CN102628062B - 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 interferon-alpha and IFN-γ

Technical field

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

Background technology

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

Interferon, rabbit has the people such as Isaacs to find from nineteen fifty-seven after, just be cloned always and express in various bio-reactors, such as Vandenbroeck etc. has carried out clone to the exon of pig PoIFN-γ gene in intestinal bacteria and in insect cell and (Vandenbroeck K is expressed in splicing, 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 transformation period of human IgG immunoglobulin (Ig) in human body can reach 19～21 days, the Fc fragment of IgG is used to connect and compose fusion rotein with IFN2 α, Fc fragment increases fusion protein molecule amount, avoid by glomerular filtration, simultaneously Fc fragment can with newborn Fc receptors bind, avoid fusion rotein to enter in lysosome and be degraded, thereby extend Half-life in vivo.Built the fusion gene of IFN2 α 2b and human IgG immunoglobulin Fc segments and in pichia spp with dimeric forms secreting, expressing, and have part glycosylation.After rat skin lower injection, in blood circulation, the transformation period reaches 65h, in blood more than retention time 120h, than the Half-life in vivo of common recombinant interferon, extend approximately 8 times, blood retention time extends 10 times, shown the potential applicability in clinical practice (Wang Lei that it is good, biotechnology journal, 2008:53-62).

Same principle, the α of pig, chicken, dog or the long-acting interferon of γ also can carry out amalgamation and expression with the Fc fragment of the IgG of corresponding animal or HAS sequence and its α or gamma interferon genes, and application the application's method can obtain corresponding long-acting interferon.

This bio-reactor of baculovirus expression system is that the eighties is set up.Since nineteen eighty-three people's the alpha-interferon that utilized first the high efficient expression of baculovirus expression system (Smith, Mol.Cell Biol., 3:2156-2165,1983), existing dozens of foreign gene has obtained high efficient expression.Insect bculovrirus expression vector system is one of current genetically engineered four large 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 is approaching natural product aspect biological activity, antigenicity and immunogenicity.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 greatly reduce production costs and make the scale operation of many valuable recombinant proteins to become possibility; 2. this expression system is eukaryotic expression system, and the exogenous protein of its expression can carry out posttranslational modification, makes it similar to natural product with biological activity etc. at biochemical property, and this provides assurance for the recombinant protein giving expression to has normal biologic activity.At present, baculovirus expression system, silkworm especially wherein (Bm)-silkworm baculovirus (BmNPV) expression system is one of individual expression system of eukaryote having most in the world business development value.One of four conventional large expression systems (being baculovirus, intestinal bacteria, yeast, mammalian cell expression system) of current genetically engineered have been become.

Baculovirus Gene group is larger, thus can only first foreign gene be connected on transfer vector by the way of homologous recombination, then by transfer vector and viral cotransfection cell or the restructuring of external enzymatic, thereby build rhabdovirus expression vector.General transfer vector is all containing virulent two homology arms, one or more bacilliform virus promoters (majority is got polyhedron promotor), after external source goal gene is inserted into promotor downstream, recombinate with baculovirus, obtain recombinant virus, again by recombinant virus purifying, infected insect cell or polypide, foreign gene along with virus copy and obtain and express.What when recombinant virus builds the earliest, build use is the wild virus of ring-type, after virus and transfer vector are recombinated, the polyhedron gene of virus is damaged, can not form polyhedron, the cell that infects this recombinant strain forms plaque under the microscope, by repeatedly repeating screening, can carry out purifying to recombinant virus, this screening process expends very large manpower and materials, and efficiency is very low, has limited to a great extent the application of baculovirus.

At present when expressing Interferon, rabbit with this bio-reactor of baculovirus expression system, all separately the Interferon, rabbit of a type to be recombinated after baculovirus to express in insect host or cell, exist low, the expressed recombinant interferon product of the expression efficiency defects such as low, poor stability of tiring, have much room for improvement.

Summary of the invention

Main purpose of the present invention is to provide a kind of expression method of new Interferon, rabbit, and it is high that this expression method has expression efficiency, the expressed recombinant interferon product advantages such as height, good stability of tiring.

Main purpose of the present invention is achieved through the following technical solutions:

An expression method for Interferon, rabbit, comprising: people or homozoic interferon-alpha and IFN-γ are combined and expressed or coexpression in insect baculovirus bio-reactor.

Wherein, people or homozoic alpha-IFN gene and gamma interferon genes are combined to express in insect baculovirus bio-reactor and comprise the following steps: (1) will restructuring to the nonessential gene locus that copies or infect of baculovirus, obtain recombinant baculovirus after people or homozoic alpha-IFN gene and gamma interferon genes gang; (2) with recombinate shape virus infection insect host or insect cell, expression alien gene, obtains the product that interferon-alpha and IFN-γ are combined expression.

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

Preferably, in step (1) in the following manner by the gene recombination of gang to the nonessential gene locus that copies or infect of baculovirus: the alpha-IFN gene of gang and gamma interferon genes are cloned on baculovirus transfer vector, obtain recombinant baculovirus transfer vector; Recombinant baculovirus transfer vector and baculovirus are recombinated in insect cell, the alpha-IFN gene of gang and IFN-γ are recombinated to copying of baculovirus or the nonessential gene locus that infects on; Wherein, described baculovirus transfer vector is preferably pVL1393 carrier; The nonessential gene locus that copies or infect of described baculovirus comprises the sites such as polyhedrosis gene site, p10, egt, p74, p26, is preferably polyhedrosis gene site.

