, and 37 ℃ of lucifuges developed the color 15 minutes.

J: stop: every hole adds stop buffer 50 μ l, termination reaction (this moment, blue upright commentaries on classics was yellow).

K: measure: with blank air-conditioning zero, the 450nm wavelength is measured the absorbancy (OD value) in each hole in regular turn.Mensuration should be after adding stop buffer be carried out with interior in 15 minutes.

L: interpretation of result: the concentration with standard substance is X-coordinate, and the OD value is an ordinate zou, on graph paper, draws typical curve, and OD value is per sample found corresponding concentration by typical curve; Multiply by extension rate again; Or the linear regression equation that calculates typical curve with the concentration and the OD value of standard substance, the OD value substitution equation with sample calculates sample concentration, multiply by extension rate again, is the actual concentrations of sample.

The mean concns that the result obtains IFN-in the silkworm blood sample is 100 mcg/ml silkworm hemolymphs, is equivalent to 8,000,000 IU/ milliliter silkworm hemolymphs approximately.

1.8.2.2 the ELISA of pig gamma interferon detects in the associating expression product

Adopt DZ046 pig gamma interferon (IFN-γ) the ELISA test kit of Beijing winter song company that the IFN-content in the silkworm blood sample is detected, detection method is with reference to above-mentioned.The mean concns that the result obtains IFN-in the silkworm blood sample is 90 mcg/ml silkworm hemolymphs, is equivalent to 6,000,000 IU/ milliliter silkworm hemolymphs approximately.

1.8.3 cytopathic-effect inhibition assay detects tiring of associating expression product

The cytopathic-effect inhibition assay (Dianzani F etc., 1989) that provides according to " Chinese biological goods rules " detects tiring of porcine alpha-IFN and IFN-associating expression product, and detection method is following:

Get 96 hole enzyme plates, every hole adds each 50 μ l of above-mentioned silkworm blood sample of 2 times of serial dilutions earlier, and adding concentration then is 2.5 * 10 ²～3.5 * 10 ³The Wish cell suspension of individual/hole (growing 24～48 hours in the 96 porocyte culture plates) is put into and is contained 5%CO ₂37 ℃ of incubators in after 11～16 hours, discard supernatant, the VSV with the KMEM nutrient solution dilution that contains 3% calf serum attacks (100TCID then ₅₀) and place and contain 5%CO ₂37 ℃ of incubators in cultivate 24～30 hours after; Discard nutrient solution and add 50 μ l violet staining liquid, discard staining fluid after 30 minutes, wash down with tap water; Blot every hole, back with paper and add the destainer of 100 μ l, be placed in 5 minutes and measure its absorption value on the ELIASA in 570 wavelength.Calculate the IU value of tiring of associating expression product sample with the quantitative response parallel method.

The detected result demonstration is on average tired and is about 2,000 ten thousand IU/ milliliter silkworm hemolymphs.

The coexpression of embodiment 2 porcine alpha-IFNs (SIFNA) and IFN-(SIFNG)

1 experimental technique

The coexpression site of porcine alpha-IFN and IFN-is respectively polyhedrosis gene site (expressing SIFNA) and the P10 gene locus (expressing SIFNG) on the ReBm-dcc-d1629.

SIFNA is through the polyhedrosis gene site on the baculovirus shuttle plasmid ReBm-dcc-d1629-d10 that recombinates by the pVL1393 carrier (construction process of ReBm-dcc-d1629 has been open in detail in CN102286534A (denomination of invention is: express insect bio-reactor and the construction process and the application of many foreign genes) the application for a patent for invention file at publication number, its microbial preservation number be CGMCC No.4914); The recombinate method of P10 gene locus of SIFNG then is to rely on the EGFP marker gene to operate in intestinal bacteria through reverse method for screening to recombinate.

1.1 the structure of reorganization universal support pSK-RE

According to the document of having delivered be published in the data on the GenBank, the type that is enhanced green fluorescent protein (EGFP) gene order (GenBank:BAL72152.1), synthetic this section sequence is used for follow-up forward and reverse screening.Goal gene is also transformed among entering intestinal bacteria TOP10 or the DH10B in the entering BmBacmid that recombinates jointly together with the EGFP gene clone, and the clone of successful reorganization can send green fluorescence under exciting light.According to pertinent literature (Kirill A.Datsenko and Barry L.Wanner; 2000) add the FRT sequence at EGFP sequence two ends; The effect of this sequence is at pCP20 plasmid (Depart of MCD biology 830KBT; Under the effect of the Flp restriction endonuclease that carries two sections FRT sequence intermediary are comprised that partly EGFP gene inscribe eliminates Yale University), make the green fluorescence just screening disappear and be used for reverse screening, synthetic the EGFP gene RE sequence that contains the FRT sequence to two ends; And adding that at two ends Hind III restriction enzyme site and Kpn I restriction enzyme site are used for the clone, sequence is seen SEQIDNo.4.

1.1.1RE the enzyme switchback of sequence and pSK carrier is received

Respectively RE sequence and pSK carrier are carried out Hind III (Buffer E) enzyme and cut with Kpn I (Buffer J) enzyme and cut, glass milk method reclaims the RE fragment.

1.1.2RE sequence connects pSK carrier and transformed into escherichia coli TOP10 competent cell

With the T4 ligase enzyme with RE sequence and the pSK carrier (Biovector Co., the LTD that cut with enzyme; Article No.: Biovector008) carry out ligation, transform and get in the intestinal bacteria TOP10 heat shock competent cell overnight cultures on flat board with Ampicillin resistance.

1.1.3 the acquisition of positive colony and evaluation

Bacterium colony on the picking resistant panel carries out cultivating in the liquid nutrient medium, then it is carried out quick method of cell disruption Preliminary detection and goes out positive colony.The plasmid of the positive colony bacterial strain that the extraction Preliminary detection goes out checks order, and obtains containing the reorganization pSK carrier (pSK-RE) of RE sequence after checking is correct, is used for the reorganization screening of shuttle plasmid.