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

Described people or homozoic alpha-IFN gene and gamma interferon genes carried out to coexpression in insect baculovirus bio-reactor comprise the following steps: (a) people or homozoic alpha-IFN gene and gamma interferon genes are recombinated respectively to copying of baculovirus or two nonessential gene locuss infecting on, obtain recombinant baculovirus; (b), by recombinate shape virus infection insect host or insect cell, expression alien gene, obtains the product of interferon-alpha and IFN-γ coexpression;

Preferably, on two nonessential gene locuss of in such a way people or homozoic alpha-IFN gene and gamma interferon genes being recombinated respectively to copying of baculovirus in step (a) or infecting, obtain recombinant baculovirus: the same EGFP of gamma interferon genes (enhanced green fluorescence protein) labelled protein gene is connected, by carry out the method for vitro recombination in intestinal bacteria, recombinated on the p10 gene locus of ReBm-dcc-d1629-d10 baculovirus shuttle vectors (silkworm baculovirus shuttle vectors ReBm-dcc-d1629-d10 carries out the external shuttle vectors that acquisition cannot normally enter viral life cycle that knocks out by the ORF1629 gene locus to BmNPV), phenotypic screen by EGFP goes out recombinant vectors, EGFP marker gene is knocked out, obtain defective type (ReBm-dcc-d1629-d10) the baculovirus shuttle vectors of the IFN-γ of having recombinated, alpha-IFN gene is recombinated in the baculovirus transfer vector that contains ORF1629 homology arm, obtain recombinant baculovirus transfer vector, by the defective type baculovirus shuttle vectors of the IFN-γ of having recombinated and recombinant baculovirus transfer vector cotransfection bombyx mori cell, the method for saving by inactivation obtains the recombinant baculovirus that ORF1629 gene is repaired, thereby in silkworm biological reactor, express the coexpression product that obtains two types of Interferon, rabbit.Wherein, when coexpression interferon-alpha and IFN-γ, the present invention preferably recombinates interferon-alpha on the polyhedrosis gene site of baculovirus, and IFN-γ is recombinated on the p10 gene locus of baculovirus.

In addition, the low antiviral vitality or the unvital situations that for fear of the various foreign interferon of expressing with this bio-reactor, due to expression environment difference, cause, the present invention is optimized by the gene of the various Interferon, rabbit to obtained, it is mainly the transformation of codon preference type and kozak sequence, make it can be suitable for giving expression to activated composition in applied bio-reactor as far as possible, by the optimization to interferon gene, the expression efficiency of Interferon, rabbit and tiring of expression product have been improved.

The present invention is optimized porcine alpha-IFN gene and gamma interferon genes, and the nucleotides sequence of the porcine alpha-IFN gene after optimization is classified as shown in SEQ ID No.1, and the nucleotides sequence of the pig gamma interferon gene after optimization is classified as shown in SEQID No.2, in addition, the present invention suddenlys change to the sequence of porcine alpha-IFN, in the situation that guaranteeing that it is active, it is transformed and makes it obtain higher protease resistant, be conducive to the time that Interferon, rabbit exists in vivo, through a large amount of point mutation experiments, the present invention finds, the effect that the 64th L-glutamic acid of porcine alpha-IFN is sported to glutamine is the best, effectively reduce the susceptibility to proteolytic ferment in blood and tissue, obtained long-acting porcine alpha-IFN mutant, the nucleotides sequence of this mutant of encoding is classified as shown in SEQ ID No.9, its aminoacid sequence is shown in SEQID No.10.

The present invention is optimized dog alpha interferon gene and gamma interferon genes, and the nucleotides sequence of the dog alpha interferon gene after optimization is classified as shown in SEQ ID No.5, and the nucleotides sequence of the dog gamma interferon genes after optimization is classified as shown in SEQID No.6; In addition, the present invention is also optimized chicken alpha interferon gene and gamma interferon genes, and the nucleotides sequence of the chicken alpha interferon gene after optimization is classified as shown in SEQ ID No.7, and the nucleotides sequence of the Chicken type Ⅱ interferon after optimization is classified as shown in 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), cabbage looper (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 copy or the nonessential gene locus that infects if the sites such as polyhedrosis gene, p10, egt, p74, p26 are by import successively Interferon box in baculovirus shuttle plasmid, realize with a recombinant virus while at host insect or a plurality of Interferon, rabbit of insect cell inner expression.

The inventive method is combined people or homozoic alpha-IFN gene and gamma interferon genes and is expressed or coexpression in same insect baculovirus bio-reactor, between expressed recombinant interferon product, there is synergistic function, can improve tiring and stability of recombinant interferon product, it is high that the inventive method has expression efficiency, the expression product advantages such as height, good stability of tiring.

Accompanying drawing explanation

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

PCR detected peaks figure after Fig. 2 SIFNG restructuring BmBacmid.

Embodiment

Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection 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, intestinal bacteria (Top10, DH10B) etc. are purchased from Invitrogen company or Japanese Riken BRC; PGL-3 carrier, luciferase test kit Luciferase Assay System are purchased from Promega; PMD-18T carrier and pMD-18T-simple carrier are purchased from TaKaRa company; Containing the BW25113/pKD46 of recombinase, DH5alpha/pCP20 is purchased from Depart of MCD biology 830KBT, Yale University.

2, enzyme and reagent: restriction enzyme, ligase enzyme are Promega company product.

3, biochemical reagents: IPTG, X-Gal, SDSWei Sigma company product.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 from Invitrogen company.

4, substratum: Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0); Insect cell substratum is TC-100.

5, the primer:

Primer sequence used in table 1 experiment

Embodiment 1 porcine alpha-IFN and IFN-γ combine expression

1 experimental technique

First to obtain the sequence of porcine alpha-IFN and IFN-γ, through transformation, the codon in the pig interferon sequence of wild-type is replaced with to the codon of preference in silkworm baculovirus, synthetic rear clone is also connected on pVL1393 carrier, in the middle of two Interferon, rabbit, by IRES sequence, be connected, can in the process with BmNPV DNA cotransfection, obtain for expressing the recombinant shuttle plasmid of target protein by the method for homologous recombination.

1.1. about the preparation of solution and substratum is referring to related documents (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998)

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

According to delivering document, and in conjunction with the data in GenBank, obtain several 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 reformation, obtain the sequence Sus scrofa interferon gamma mRNA (GenBank:GU433229.1) of pig gamma interferon.

According to the gene order obtaining and as the silkworm of bio-reactor and the feature of Bombyx mori nuclear polyhydrosis virus, two gene orders are carried out to the sub-Preference transformation of translation cipher (Codon usage bias adjustment), GC content transformation (GC Content Adjustment) in sequence, the removal of conventional restriction endonuclease sites etc., obtain the improved sequence of two Interferon, rabbit, in addition according to will with carrier pVL1393 sequence on the sequence that obtains after 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, SIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.IRES sequence is shown in SEQIDNo.3, and upstream and downstream restriction enzyme site is Xba I and EcoR I, and these three sections of sequences are synthesized respectively for subsequent operations; Above-mentioned several gene is connected to above pUC57 carrier after all synthesizing.

1.3IRES sequence is connected on pVL1393 carrier

Fig. 1 is shown in by the pVL1393 carrier schematic diagram that contains ORF1629 homology arm.Its main element is as follows: upstream homology arm is Recombination sequence (ORF603+): bases 1-3997, polyhedron upstream promoter is Polyhedrin promoter:bases 3998-4092, polyhedrosis gene is Polyhedrin gene:bases 4093-4738, multiple clone site is Multiple cloning site:bases 4128-4179, downstream homology arm is Recombinationsequence (ORF1629+): bases 4738-7002, intestinal bacteria replication origin is ColE1 origin:bases8029-7356, resistance screening gene is ammonia benzyl mycin resistant gene Ampicillin resistance gene:bases8965-8177.

1.3.1IRES sequence enzyme is cut

Enzyme is cut system in Table 2

Table 2 enzyme is cut system

10 * damping fluid H, 5 μ L are containing 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 ℃ are reacted 2.5 hours.

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

1.3.3pVL1393 carrier enzyme is cut

Enzyme is cut system in Table 3.

Table 3 enzyme is cut system

10 * damping fluid H, 5 μ L are containing 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, after 1.5 hours, take a morsel and carry out agarose gel electrophoresis, and check enzyme adds 1 μ L EcoR I reaction 2 hours again after cutting entirely, and then 65 ℃ of temperature are bathed 15 minutes by enzyme-deactivating, obtain the carrier that enzyme cuts.

1.3.4IRES endonuclease bamhi connects pVL1393 carrier

Linked system is in Table 4.

Table 4 linked system

16 ℃ of reactions are spent the night.

1.3.5 connect product and transform TOP10 competent cell

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

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

(1) plasmid DNA 20～100ng or connection mixture 3 μ L are added in the competent cell of the above-mentioned preparation of 100 μ L, mix gently ice bath 30min;

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

(3) slightly centrifugal, go resuspended precipitation after part supernatant, coat several containing on corresponding antibiotic LB flat board.Be inverted overnight incubation for 37 ℃.

1.3.6 the evaluation of recombinant plasmid

Disrupt red cell Rapid identification

Plasmid after restructuring and original plasmid molecule amount can detect recon (Beuken, et al., Biotechniques, 72:3827-3836,1998) by this method while having certain difference.

(a) a plurality of single colony transformation of picking are inoculated in 4mL containing 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 Eppendorf pipe, the centrifugal 10sec of 12,000g, abandons supernatant, adds 30 μ L damping fluids (6% sucrose, 0.1% tetrabromophenol sulfonphthalein), then adds 20 μ L phenol/chloroforms (1: 1), and fully thalline is upspring in vibration;

(c) 12, the centrifugal 5min of 000g, gets supernatant loading electrophoresis, 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 plasmid DNA

(2) extraction of Bacmid plasmid 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 in Table 5.

Table 5 identification system

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

Enzyme 0.5 μ L ddH ₂o 11 μ L

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

If the buffer difference of two kinds of enzymes is first used single endonuclease digestion 1～2h, add the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes precipitation DNA, remove supernatant and carry out again second enzyme and cut.After identifying correctly, obtain being connected with the pVL1393-IRES vector plasmid of IRES sequence.

1.4 porcine alpha-IFNs (SIFNA) are connected on pVL1393-IRES carrier

1.4.1SIFNA cut back to close with the enzyme of pVL1393-IRES

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

1.4.2SIFNA be connected to that pVL1393-IRES goes up and transform intestinal bacteria TOP10 competent cell linked system and method is shown in 1.3.4, connection product is transformed in TOP10 competent cell.

1.4.3 the detection of recombinant plasmid

The positive bacterium colony of antibiotic-screening after picking transforms, slightly carries and picks out recombinant clone and extract plasmid and carry out enzyme and cut evaluation, and method is shown in 1.3.6.After detecting correctly, obtain plasmid pVL1393-SA-IRES.

1.5 pig gamma interferons (SIFNG) are connected on pVL1393-SA-IRES carrier

The plasmid that contains SIFNG and pVL1393-SA-IRES carrier are carried out to EcoR I (BufferH) and Bgl II (BufferD) double digestion, because endonuclease reaction Buffer is different, so first carry out an endonuclease reaction, after 1-2 hour, detect enzyme and cut full postprecipitation plasmid, carry out the second enzyme and cut, method is shown in 1.3.6.

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

1.6 interferon genes and BmNPV DNA cotransfection bombyx mori cell carry out virus restructuring

PVL1393-SAIG plasmid, with liposome embedded cotransfection bombyx mori cell for BmNPV DNA, is carried out to the pVL1393 plasmid of homology arm and recombinates due to BmNPV DNA, the cell culture obtaining without being the baculovirus after restructuring in polyhedron plaque.

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

The screening of 1.7 recombinant virus Bm-SAIG, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, adding X-gal makes its final concentration reach 150 μ g/mL, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 33 spots is all the recombinant virus Bm-SAIG containing porcine alpha-IFN gene and pig gamma interferon gene, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection.

1.8 recombinant virus Bm-SAIG combining of α and IFN-γ 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 to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains porcine alpha-IFN and the IFN-γ associating expression product in silkworm.