1.2 pig gamma interferon (SIFNG) is connected in the pSK-RE carrier

Again design the pig gamma interferon that obtains among primer (RSIFNG-F and RSIFNG-R) the amplification embodiment 1 its upstream and downstream restriction enzyme site is revised as XbaI and BamHI; The PCR product is connected in the pMD-18T carrier order-checking to carry out enzyme with above-mentioned two kinds of enzymes respectively after correct and cuts; Reclaim the purpose fragment, be connected and obtain to contain the recombinant vectors pSK-RESG of pig gamma interferon with the pSK-RE carrier of cutting with same enzyme.

1.3 pig gamma interferon is recombinated on the ReBm-dcc-d1629-d10 shuttle plasmid

1.3.1 contain the competent preparation of electric shock of red recombinase and ReBm-dcc-d1629-d10 plasmid

The red recombinase gene is present on the PKD46 plasmid; RepA101 (ts) gene is arranged on this plasmid; Cultivation can be lost plasmid under 37 ℃ of conditions, and the intestinal bacteria that contain the PKD46 plasmid are here cultivated under 30 ℃ of conditions, and the red recombinase gene is to induce down at L-arabinose to express; Therefore, the sharp competent preparation process of this electricity is following:

(1) with sterilization toothpick picking mono-clonal bacterium colony, be inoculated in the 4ml LB liquid nutrient medium 37 ℃, 250rpm, wave and culture spends the night.

(2) above-mentioned bacterium liquid was inoculated in the 400ml LB liquid nutrient medium with 1: 100, and 37 ℃, 250rpm, the about 1.5-2h of wave and culture reaches about 0.3 to bacterium liquid OD600 value, and adding L-arabinose to final concentration is 10mM.

(3) 37 ℃, 250rpm, inducing culture 1h.

(4) with bacterium liquid at precooling 5min on ice, after transfer in the Centrifuge Cup of 400ml precooling, 4 ℃, 7000rpm, 1min.

(5) abandon supernatant, add a small amount of ddH in the Centrifuge Cup ₂O with the piping and druming of pipettor head, suspends deposition, again with ddH ₂O adds to about 400ml, and 4 ℃, 9,000rpm, 1min.

(6) abandon supernatant, add a small amount of aqua sterilisa, resuspended thalline is again with ddH ₂O adds to about 400ml, and 4 ℃, 10,000rpm, 1min.

(7) abandon supernatant, add 10% glycerine of a small amount of precooling, resuspended thalline is added 10% glycerine again to 40ml, and 4 ℃ 11,000rpm, 1.5min.

(8) abandon supernatant, add the glycerine of 1ml 10%, the deposition that suspends, the 80ul/ pipe is sub-packed in the centrifuge tube of 1.5ml.These intestinal bacteria contain the BmBacmid plasmid in advance, have obtained needed electric shock competence.

The entering shuttle vectors 1.3.2 pig gamma interferon gene and screening-gene EGFP recombinate simultaneously

With design primer RP10SK-S, RP10SK-X, the primer outside is used for homologous recombination for the p10 dna homolog arm about 50bp respectively, and the inboard is used for amplifying target genes and marker gene for the amplimer sequence about 20bp.

With above-mentioned two pairs of primers pSK-RE is carried out pcr amplification, get in the above-mentioned competence that contains the Red recombinase reclaiming the product electric shock.

Electricity swashs conversion process reference relevant document (Joseph et al., the molecular cloning experiment guide third edition, 2002):

That on resistant panel, can grow can tentatively be decided to be recombinant bacterial strain, and the bacterium colony of green fluorescence is sent in observation when excitation wavelength Em.=510 under fluorescent microscope, tentatively is decided to be the bacterial strain that contains recombinant plasmid (BmBacmid-SIFNG).

1.3.3EGFP knocking out of screening sequence

The coli strain preparation that will contain above-mentioned recombinant plasmid (BmBacmid-SIFNG) becomes the heat shock competence, method reference implementation example 1; PCP20 plasmid (Depart ofMCD biology 830KBT, Yale University) heat shock is transformed in the competence.The pCP20 plasmid can be expressed the Flp restriction endonuclease under 42 ℃ culture condition; So the intestinal bacteria that will contain pCP20 plasmid and BmBacmid-SIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivated 2 hours, the EGFP gene above the recombinant plasmid is excised by inscribe.Intestinal bacteria behind the inducing culture are coated on incubated overnight on the resistant panel, grow bacterium and fall behind in that to pick out the bacterium colony that does not have green fluorescence under the above-mentioned fluorescence excitation optical condition be the recombinant plasmid clone Bm-SIFNG that knocks out the EGFP gene order.

1.3.4 the detection of recombinant plasmid Bm-SIFNG

Design upstream and downstream and gene inboard that two pairs of primers lay respectively at two ends homologous recombination arm; Homology arm upstream and downstream primer is respectively JRP10-F and JRP10-R, and the inboard primer of SIFNG gene is respectively JIFNG-SR and JIFNG-XF, and using JP10-F and JSIFNG-SR respectively is a pair of primer; JSIFNG-XF and JRP10-R are that a pair of primer carries out the detection of bacterium liquid fast PCR to above-mentioned positive colony; Obtain predicting the fragment of size, further the plasmid (Bm-SIFNG) after obtaining recombinating is identified in order-checking, and the PCR product is reclaimed and checks order; Sequencing primer gets final product with amplimer, and the result sees Fig. 2.

Two figure are respectively the PCR detection case of SIFNG upstream and downstream about from Fig. 2, and in the green frame is the sequence in the SIFNG gene, and in the red frame is the sequence of P10 gene among the Bmbacmid.Can draw SIFNG from above-mentioned analysis and successfully recombinate above the Bmbacmid shuttle plasmid, obtain detecting correct recombinant plasmid Bm-SIFNG.

1.4 porcine alpha-IFN gene is connected on the pVL1393 carrier

Detailed method changes the pVL1393-IRES carrier into pVL1393 and gets final product with reference to described in the embodiment 1, obtains pVL1393-SIFNA.

1.5 porcine alpha-IFN gene is recombinated with Bm-SIFNG cotransfection bombyx mori cell

With the pVL1393-SIFNA plasmid with Bm-SIFNG with liposome embedded cotransfection bombyx mori cell.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned Bm-SIFNG, pVL1393-SIFNA DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%; Seal; Cultivate 4～5d for 27 ℃, after floating to cell, collect screening and expression that supernatant is used for recombinant virus.

1.6 the screening of recombinant virus BmSAG, purifying and amplification

Inoculate an amount of Bm (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths, be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ L TC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 25 spots all is the coexpression recombinant virus that contains porcine alpha-IFN and porcine beta interferon gene, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of 100pL recombinant virus infection normal growth respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations in order to injection, obtained the porcine alpha-IFN of purifying and the recombinant virus BmSAG of IFN-coexpression.

1.7 the coexpression and the detection of recombinant virus BmSAG α and IFN-in silkworm

1.7.1 the expression of recombinant virus BmSAG

Above-mentioned 25 recombinant virus nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain porcine alpha-IFN and the IFN-associating expression product in silkworm.

1.7.2 the ELISA of expression product detects

Content to porcine alpha-IFN in the above-mentioned silkworm blood and pig gamma interferon carries out the ELISA detection; Method and operation steps are with reference to embodiment 1; The concentration that obtains porcine alpha-IFN in the silkworm blood is 140 mcg/ml silkworm hemolymphs, is equivalent to about 1,000 ten thousand IU/ milliliter silkworm hemolymphs; The concentration of pig gamma interferon is 120 mcg/ml silkworm hemolymphs, is equivalent to about 9,000,000 IU/ milliliter silkworm hemolymphs.

1.7.3 the activity of expression product detects

With cytopathic-effect inhibition assay the product of porcine alpha-IFN and pig gamma interferon coexpression in the above-mentioned silkworm blood being carried out activity detects; Working method and step be with reference to embodiment 1, obtains in this silkworm blood sample the Interferon, rabbit MV of tiring at last and reach 2,500 ten thousand IU/ milliliter silkworm hemolymphs.

Embodiment 3 canine interferon alphas and IFN-unite expression

1 experimental technique

At first to obtain the sequence of canine interferon alpha (abbreviating CIFNA as behind the Canis lupus familiaris IFNA) and IFN-(abbreviating CIFNG as behind the Canis lupus familiaris IFNG); Through transforming the codon that the codon in the Interferon, rabbit sequence of wild-type is replaced with preference in the silkworm baculovirus; With the synthetic rear clone of two sections sequences and be connected on the pVL1393 carrier; Link to each other through the IRES sequences in the middle of two Interferon, rabbit, method that then can be through homologous recombination with the process of BmNPV DNA cotransfection in the baculovirus that obtains recombinating be used for infected silkworm and needing obtain expressed proteins.

1.1 the transformation of canine interferon alpha and dog IFN-sequence and synthetic

Consult domestic and international in recent years document about canine interferon alpha and dog IFN-report; Sequence according to the synthetic two kinds of Interferon, rabbit of the sequence among the NCBI; The sequence number of institute's basis is respectively that GenBank:HQ680864.1 (CIFNA) and NCBI Reference Sequence:NM_001003174.1 (CIFNG) transform; According to the gene order that obtains and as the silkworm of bio-reactor and the characteristics of Bombyx mori nuclear polyhydrosis virus two gene orders are carried out GC content transformation (GC Content Adjustment) in the sub-preference property transformation of translation cipher (Codon usage bias adjustment), the sequence, used the removal of restriction endonuclease sites etc. always; Obtain the improved sequence of two Interferon, rabbit; In addition according to will with carrier pVL1393 sequence on the sequence that obtains behind two gene optimizations of polyclone restriction enzyme site design be respectively CIFNA (SEQ ID No.5) and CIFNG (SEQ ID No.6); CIFNA upstream and downstream restriction enzyme site is BamH I and Xba I, and CIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.

1.2 canine interferon alpha (CIFNA) is connected on the pVL1393-IRES carrier

1.2.1CIFNA receive with the enzyme switchback of pVL1393-IRES

To CIFNA with pVL1393-IRES carries out BamH I (Buffer E) respectively and Xba I (Buffer E) enzyme is cut, glass milk method reclaims the endonuclease bamhi that obtains CIFNA and pVL1393-IRES, directly uses the prepared pVL1393-IRES plasmid of embodiment 1.

1.2.2CIFNA upward also transformed into escherichia coli TOP10 competent cell linked system and method will connect product and be transformed in the TOP10 competent cell with embodiment 1 to be connected to pVL1393-IRES.

1.2.3 the detection of recombinant plasmid

Picking transforms the positive bacterium colony of back antibiotic-screening, slightly carries picking out recombinant clone and extracting plasmid and carry out enzyme and cut evaluation, and method is seen embodiment 1, detects to obtain plasmid pVL1393-CA-IRES after correct.

1.3 dog IFN-(CIFNG) is connected on the pVL1393-CA-IRES carrier

The plasmid and the pVL1393-CA-IRES carrier that will contain CIFNG carry out EcoR I (BufferH) and Bgl II (BufferD) double digestion; Because the endonuclease reaction damping fluid is different; So carry out an endonuclease reaction earlier, detect enzyme after 1-2 hour and cut full postprecipitation plasmid, carry out second kind of enzyme and cut.

Enzyme is cut evaluation after filtering out positive colony, and the sequence of three fragment genes that add in the pVL1393 carrier polyclone restriction enzyme site is measured in the plasmid that the obtains detection of checking order, and obtains pVL1393-CAIG after correct.

1.4 interferon gene and BmNPV DNA cotransfection bombyx mori cell carry out the virus reorganization

With the pVL1393-CAIG plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-SAIG DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%; Seal; Cultivate 4～5d for 27 ℃, after floating to cell, collect screening and expression that supernatant is used for recombinant virus.

1.5 the screening of recombinant virus, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths; Be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, add X-gal and make its final concentration reach 150 μ g/mL, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ L TC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 37 spots all is the recombinant virus Bm-CAIG that contains canine interferon alpha and dog IFN-gene, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations in order to injection, obtained uniting of canine interferon alpha and IFN-expressing carrier B m-CAIG.