1.8.2ELISA method detects

1.8.2.1 the ELISA that combines porcine alpha-IFN in expression product detects

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

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

B: application of sample: establish respectively blank well (blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole.On the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, 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 by sample, does not touch hole wall as far as possible, rocks and mixes gently.

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

D: dosing: (48T 20 times) distilled water 30 times for concentrated cleaning solution by 30 (48T 20 times) is doubly standby after dilution.

E: washing: carefully take shrouding film off, discard liquid, dry, washings is filled it up with in every hole, discards after standing 30 seconds, so repeats 5 times, pats dry.

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

G: incubation: operate same C.

H: washing: operate same E.

I: colour developing: every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 ℃ of lucifuges develop the color 15 minutes.

J: stop: every hole adds stop buffer 50 μ l termination reaction (the now blue vertical yellow that turns).

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

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

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

1.8.2.2 the ELISA that combines pig gamma interferon in expression product detects

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

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

Cytopathic-effect inhibition assay (the Dianzani F etc. that provide according to < < Products in China rules > >, 1989) detect tiring of porcine alpha-IFN and IFN-γ associating expression product, detection method is as follows:

Get 96 hole enzyme plates, every hole first adds each 50 μ l of above-mentioned silkworm blood sample of 2 times of serial dilutions, and then adding concentration is 2.5 * 10 ²～3.5 * 10 ³the Wish cell suspension in individual/hole (growing 24～48 hours in 96 porocyte culture plates), puts into containing 5%CO ₂37 ℃ of incubators in after 11～16 hours, discard supernatant, then with the VSV of the KMEM nutrient solution dilution containing 3% calf serum, attack (100TCID ₅₀) and be placed in containing 5%CO ₂37 ℃ of incubators in cultivate after 24～30 hours, discard nutrient solution and add 50 μ l violet staining liquid, after 30 minutes, discard staining fluid, with tap water, wash down, paper using blots the destainer that rear every hole adds 100 μ l, within 5 minutes, is placed in microplate reader and measures it in the absorption value at 570 wavelength places.With quantitative response parallel method, calculate the IU value of tiring of associating expression product sample.

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-γ (SIFNG)

1 experimental technique

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

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

The structure of 1.1 restructuring universal support pSK-RE

According to the document of having delivered and be published in the data on GenBank, the type that is enhanced green fluorescent protein (EGFP) gene order (GenBank:BAL72152.1), synthetic this section of sequence is for follow-up forward and reverse screening.Goal gene is entered in intestinal bacteria TOP10 or DH10B to jointly recombinating together to enter in BmBacmid and transform with EGFP gene clone, and the clone of success restructuring can send green fluorescence under exciting light.According to pertinent literature (Kirill A.Datsenko and Barry L.Wanner, 2000) at EGFP sequence two ends, add FRT sequence, the effect of this sequence is at pCP20 plasmid (Depart of MCD biology 830KBT, under the effect of the Flp restriction endonuclease carrying, the part in the middle of two sections of FRT sequences is comprised to EGFP gene inscribe eliminates Yale University), the green fluorescence just screening is disappeared for reverse screening, synthetic the EGFP gene RE sequence that contains FRT sequence to two ends, and at two ends, add that Hind III restriction enzyme site and Kpn I restriction enzyme site are for clone, sequence is shown in SEQIDNo.4.

1.1.1RE the enzyme of sequence and pSK carrier cuts back to close

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

1.1.2RE sequence connects pSK carrier and transforms intestinal bacteria TOP10 competent cell

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

1.1.3 the acquisition of positive colony and evaluation

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

1.2 pig gamma interferons (SIFNG) are connected in pSK-RE carrier

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

1.3 pig gamma interferons are recombinated on ReBm-dcc-d1629-d10 shuttle plasmid

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

Red recombinase gene is present on PKD46 plasmid, on this plasmid, there is repA101 (ts) gene, under 37 ℃ of conditions, cultivate and can lose plasmid, here the intestinal bacteria that contain PKD46 plasmid are cultivated under 30 ℃ of conditions, and red recombinase gene is to express under L-arabinose induction, therefore, the sharp competent preparation process of this electricity is as follows:

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

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

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

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

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

(6) abandon supernatant, add a small amount of aqua sterilisa, resuspended thalline, then by 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, then add 10% glycerine to 40ml, and 4 ℃ 11,000rpm, 1.5min.

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

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

Respectively with design primer RP10SK-S, RP10SK-X, primer outside be the p10 DNA homolog arm of 50bp left and right for homologous recombination, inner side is that amplimer sequence about 20bp is for amplifying target genes and marker gene.

With above-mentioned two couples of primer pair pSK-RE, carry out pcr amplification, by reclaiming product electric shock, enter in the competence of the above-mentioned Red of containing recombinase.

Electroporation process is with reference to related documents (Joseph et al., the molecular cloning experiment guide third edition, 2002):

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

1.3.3EGFP screen knocking out of sequence

The coli strain preparation that contains above-mentioned recombinant plasmid (BmBacmid-SIFNG) is become to heat shock competence, method reference example 1; PCP20 plasmid (Depart ofMCD biology 830KBT, Yale University) heat shock is transformed in competence.PCP20 plasmid can be expressed Flp restriction endonuclease under the culture condition of 42 ℃; so by the intestinal bacteria that contain pCP20 plasmid and BmBacmid-SIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivate 2 hours, the EGFP gene above recombinant plasmid is excised by inscribe.Intestinal bacteria after inducing culture are coated on to incubated overnight in resistant panel, grow bacterium and fall behind in and under above-mentioned fluorescence excitation optical condition, pick out that there is no the bacterium colony of green fluorescence be the recombinant plasmid clone Bm-SIFNG that knocks out EGFP gene order.

1.3.4 the detection of recombinant plasmid Bm-SIFNG

Design upstream and downstream and gene inner side 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, inside SIFNG gene, primer is respectively JIFNG-SR and JIFNG-XF, with JP10-F and JSIFNG-SR, be pair of primers respectively, JSIFNG-XF and JRP10-R are that pair of primers carries out the detection of bacterium liquid fast PCR to above-mentioned positive colony, obtain predicting big or small fragment, further the plasmid (Bm-SIFNG) after being recombinated is identified in order-checking, PCR product is reclaimed and checked order, amplimer for sequencing primer, the results are shown in Figure 2.