1.6 canine interferon alpha and the uniting of IFN-of recombinant virus Bm-CAIG in silkworm expresses and the product detection

1.6.1 recombinant virus Bm-CAIG unites expression

Above-mentioned 37 recombinant virus Bm-CAIG nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain canine interferon alpha and the IFN-associating expression product in silkworm.

1.6.2 the ELISA of associating expression product detects

Dog IFN-(IFN-γ) the ELISA test kit that DQ003 canine interferon alpha (IFN-α) ELISA test kit that provides with Beijing winter song Bioisystech Co., Ltd and the emerging biotechnology of Shanghai section provide detects above-mentioned silkworm blood sample; Working method and step are with reference to embodiment 1; The concentration that obtains canine interferon alpha in the associating expression product is 80 mcg/ml silkworm hemolymphs; Be equivalent to 7,000,000 IU/ milliliter silkworm hemolymphs approximately; The concentration of dog IFN-is 70 mcg/ml silkworm hemolymphs, is equivalent to 5,000,000 IU/ milliliter silkworm hemolymphs approximately.

1.6.3 the activity of associating expression product detects

With cytopathic-effect inhibition assay the product of canine interferon alpha in the above-mentioned silkworm blood and dog IFN-coexpression being carried out activity detects; Working method and step be with reference to embodiment 1, obtains in this silkworm blood sample the Interferon, rabbit MV of tiring at last and reach 1,500 ten thousand IU/ milliliter silkworm hemolymphs.

The coexpression of embodiment 4 canine interferon alphas (CIFNA) and IFN-(CIFNG)

1 experimental technique

The coexpression site of canine interferon alpha and IFN-is respectively polyhedrosis gene site (expressing CIFNA) and the P10 gene locus (expressing CIFNG) on the ReBm-dcc-d1629.CIFNA is through the polyhedrosis gene site on the ReBm-dcc-d1629 that recombinates by the pVL1393 carrier; Method is with embodiment 1, and the recombinate method of P10 gene locus of CIFNG then is to rely on the EGFP marker gene to operate in intestinal bacteria through reverse method for screening to recombinate.

1.1 dog IFN-(CIFNG) is connected in the pSK-RE carrier

Again design the dog IFN-that obtains among primer (RCIFNG-F and RCIFNG-R) the amplification embodiment 3; Its upstream and downstream restriction enzyme site is revised as XbaI and BamHI; The PCR product is connected in the pMD-18T carrier order-checking to carry out enzyme with above-mentioned two kinds of enzymes respectively after correct and cuts; Reclaim the purpose fragment, be connected and obtain to contain the reorganization pSK-RE carrier of dog IFN-with the pSK-RE carrier of cutting with same enzyme.

The entering shuttle vectors 1.2 dog IFN-gene and screening-gene EGFP gene are recombinated simultaneously

With two among the embodiment 2 general homology arm amplimer RP10SK-S, RP10SK-X amplifying target genes and reverse screening-gene; Contain in the intestinal bacteria Top10 competence of Red recombinase and Bmbacmid shuttle plasmid reclaiming product electric shock conversion entering; Screen the BmBacmid of obtained recombinating dog IFN-gene and EGFP gene, concrete experimental technique is with embodiment 2.

1.3 the detection of recombinant plasmid knocks out with reverse screening-gene

Method with embodiment 2; Design upstream and downstream and gene inboard that two pairs of primers lay respectively at two ends homologous recombination arm; Use JRP10-F and JCIFNG-SR to detect primer, JCIFNG-XF and JRP10-R respectively and detect primer as the downstream homology arm as upper reaches homology arm; Above-mentioned positive colony is carried out bacterium liquid fast PCR detect, obtain predicting the fragment of size, further the plasmid (BmBacmid-CIFNG) after obtaining recombinating is identified in order-checking.

The coli strain preparation that will contain above-mentioned recombinant plasmid (BmBacmid-CIFNG) becomes the heat shock competence, and method reference implementation example 1 transforms the heat shock of pCP20 plasmid in the competence.The pCP20 plasmid can be expressed the Flp restriction endonuclease under 42 ℃ culture condition; So the intestinal bacteria that will contain pCP20 plasmid and BmBacmid-CIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivated 2 hours, the reverse screening-gene above the recombinant plasmid is excised by inscribe.Intestinal bacteria behind the inducing culture are coated on incubated overnight on the flat board that contains streptomycin resistance, the clone who obtains is the Bm-CIFNG that cuts away screening-gene.

1.4 dog alpha interferon gene is connected on the pVL1393 carrier

Detailed method changes the pVL1393-IRES carrier into pVL1393 and gets final product with reference to described in the embodiment 1, obtains pVL1393-CIFNA.

1.5 dog alpha interferon gene and Bm-CIFNG cotransfection bombyx mori cell are recombinated

With the pVL1393-CIFNA plasmid with Bm-CIFNG with liposome embedded cotransfection bombyx mori cell, cultural method is seen embodiment 1, final primary dcreening operation obtains coexpression recombinant viral vector BmCAG.

1.6 the screening of recombinant virus BmCAG, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths, be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ LTC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 30 spots all is the coexpression recombinant virus BmCAG that contains canine interferon alpha and dog IFN-, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations in order to injection, obtained recombinant virus BmCAG.

1.7 the coexpression and the product of recombinant virus BmCAG canine interferon alpha and IFN-in silkworm detect

1.7.1 the expression of recombinant virus BmCAG

Above-mentioned 30 recombinant virus nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain canine interferon alpha and the dog IFN-coexpression product in silkworm.

1.7.2 the ELISA of expression product detects

Respectively the above-mentioned silkworm blood that contains the coexpression product being carried out ELISA with the test kit among the embodiment 3 detects; Working method and step are with reference to embodiment 3; The concentration that obtains the canine interferon alpha in the above-mentioned silkworm blood is 100 mcg/ml silkworm hemolymphs, is equivalent to 8,000,000 IU/ milliliter silkworm hemolymphs approximately; The concentration of dog IFN-is 80 mcg/ml silkworm hemolymphs, is equivalent to 6,000,000 IU/ milliliter silkworm hemolymphs approximately.

1.7.3 the activity of expression product detects

Adopt cytopathic-effect inhibition assay that the Interferon, rabbit in the above-mentioned silkworm blood sample is tired and detect, working method and step be with reference to embodiment 1, and obtaining finally in the above-mentioned silkworm blood that dog interferon on average tires is 2,200 ten thousand IU/ milliliter silkworm hemolymphs.Embodiment 5 chicken alpha-interferons and IFN-unite expression

1 experimental technique

At first obtain the sequence of chicken alpha-interferon (abbreviating GIFNA as behind the Gallus gallus IFNA) and IFN-(abbreviating GIFNG as behind the Gallus gallus IFNG); Through transforming the codon that the codon in the Interferon, rabbit sequence of wild-type is replaced with preference in the silkworm baculovirus; With the synthetic rear clone of two sections sequences and be connected on the pVL1393 carrier; Link to each other through the IRES sequences in the middle of two Interferon, rabbit, method that then can be through homologous recombination with the process of BmNPV cotransfection in the baculovirus that obtains recombinating be used for infected silkworm and needing obtain expressed proteins.

1.1 the transformation of chicken alpha-interferon and chicken IFN-sequence and synthetic

Consult domestic and international in recent years document about chicken alpha-interferon and chicken IFN-report; Sequence according to the synthetic two kinds of Interferon, rabbit of the sequence among the NCBI; The sequence number of institute's basis is respectively that GIFNA is NCBI Reference Sequence:NM_205427.1 and GenBank:AM049251.1; GIFNG is that NCBI Reference Sequence:NM_205149.1 transforms; According to the gene order that obtains and as the silkworm of bio-reactor and the characteristics of Bombyx mori nuclear polyhydrosis virus two gene orders are carried out GC content transformation (GC Content Adjustment) in the sub-preference property transformation of translation cipher (Codon usage bias adjustment), the sequence, used the removal of restriction endonuclease sites etc. always; Obtain the improved sequence of two Interferon, rabbit; In addition according to will with carrier pVL1393 sequence on the sequence that obtains behind two gene optimizations of polyclone restriction enzyme site design ask GIFNA (SEQIDNo.7) and GIFNG (SEQIDNo.8) respectively; GIFNA upstream and downstream restriction enzyme site is BamH I and XbaI, and GIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.

1.2 chicken alpha-interferon (GIFNA) is connected on the pVL1393-IRES carrier

1.2.1GIFNA receive with the enzyme switchback of pVL1393-IRES

To GIFNA with pVL1393-IRES carries out BamH I (Buffer E) respectively and Xba I (Buffer E) enzyme is cut; Method is seen embodiment 1; Glass milk method reclaims the endonuclease bamhi that obtains GIFNA and pVL1393-IRES, directly uses the pVL1393-IRES plasmid of preparation among the embodiment 1.

1.2.2GIFNA being connected to pVL1393-IRES goes up and transformed into escherichia coli TOP10 competent cell

Linked system and method are seen embodiment 1, will connect product and be transformed in the TOP10 competent cell.

1.3 the detection of recombinant plasmid

Picking transforms the positive bacterium colony of back antibiotic-screening, slightly carries picking out recombinant clone and extracting plasmid and carry out enzyme and cut evaluation, and method is seen embodiment 1.Obtain plasmid pVL1393-GA-IRES after detecting correctly.

1.4 chicken IFN-(GIFNG) is connected on the pVL1393-GA-IRES carrier

The plasmid and the pVL1393-GA-IRES carrier that will contain GIFNG carry out EcoR I (BufferH) and Bgl II (BufferD) double digestion; Because the endonuclease reaction damping fluid is different; So carry out an endonuclease reaction earlier, detect enzyme after 1-2 hour and cut full postprecipitation plasmid, carry out second kind of enzyme and cut.

Enzyme is cut evaluation after filtering out positive colony, and the sequence of three fragment genes that add in the pVL1393 carrier polyclone restriction enzyme site is measured in the plasmid that the obtains detection of checking order, and obtains pVL1393-GAIG after correct.

1.5 interferon gene and BmNPV DNA cotransfection bombyx mori cell carry out the virus reorganization

With the pVL1393-GAIG plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-SAIG DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃; Add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%, seals; After 27 ℃ of cultivation 4～5d float to cell, collect screening and expression that supernatant is used for recombinant virus.

1.6 the screening of recombinant virus Bm-GAIG, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths; Be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, add X-gal and make its final concentration reach 150 μ g/mL, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ L TC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 28 spots all is the recombinant virus Bm-GAIG that contains chicken alpha-interferon and chicken IFN-gene, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations were in order to injection.

1.7 recombinant virus Bm-GAIG uniting of chicken alpha-interferon and IFN-in silkworm expresses and product detects

1.7.1 the expression of recombinant virus

Above-mentioned 28 recombinant virus Bm-GAIG nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain porcine alpha-IFN and the IFN-associating expression product in silkworm.

1.7.2 the ELISA of expression product detects

DJ008 chicken alpha-interferon (IFN-α) the ELISA test kit that provides with Beijing winter song Bioisystech Co., Ltd and DJ011 chicken IFN-(IFN-γ) ELISA test kit are respectively to the IFN-in the above-mentioned silkworm blood sample detection associating expression product and the concentration of IFN-; Working method and step are seen embodiment 1; The concentration that is respectively IFN-is 90 mcg/ml silkworm hemolymphs, is equivalent to 8,000,000 IU/ milliliter silkworm hemolymphs approximately; The concentration of IFN-is 70 mcg/ml silkworm hemolymphs, is equivalent to 5,000,000 IU/ milliliter silkworm hemolymphs approximately.

1.7.3 the activity of expression product detects

Adopt cytopathic-effect inhibition assay to detect tiring of chicken interferon in the above-mentioned silkworm blood, working method and step are with reference to embodiment 1, and the tiring of chicken interferon that finally obtains the associating expression is 1,800 ten thousand IU/ milliliter silkworm hemolymphs.