From Fig. 2, upper and lower two figure are respectively the PCR detection case of SIFNG upstream and downstream, and in green frame is the sequence in SIFNG gene, and in red frame is the sequence of P10 gene in Bmbacmid.From above-mentioned analysis, can show that SIFNG has successfully recombinated above Bmbacmid shuttle plasmid, obtain detecting correct recombinant plasmid Bm-SIFNG.

1.4 porcine alpha-IFN genes are connected on pVL1393 carrier

Detailed method, with reference to described in embodiment 1, changes pVL1393-IRES carrier into pVL1393, obtains pVL1393-SIFNA.

1.5 porcine alpha-IFN genes are recombinated with Bm-SIFNG cotransfection bombyx mori cell

By pVL1393-SIFNA plasmid with liposome embedded cotransfection bombyx mori cell for Bm-SIFNG.

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

The screening of 1.6 recombinant virus BmSAG, purification and amplification

Inoculate appropriate Bm (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 25 spots is all the coexpression recombinant virus containing porcine alpha-IFN and porcine beta interferon gene, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of 100pL recombinant virus infection normal growth, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection, obtain the porcine alpha-IFN of purifying and the recombinant virus BmSAG of IFN-γ coexpression.

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

1.7.1 the expression of recombinant virus BmSAG

Above-mentioned 25 recombinant virus nutrient solutions are pressed to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains porcine alpha-IFN and the IFN-γ associating expression product in silkworm.

1.7.2 the ELISA of expression product detects

Content to the porcine alpha-IFN in above-mentioned silkworm blood and pig gamma interferon carries out ELISA detection, method and operation steps are with reference to embodiment 1, the concentration that obtains porcine alpha-IFN in silkworm blood is 140 mcg/ml silkworm hemolymphs, is equivalent to approximately 1,000 ten thousand IU/ milliliter silkworm hemolymphs; The concentration of pig gamma interferon is 120 mcg/ml silkworm hemolymphs, is equivalent to approximately 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 above-mentioned silkworm blood being carried out to activity detects, working method and step be with reference to embodiment 1, finally obtains in this silkworm blood sample the Interferon, rabbit mean value of tiring and reach 2,500 ten thousand IU/ milliliter silkworm hemolymphs.

Embodiment 3 canine interferon alphas and IFN-γ combine expression

1 experimental technique

First to obtain the sequence of canine interferon alpha (after Canis lupus familiaris IFNA referred to as CIFNA) and IFN-γ (after Canis lupus familiaris IFNG referred to as CIFNG), through transformation, the codon in the Interferon, rabbit sequence of wild-type is replaced with to the codon of preference in silkworm baculovirus, two sections of sequences are synthesized to rear clone and are connected on pVL1393 carrier, in the middle of two Interferon, rabbit, by IRES sequence, be connected, the baculovirus that can obtain by the method for homologous recombination restructuring in the process with BmNPV DNA cotransfection obtains needing the albumen of expression for infected silkworm.

The transformation of 1.1 canine interferon alphas and dog interferon-β sequence and synthetic

Consult recent domestic about the document of canine interferon alpha and dog interferon-β report, according to the sequence of the synthetic two kinds of Interferon, rabbit of the sequence in 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 obtaining and as the silkworm of bio-reactor and the feature of Bombyx mori nuclear polyhydrosis virus, two gene orders are carried out to the sub-Preference transformation of translation cipher (Codon usage bias adjustment), GC content transformation (GC Content Adjustment) in sequence, the removal of conventional restriction endonuclease sites etc., obtain the improved sequence of two Interferon, rabbit, in addition according to will with carrier pVL1393 sequence on the sequence that obtains after 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, CIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.

1.2 canine interferon alphas (CIFNA) are connected on pVL1393-IRES carrier

1.2.1CIFNA cut back to close with the enzyme of pVL1393-IRES

To CIFNA with pVL1393-IRES carries out respectively BamH I (Buffer E) 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 be connected to pVL1393-IRES and go up and transform intestinal bacteria TOP10 competent cell linked system and method with embodiment 1, connection product is transformed in TOP10 competent cell.

1.2.3 the detection of recombinant plasmid

The positive bacterium colony of antibiotic-screening after picking transforms, slightly carries and picks out recombinant clone and extract plasmid and carry out enzyme and cut evaluation, and method is shown in embodiment 1, detects after correct and obtains plasmid pVL1393-CA-IRES.

1.3 dog IFN-γ (CIFNG) are connected on pVL1393-CA-IRES carrier

The plasmid that contains CIFNG and pVL1393-CA-IRES carrier are carried out to EcoR I (BufferH) and Bgl II (BufferD) double digestion, because endonuclease reaction damping fluid is different, so first carry out an endonuclease reaction, after 1-2 hour, detect enzyme and cut full postprecipitation plasmid, carry out the second enzyme and cut.

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

1.4 interferon genes and BmNPV DNA cotransfection bombyx mori cell carry out virus restructuring

By pVL1393-CAIG plasmid with liposome embedded cotransfection bombyx mori cell for BmNPV DNA.

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

The screening of 1.5 recombinant viruses, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, adding X-gal makes its final concentration reach 150 μ g/mL, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 37 spots is all the recombinant virus Bm-CAIG containing canine interferon alpha and dog gamma interferon genes, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection, obtain combining of canine interferon alpha and IFN-γ expressing carrier B m-CAIG.

Canine interferon alpha and the combining of IFN-γ of 1.6 recombinant virus Bm-CAIG in silkworm expresses and product detection

1.6.1 recombinant virus Bm-CAIG's combines expression

Above-mentioned 37 recombinant virus Bm-CAIG nutrient solutions are pressed to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains canine interferon alpha and the IFN-γ associating expression product in silkworm.