The coexpression of embodiment 6 chicken alpha-interferons (GIFNA) and IFN-(GIFNG)

1 experimental technique

The coexpression site of chicken alpha-interferon and IFN-is respectively polyhedrosis gene site (expressing GIFNA) and the P10 gene locus (expressing GIFNG) on the ReBm-dcc-d1629-d10 shuttle vectors.

GIFNA is through the polyhedrosis gene site on the ReBm-dcc-d1629-d10 shuttle vectors of recombinating by the pVL1393 carrier; Method similar embodiment 1, the recombinate method of P10 gene locus of GIFNG then is to rely on the EGFP marker gene to operate in intestinal bacteria through reverse method for screening to recombinate.

1.1 chicken IFN-(GIFNG) is connected in the pSK-RE carrier

Again design the pig gamma interferon that obtains among primer (RGIFNG-F and RGIFNG-R) the amplification embodiment 1 its upstream and downstream restriction enzyme site is revised as XbaI and BamHI; The PCR product is connected in the pMD-18T carrier order-checking to carry out enzyme with above-mentioned two kinds of enzymes respectively after correct and cuts; Reclaim the purpose fragment, be connected and obtain to contain the reorganization pSK-RE carrier of pig gamma interferon with the pSK-RE carrier of cutting with same enzyme.

1.2 the chicken IFN-is recombinated on the ReBm-dcc-d1629-d10 shuttle vectors

1.2.1 contain competent preparation manipulation method of the electric shock of red recombinase and ReBm-dcc-d1629-d10 plasmid and step with reference to embodiment 2.

The entering shuttle vectors 1.2.2 chicken IFN-gene and reverse screening-gene EGFP gene are recombinated simultaneously

With design primer RP10SK-S, RP10SK-X, the primer outside is used for homologous recombination for the p10 dna homolog arm about 50bp respectively, and the inboard is used for amplifying target genes and reverse screening-gene for the amplimer sequence about 20bp.With above-mentioned two pairs of primers pSK-RE is carried out pcr amplification, will reclaim the product electric shock and get in the above-mentioned competence.Sharp conversion process working method of electricity and step are with reference to embodiment 2.

1.2.3 the detection of recombinant plasmid

(JRP10-F and JGIFNG-SR are the upstream detection primer to design two pairs of primers; JGIFNG-XF and JRP10-R are the detected downstream primer) upstream and downstream and the gene that lay respectively at two ends homologous recombination arm be inboard; Above-mentioned positive colony is carried out bacterium liquid fast PCR to be detected; Obtain predicting the fragment of size, further the plasmid (BmBacmid-GIFNG) after obtaining recombinating is identified in order-checking.

1.24 oppositely screen pounding out of sequence

The coli strain preparation that will contain above-mentioned recombinant plasmid (BmBacmid-GIFNG) becomes the heat shock competence, and method reference implementation example 1 transforms the heat shock of pCP20 plasmid in the competence.The pCP20 plasmid can be expressed the Flp restriction endonuclease under 42 ℃ culture condition; So the intestinal bacteria that will contain pCP20 plasmid and BmBacmid-GIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivated 2 hours; Screening-gene above the recombinant plasmid is excised by inscribe, filters out the recombinant plasmid Bm-GIFNG of the GIFNG that recombinated.

1.3 chicken alpha interferon gene is connected on the pVL1393 carrier

Detailed method changes the pVL1393-IRES carrier into pVL1393 and gets final product with reference to described in the embodiment 1, obtains pVL1393-GIFNA.

1.4 chicken alpha interferon gene and Bm-GIFNG cotransfection bombyx mori cell are recombinated

With the pVL1393-GIFNA plasmid with Bm-GIFNG with liposome embedded cotransfection bombyx mori cell.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmBacmid-GIFNG, pVL1393-GIFNA DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%; Seal; Cultivate 4～5d for 27 ℃, after floating to cell, collect screening and expression that supernatant is used for recombinant virus.

1.5 the screening of recombinant virus BmGAG, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths, be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ LTC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 25 spots all is the coexpression recombinant virus BmGAG that contains chicken alpha-interferon and chicken IFN-, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations were in order to injection.

1.6 the coexpression and the detection of recombinant virus BmGAG chicken alpha-interferon and IFN-in silkworm

1.6.1 the expression of recombinant virus BmGAG

Above-mentioned 25 recombinant virus nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain chicken alpha-interferon and the IFN-coexpression product in silkworm.

1.6.2 the ELISA of expression product detects

Respectively the above-mentioned silkworm blood that contains the coexpression product being carried out ELISA with the test kit among the embodiment 3 detects; Working method and step are with reference to embodiment 3; The concentration that obtains the chicken alpha-interferon in the above-mentioned silkworm blood is 100 mcg/ml silkworm hemolymphs; Be equivalent to 8,000,000 IU/ milliliter silkworm hemolymphs, the concentration of chicken IFN-is 90 mcg/ml silkworm hemolymphs, is equivalent to 7,000,000 IU/ milliliter silkworm hemolymphs.

1.6.3 the activity of expression product detects

Adopt cytopathic-effect inhibition assay that the Interferon, rabbit in the above-mentioned silkworm blood sample is tired and detect, working method and step be with reference to embodiment 1, and obtaining finally in the above-mentioned silkworm blood that chicken interferon on average tires is 2,000 ten thousand IU/ milliliter silkworm hemolymphs.The expression of embodiment 7 porcine alpha-IFN two mutants

1 experimental technique

1.1 the acquisition of porcine alpha-IFN mutant gene

This experiment suddenlys change to the sequence of porcine alpha-IFN; It is obtained than higher protease resistant guaranteeing under its active situation it is transformed; Help the time that Interferon, rabbit exists in vivo, through a large amount of point mutation experiment, wherein the 64th L-glutamic acid effect of sporting Stimulina is the best; Reduced long-acting porcine alpha-IFN two mutants to the susceptibility of proteolytic ferment in blood and the tissue; Its nucleotide sequence is seen SEQ ID No.9, and coded aminoacid sequence is SEQ ID No.10, and two ends add that BamH I and Xba I restriction enzyme site are used for follow-up clone operations.