1.6.2 the ELISA that combines expression product detects

Dog IFN-γ (IFN-γ) the ELISA test kit that the DQ003 canine interferon alpha providing with Beijing Dong Ge Bioisystech Co., Ltd (IFN-α) ELISA test kit 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 associating expression product is 80 mcg/ml silkworm hemolymphs, approximately be equivalent to 7,000,000 IU/ milliliter silkworm hemolymphs, the concentration of dog IFN-γ is 70 mcg/ml silkworm hemolymphs, is approximately equivalent to 5,000,000 IU/ milliliter silkworm hemolymphs.

The activity of 1.6.3 combining expression product detects

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

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

1 experimental technique

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

1.1 dog IFN-γ (CIFNG) are connected in pSK-RE carrier

Redesign the dog IFN-γ obtaining in primer (RCIFNG-F and RCIFNG-R) amplification embodiment 3, its upstream and downstream restriction enzyme site is revised as to XbaI and BamHI, PCR product is connected in pMD-18T carrier to order-checking to carry out enzyme with above-mentioned two kinds of enzymes respectively after correct and cuts, reclaim object fragment, be connected with the pSK-RE carrier of cutting with same enzyme and obtain the restructuring pSK-RE carrier that contains dog IFN-γ.

1.2 dog gamma interferon genes and screening-gene EGFP gene are recombinated and are entered shuttle vectors simultaneously

With two in embodiment 2 general homology arm amplimer RP10SK-S, RP10SK-X amplifying target genes and reverse screening-gene, by reclaiming the conversion of product electric shock, enter in the intestinal bacteria Top10 competence that contains Red recombinase and Bmbacmid shuttle plasmid, the BmBacmid that screens obtained recombinating dog gamma interferon genes and EGFP gene, specific experiment method is with embodiment 2.

The detection of 1.3 recombinant plasmids and oppositely knocking out of screening-gene

Method with embodiment 2, design upstream and downstream and gene inner side that two pairs of primers lay respectively at two ends homologous recombination arm, with JRP10-F and JCIFNG-SR, be that upstream homology arm detection primer, JCIFNG-XF and JRP10-R are that downstream homology arm detects primer respectively, above-mentioned positive colony is carried out to bacterium liquid fast PCR to be detected, obtain predicting big or small fragment, further the plasmid (BmBacmid-CIFNG) after being recombinated is identified in order-checking.

The coli strain preparation that contains above-mentioned recombinant plasmid (BmBacmid-CIFNG) is become to heat shock competence, and method reference example 1, is transformed into the heat shock of pCP20 plasmid in competence.PCP20 plasmid can be expressed Flp restriction endonuclease under the culture condition of 42 ℃; so by the intestinal bacteria that contain pCP20 plasmid and BmBacmid-CIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivate 2 hours, the reverse screening-gene above recombinant plasmid is excised by inscribe.Intestinal bacteria after inducing culture are coated on to 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 genes are connected on pVL1393 carrier

Detailed method, with reference to described in embodiment 1, changes pVL1393-IRES carrier into pVL1393, obtains pVL1393-CIFNA.

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

By pVL1393-CIFNA plasmid, with liposome embedded cotransfection bombyx mori cell for Bm-CIFNG, cultural method is shown in embodiment 1, and final primary dcreening operation obtains coexpression recombinant viral vector BmCAG.

The screening of 1.6 recombinant virus BmCAG, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ LTC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 30 spots is all the coexpression recombinant virus BmCAG containing canine interferon alpha and dog interferon-β, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection, obtain recombinant virus BmCAG.

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

1.7.1 the expression of recombinant virus BmCAG

Above-mentioned 30 recombinant virus nutrient solutions are pressed to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains canine interferon alpha and the dog IFN-γ coexpression product in silkworm.

1.7.2 the ELISA of expression product detects

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

1.7.3 the activity of expression product detects

Adopt cytopathic-effect inhibition assay that the Interferon, rabbit in above-mentioned silkworm blood sample is tired and detected, working method and step be with reference to embodiment 1, and finally obtaining in 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-γ combine expression

1 experimental technique

First obtain the sequence of chicken alpha-interferon (after Gallus gallus IFNA referred to as GIFNA) and IFN-γ (after Gallus gallus IFNG referred to as GIFNG), through transformation, the codon in the Interferon, rabbit sequence of wild-type is replaced with to the codon of preference in silkworm baculovirus, two sections of sequences are synthesized to rear clone and are connected on pVL1393 carrier, in the middle of two Interferon, rabbit, by IRES sequence, be connected, the baculovirus that can obtain by the method for homologous recombination restructuring in the process with BmNPV cotransfection obtains the albumen that need to express for infected silkworm.

The transformation of 1.1 chicken alpha-interferons and chicken interferon-β sequence and synthetic

Consult recent domestic about the document of chicken alpha-interferon and chicken interferon-β report, according to the sequence of the synthetic two kinds of Interferon, rabbit of the sequence in 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 obtaining and as the silkworm of bio-reactor and the feature of Bombyx mori nuclear polyhydrosis virus, two gene orders are carried out to the sub-Preference transformation of translation cipher (Codon usage bias adjustment), GC content transformation (GC Content Adjustment) in sequence, the removal of conventional restriction endonuclease sites etc., obtain the improved sequence of two Interferon, rabbit, in addition according to will with carrier pVL1393 sequence on the sequence that obtains after two gene optimizations of polyclone restriction enzyme site design ask respectively GIFNA (SEQIDNo.7) and GIFNG (SEQIDNo.8), GIFNA upstream and downstream restriction enzyme site is BamH I and XbaI, GIFNG upstream and downstream restriction enzyme site is EcoR I and Bgl II.

1.2 chicken alpha-interferons (GIFNA) are connected on pVL1393-IRES carrier

1.2.1GIFNA cut back to close with the enzyme of pVL1393-IRES

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

1.2.2GIFNA be connected to pVL1393-IRES and go up and transform intestinal bacteria TOP10 competent cell

Linked system and method are shown in embodiment 1, and connection product is transformed in TOP10 competent cell.

The detection of 1.3 recombinant plasmids

The positive bacterium colony of antibiotic-screening after picking transforms, slightly carries and picks out recombinant clone and extract plasmid and carry out enzyme and cut evaluation, and method is shown in embodiment 1.After detecting correctly, obtain plasmid pVL1393-GA-IRES.

1.4 chicken IFN-γ (GIFNG) are connected on pVL1393-GA-IRES carrier

The plasmid that contains GIFNG and pVL1393-GA-IRES carrier are carried out to EcoR I (BufferH) and Bgl II (BufferD) double digestion, because endonuclease reaction damping fluid is different, so first carry out an endonuclease reaction, after 1-2 hour, detect enzyme and cut full postprecipitation plasmid, carry out the second enzyme and cut.

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

1.5 interferon genes and BmNPV DNA cotransfection bombyx mori cell carry out virus restructuring

By pVL1393-GAIG plasmid with liposome embedded cotransfection bombyx mori cell for BmNPV DNA.

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

The screening of 1.6 recombinant virus Bm-GAIG, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, adding X-gal makes its final concentration reach 150 μ g/mL, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 28 spots is all the recombinant virus Bm-GAIG containing chicken alpha-interferon and Chicken type Ⅱ interferon, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection.

1.7 recombinant virus Bm-GAIG combining of chicken alpha-interferon and IFN-γ 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 to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains porcine alpha-IFN and the IFN-γ associating expression product in silkworm.

1.7.2 the ELISA of expression product detects

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

1.7.3 the activity of expression product detects

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

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

1 experimental technique

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

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

1.1 chicken IFN-γ (GIFNG) are connected in pSK-RE carrier

Redesign the pig gamma interferon obtaining in primer (RGIFNG-F and RGIFNG-R) amplification embodiment 1 its upstream and downstream restriction enzyme site is revised as to XbaI and BamHI, PCR product is connected in pMD-18T carrier to order-checking to carry out enzyme with above-mentioned two kinds of enzymes respectively after correct and cuts, reclaim object fragment, be connected with the pSK-RE carrier of cutting with same enzyme and obtain the restructuring pSK-RE carrier that contains pig gamma interferon.

1.2 chicken IFN-γ are recombinated on ReBm-dcc-d1629-d10 shuttle vectors

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

1.2.2 Chicken type Ⅱ interferon and oppositely screening-gene EGFP gene are recombinated and are entered shuttle vectors simultaneously

Respectively with design primer RP10SK-S, RP10SK-X, primer outside be the p10 DNA homolog arm of 50bp left and right for homologous recombination, inner side is that the amplimer sequence of 20bp left and right is for amplifying target genes and reverse screening-gene.With above-mentioned two couples of primer pair pSK-RE, carry out pcr amplification, recovery product electric shock is entered in above-mentioned competence.Electroporation process operation method and step are with reference to embodiment 2.

1.2.3 the detection of recombinant plasmid

(JRP10-F and JGIFNG-SR are upstream detection primer to design two pairs of primers, JGIFNG-XF and JRP10-R are detected downstream primer) lay respectively at the upstream and downstream of two ends homologous recombination arm and gene inner side, above-mentioned positive colony is carried out to bacterium liquid fast PCR to be detected, obtain predicting big or small fragment, further the plasmid (BmBacmid-GIFNG) after being recombinated is identified in order-checking.

1.24 oppositely screen pounding out of sequence

The coli strain preparation that contains above-mentioned recombinant plasmid (BmBacmid-GIFNG) is become to heat shock competence, and method reference example 1, is transformed into the heat shock of pCP20 plasmid in competence.PCP20 plasmid can be expressed Flp restriction endonuclease under the culture condition of 42 ℃; so by the intestinal bacteria that contain pCP20 plasmid and BmBacmid-GIFNG plasmid in 42 ℃ of inducing culture after 30 minutes 37 ℃ cultivate 2 hours; screening-gene above recombinant plasmid is excised by inscribe, filters out the recombinant plasmid Bm-GIFNG of the GIFNG that recombinated.

1.3 chicken alpha interferon genes are connected on pVL1393 carrier

Detailed method, with reference to described in embodiment 1, changes pVL1393-IRES carrier into pVL1393, obtains pVL1393-GIFNA.

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

By pVL1393-GIFNA plasmid with liposome embedded cotransfection bombyx mori cell for Bm-GIFNG.

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

The screening of 1.5 recombinant virus BmGAG, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ LTC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 25 spots is all the coexpression recombinant virus BmGAG containing chicken alpha-interferon and chicken IFN-γ, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final pure recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection.

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

1.6.1 the expression of recombinant virus BmGAG

Above-mentioned 25 recombinant virus nutrient solutions are pressed to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains chicken alpha-interferon and the IFN-γ coexpression product in silkworm.

1.6.2 the ELISA of expression product detects

With the test kit in embodiment 3, respectively the above-mentioned silkworm blood that contains coexpression product is carried out to ELISA detection, working method and step are with reference to embodiment 3, the concentration that obtains the chicken alpha-interferon in 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-γ 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 above-mentioned silkworm blood sample is tired and detected, working method and step be with reference to embodiment 1, and finally obtaining in 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 mutant

1 experimental technique

The acquisition of 1.1 porcine alpha-IFN mutant genes

This experiment suddenlys change to the sequence of porcine alpha-IFN, in the situation that guaranteeing that it is active, it is transformed and makes it obtain higher protease resistant, be conducive to the time that Interferon, rabbit exists in vivo, through a large amount of point mutation experiments, the effect that wherein the 64th L-glutamic acid sports glutamine is the best, reduced the long-acting porcine alpha-IFN mutant to the susceptibility of proteolytic ferment in blood and tissue, its nucleotide sequence is shown in SEQ ID No.9, coded aminoacid sequence is SEQ ID No.10, two ends add that BamH I and Xba I restriction enzyme site are for follow-up clone operations.