1.2 porcine alpha-IFN mutant gene (SIFNA-MUT) is cloned on the pVL1393 carrier.

Working method obtains pVL1393-SIFNA-MUT with reference to embodiment 1.

The BmNPV DNA 1.3 the cotransfection bombyx mori cell obtains recombinating.

With the pVL1393-SIFNA-MUT plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell.

Inoculate about 0.5～1 * 10 ⁶The Bm-N cell is in 15cm ²In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary, add the 1mL serum free medium again with serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-SIFNA-MUT DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that 4～6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%; Seal; Cultivate 4～5d for 27 ℃, after floating to cell, collect screening and expression that supernatant is used for recombinant virus.

1.4 the screening of recombinant virus, purifying and amplification

Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, inhale and remove substratum, with the cotransfection supernatant by 10 ^-3～10 ^-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h; 2% low melting point sepharose is melted in 60 ℃ of water-baths, be chilled to 40 ℃ of 2X TC-100 substratum (containing 20%FBS) and mix, glue is slowly poured into along little dish edge with 40 ℃ of preheatings of equal-volume warp; Solidify the back and seal, be inverted for 27 ℃ and cultivate 4～7d with Parafilm.Microscopically is chosen no polyhedron recombinant virus plaque, gets the recombinant virus spot with the Tip choicest in the super clean bench, is dissolved in the 400 μ L TC-100 substratum; Placing 4h for 4 ℃ discharges virus particle; Get 100 μ L virus liquid inductance and dye the cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare virus genom DNA fast by alkaline denaturation; Carry out the pcr amplification analysis; The virus of finding 29 spots all is the recombinant virus Bm-SAM that contains the porcine alpha-IFN two mutants, can amplify the segmental viral supernatant of purpose shop spot and carry out the screening first time, the final recombinant virus that obtains to contain goal gene.Get the Bm-5 attached cell of 100pL recombinant virus infection normal growth respectively, after about 5d treated that cell floats after the infection, 4 ℃ of preservations in order to injection, obtained the recombinant virus Bm-SAM that contains the porcine alpha-IFN two mutants of purifying.

1.5 the porcine alpha-IFN two mutants of recombinant virus Bm-SAM in silkworm expressed and product detects

1.5.1 the expression of recombinant virus Bm-SAM

Above-mentioned 29 recombinant virus nutrient solutions are pressed 10 ⁵The pfu/ head is injected five ages childrens silkworm, treat the silkworm morbidity after, cut foot, collect silkworm blood ,-20 ℃ are frozen, obtain the coexpression product of porcine alpha-IFN two mutants in silkworm.

1.5.2 the ELISA of expression product detects

With the porcine alpha-IFN ELISA detection kit among the embodiment 1 the above-mentioned silkworm blood that contains expression product is carried out ELISA and detect, working method and step are with reference to embodiment 1, and the concentration that obtains the porcine alpha-IFN in the above-mentioned silkworm blood is 110 mcg/ml silkworm hemolymphs.

1.5.3 the activity of expression product detects

Adopting cytopathic-effect inhibition assay that the Interferon, rabbit in the above-mentioned silkworm blood sample is tired detects; Working method and step are with reference to embodiment 1; Obtaining finally in the above-mentioned silkworm blood that porcine alpha-IFN on average tires is 1,500 ten thousand IU/ milliliter silkworm hemolymphs, and preliminary feeding animal experiment shows that the intravital transformation period improved 5 times.

< 110>expression method of animal IFN-and IFN-

<120> DQXL-0026

< 130>Biological Technology institute, Chinese Academy of Agricultural Sciences

<160> 10

<170> PatentIn version 3.5

<210> 1

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<212> DNA

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<220>

<221> CDS

<222> (1)..(582)