1.2 porcine alpha-IFN mutant genes (SIFNA-MUT) are cloned on pVL1393 carrier.

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

The 1.3 cotransfection bombyx mori cells BmNPV DNA that obtains recombinating.

By pVL1393-SIFNA-MUT plasmid with liposome embedded cotransfection bombyx mori cell for BmNPV DNA.

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

The screening of 1.4 recombinant viruses, purification and amplification

Inoculate appropriate Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, by cotransfection supernatant liquor by 10 ^-3～10 ^-5do, after suitably dilution, to get 1mL diluent and be added in attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, being chilled to 40 ℃ mixes through the 2X of 40 ℃ of preheatings TC-100 substratum (containing 20%FBS) with equal-volume, along little dish edge, glue is slowly poured into, solidify afterwards with Parafilm sealing, be inverted for 27 ℃ and cultivate 4～7d.Under microscope, choose without polyhedron recombinant virus plaque, in super clean bench, with Tip choicest, get recombinant virus spot, be dissolved in 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect the cell in 24 orifice plates, cultivate after 3d for 27 ℃, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out pcr amplification analysis, the virus of finding 29 spots is all the recombinant virus Bm-SAM containing porcine alpha-IFN mutant, the viral supernatant liquor paving spot that can amplify object fragment screens for the first time, the final recombinant virus obtaining containing goal gene.Get respectively the Bm-5 attached cell of 100pL recombinant virus infection normal growth, after infection, about 5d is after cell floats, and 4 ℃ of preservations, in order to injection, obtain the recombinant virus Bm-SAM that contains porcine alpha-IFN mutant of purifying.

The porcine alpha-IFN mutant of 1.5 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 to 10 ⁵pfu/ head is injected young silkworm in five ages, after silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, obtains the coexpression product of porcine alpha-IFN mutant in silkworm.

1.5.2 the ELISA of expression product detects

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

1.5.3 the activity of expression product detects

Adopt cytopathic-effect inhibition assay that the Interferon, rabbit in above-mentioned silkworm blood sample is tired and detected, working method and step are with reference to embodiment 1, finally obtaining in 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 transformation period in body improved 5 times.

The expression method of <110> animal interferon-alpha and IFN-γ

<120> DQXL-0026

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

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 585

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<213> artifical sequence

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ggatccaaaa tggccccgac ctccgctttt ctcactgtgc tcgtgctgct gtcatgtaat 60

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<213> artifical sequence

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<213> artifical sequence

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<222> (1)..(582)

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

20 25 30

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35 40 45

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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 (5)

1. an expression method for Interferon, rabbit, is characterized in that, a Interferon, rabbit of pig, dog or chicken and IFN-γ are combined to expression or coexpression in insect baculovirus bio-reactor; Described method of combining expression comprises: (1) by a interferon gene of pig, dog or chicken and gamma interferon genes by the nucleotide sequence gang shown in SEQ ID No.3 after restructuring to the nonessential gene locus that copies or infect of baculovirus, obtain recombinant baculovirus; (2) with recombinate shape virus infection insect host or insect cell, expression alien gene, obtains the product that a Interferon, rabbit and IFN-γ are combined expression; The method of described coexpression comprises: (1) a interferon gene of pig, dog or chicken and gamma interferon genes are recombinated respectively to copying of baculovirus or two nonessential gene locuss infecting on, obtain recombinant baculovirus; (2), by recombinate shape virus infection insect host or insect cell, expression alien gene, obtains the product of a Interferon, rabbit and IFN-γ coexpression; The nucleotides sequence of wherein said pig a interferon gene is classified as shown in SEQ ID No.1, and the nucleotides sequence of pig gamma interferon is classified as shown in SEQ ID No.2; The nucleotides sequence of dog a Interferon, rabbit is classified as shown in SEQ ID No.5, and the nucleotides sequence of dog IFN-γ is classified as shown in SEQ ID No.6; The nucleotides sequence of chicken a Interferon, rabbit is classified as shown in SEQ ID No.7, and the nucleotides sequence of chicken IFN-γ is classified as shown in SEQ ID No.8.

2. according to associating expression method claimed in claim 1, it is characterized in that, in step (1) in the following manner by the gene recombination of gang to the nonessential gene locus that copies or infect of baculovirus: a interferon gene of gang and gamma interferon genes are cloned on baculovirus transfer vector, obtain recombinant baculovirus transfer vector; Recombinant baculovirus transfer vector and baculovirus are recombinated in insect cell, a interferon gene of gang and IFN-γ are recombinated to copying of baculovirus or the nonessential gene locus that infects on.

3. according to expression method claimed in claim 2, it is characterized in that: described baculovirus transfer vector is pVL1393 carrier; The a interferon gene of gang and IFN-γ are recombinated on the polyhedrosis gene site of baculovirus.

4. according to coexpression method claimed in claim 1, it is characterized in that, on two nonessential gene locuss of in such a way a interferon gene of pig, dog or chicken and gamma interferon genes being recombinated respectively to copying of baculovirus in step (1) or infecting, obtain recombinant baculovirus: gamma interferon genes is connected with enhanced green fluorescence protein marker gene, recombinated on the p10 gene locus of Ream-dcc-d1629-d10 baculovirus shuttle vectors, the phenotypic screen by EGFP goes out recombinant vectors; EGFP marker gene is knocked out, obtain the defective type baculovirus shuttle vectors of the IFN-γ of having recombinated; A interferon gene is recombinated in the baculovirus transfer vector that contains ORF1629 homology arm, obtain recombinant baculovirus transfer vector; By the defective type baculovirus shuttle vectors of the IFN-γ of having recombinated and recombinant baculovirus transfer vector cotransfection bombyx mori cell, obtain recombinant baculovirus.

5. according to expression method claimed in claim 4, it is characterized in that: a Interferon, rabbit is recombinated on the polyhedrosis gene site of baculovirus, IFN-γ is recombinated on the p10 gene locus of baculovirus.