<400> 9

gga tcc atg gcc ccg acc tcc gct ttt ctc act gtg ctc gtg ctg ctg 48

Gly Ser Met Ala Pro Thr Ser Ala Phe Leu Thr Val Leu Val Leu Leu

1 5 10 15

tca tgt aat gct att tgt tgc ctt ggc tgc gac ctg cct cag acg cac 96

Ser Cys Asn Ala Ile Cys Cys Leu Gly Cys Asp Leu Pro Gln Thr His

20 25 30

tca tta gca cat aca aga gcg ctt cgc ctg ttg gct caa atg aga cgc 144

Ser Leu Ala His Thr Arg Ala Leu Arg Leu Leu Ala Gln Met Arg Arg

35 40 45

ata agc cct ttc tcc tgc ctc gac cac cgt agg gat ttt ggt agc cca 192

Ile Ser Pro Phe Ser Cys Leu Asp His Arg Arg Asp Phe Gly Ser Pro

50 55 60

cat caa gcc tta ggt gga aac caa gtc cag aaa gcg caa gct atg gcc 240

His Gln Ala Leu Gly Gly Asn Gln Val Gln Lys Ala Gln Ala Met Ala

65 70 75 80

ttg gta cac gag atg ctc caa cag aca ttc cag ctg ttt tcg act gaa 288

Leu Val His Glu Met Leu Gln Gln Thr Phe Gln Leu Phe Ser Thr Glu

85 90 95

gga agt gct gcc gca tgg gac gag tca ctc tta cat caa ttc tgc acc 336

Gly Ser Ala Ala Ala Trp Asp Glu Ser Leu Leu His Gln Phe Cys Thr

100 105 110

ggc ctg gac caa cag ctt aga gat ctg gaa gca tgt gtg atg cag gaa 384

Gly Leu Asp Gln Gln Leu Arg Asp Leu Glu Ala Cys Val Met Gln Glu

115 120 125

gtt ggc ctg gag ggt acc ccg ctt ctg gaa gag gat tct atc ttg gct 432

Val Gly Leu Glu Gly Thr Pro Leu Leu Glu Glu Asp Ser Ile Leu Ala

130 135 140

gtg cgt aaa tac ttt cac agg ttg acg ctc tac tta cag gag aag tca 480

Val Arg Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Ser

145 150 155 160

tat tct cct tgt gct tgg gaa att gtc cgc gcc gag gta atg cgt gtg 528

Tyr Ser Pro Cys Ala Trp Glu Ile Val Arg Ala Glu Val Met Arg Val

165 170 175

ttt agt tca agc acc aac ctg caa gac aga ctc aga aag aag gaa taa 576

Phe Ser Ser Ser Thr Asn Leu Gln Asp Arg Leu Arg Lys Lys Glu

180 185 190

tct aga 582

Ser Arg

<210> 10

<211> 191

<212> PRT

<213> artifical sequence

<400> 10

Gly Ser Met Ala Pro Thr Ser Ala Phe Leu Thr Val Leu Val Leu Leu

1 5 10 15

Ser Cys Asn Ala Ile Cys Cys Leu Gly Cys Asp Leu Pro Gln Thr His

20 25 30

Ser Leu Ala His Thr Arg Ala Leu Arg Leu Leu Ala Gln Met Arg Arg

35 40 45

Ile Ser Pro Phe Ser Cys Leu Asp His Arg Arg Asp Phe Gly Ser Pro

50 55 60

His Gln Ala Leu Gly Gly Asn Gln Val Gln Lys Ala Gln Ala Met Ala

65 70 75 80

Leu Val His Glu Met Leu Gln Gln Thr Phe Gln Leu Phe Ser Thr Glu

85 90 95

Gly Ser Ala Ala Ala Trp Asp Glu Ser Leu Leu His Gln Phe Cys Thr

100 105 110

Gly Leu Asp Gln Gln Leu Arg Asp Leu Glu Ala Cys Val Met Gln Glu

115 120 125

Val Gly Leu Glu Gly Thr Pro Leu Leu Glu Glu Asp Ser Ile Leu Ala

130 135 140

Val Arg Lys Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Ser

145 150 155 160

Tyr Ser Pro Cys Ala Trp Glu Ile Val Arg Ala Glu Val Met Arg Val

165 170 175

Phe Ser Ser Ser Thr Asn Leu Gln Asp Arg Leu Arg Lys Lys Glu

180 185 190

Claims (10)

1. the expression method of an Interferon, rabbit is characterized in that, comprising: people or homozoic IFN-and IFN-are united in the insect baculovirus bio-reactor express or coexpression.

2. according to the described expression method of claim 1; It is characterized in that; The method of people or homozoic IFN-and IFN-being united expression in the insect baculovirus bio-reactor comprises: recombinate after with people or homozoic alpha-IFN gene and IFN-gene gang on the nonessential gene locus that duplicates or infect of baculovirus (1), obtains recombinant baculovirus; (2) with recombinate shape virus infection insect host or insect cell, expression alien gene obtains IFN-and IFN-associating expressed products.

3. according to the described expression method of claim 2, it is characterized in that: through the nucleotide sequence shown in the SEQ ID No.3 people or homozoic alpha-IFN gene and IFN-gene are connected together in the step (1).

4. according to the described expression method of claim 2; It is characterized in that; In the step (1) in the following manner with the recombination of gang to the nonessential gene locus that duplicates or infect of baculovirus: the alpha-IFN gene of gang and IFN-gene clone to baculovirus transfer vector, are obtained the recombinant baculovirus transfer vector; Recombinant baculovirus transfer vector and baculovirus are recombinated in insect cell, with the alpha-IFN gene of gang and IFN-recombinate duplicating of baculovirus or the nonessential gene locus that infects on.

5. according to the described expression method of claim 4, it is characterized in that: described baculovirus transfer vector is the pVL1393 carrier; The alpha-IFN gene of gang and IFN-are recombinated on the polyhedrosis gene site of baculovirus.

6. according to the described expression method of claim 1; It is characterized in that; Described people or homozoic alpha-IFN gene and IFN-gene are carried out coexpression in the insect baculovirus bio-reactor method may further comprise the steps: (1) people or homozoic alpha-IFN gene and IFN-gene are recombinated duplicating of baculovirus respectively or two nonessential gene locuss infecting on, obtain recombinant baculovirus; (2) with recombinate shape virus infection insect host or insect cell, expression alien gene obtains the product of IFN-and IFN-coexpression.

7. according to the described expression method of claim 6; It is characterized in that; On two nonessential gene locuss of in such a way people or homozoic alpha-IFN gene and IFN-gene being recombinated duplicating of baculovirus respectively in the step (1) or infecting; Obtain recombinant baculovirus: the IFN-gene is connected with the enhanced green fluorescence protein marker gene; On the p10 gene locus with its ReBm-dcc-d1629-d10 baculovirus shuttle vectors of recombinating, the phenotypic screen through EGFP goes out recombinant vectors; The EGFP marker gene is knocked out the defective type baculovirus shuttle vectors of the IFN-that obtains to have recombinated; Alpha-IFN gene recombinated to contain in the baculovirus transfer vector of ORF1629 homology arm, obtain the recombinant baculovirus transfer vector; With the defective type baculovirus shuttle vectors and the recombinant baculovirus transfer vector cotransfection bombyx mori cell of the IFN-of having recombinated, obtain recombinant baculovirus.

8. according to claim 6 or 7 described expression methods, it is characterized in that: IFN-is recombinated on the polyhedrosis gene site of baculovirus, IFN-is recombinated on the p10 gene locus of baculovirus.

9. according to the described expression method of claim 1, it is characterized in that: described animal comprises pig, dog or chicken; Preferably, the nucleotides sequence of said porcine alpha-IFN gene is classified as shown in SEQ ID No.1 or the SEQ ID No.9, and the nucleotides sequence of pig gamma interferon is classified as shown in the SEQ ID No.2; The nucleotides sequence of canine interferon alpha is classified as shown in the SEQ ID No.5, and the nucleotides sequence of dog IFN-is classified as shown in the SEQ ID No.6; The nucleotides sequence of chicken alpha-interferon is classified as shown in the SEQID No.7, and the nucleotides sequence of chicken IFN-is classified as shown in the SEQ ID No.8.

10. porcine alpha-IFN two mutants, it is characterized in that: its aminoacid sequence is shown in the SEQ ID No.10.

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