CN102994550A - Method for expressing exogenous gene in animal cell or animal tissue - Google Patents
Method for expressing exogenous gene in animal cell or animal tissue Download PDFInfo
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Abstract
The invention discloses a method for expressing an exogenous gene in an animal cell or animal tissue. The method provides a silkworm baculovirus vector for introducing the exogenous gene into a mammal cell or tissue for carrying out gene presentation. The silkworm baculovirus vector comprises a framework vector silkworm baculovirus BmNPV and an expression cassette of the exogenous vector, which is recombined to a framework vector, wherein the expression cassette of the exogenous vector comprises a mammal promoter, the exogenous gene and a terminator. The invention further discloses a method for expressing the exogenous gene in the animal cell or animal tissue. The method comprises the following steps: infecting the constructed silkworm baculovirus vector with an insect host or insect cell; cultivating the infected insect host or insect cell for amplifying the silkworm baculovirus vector; and harvesting and purifying the amplified product. A silkworm baculovirus gene representation vector constructed by adopting the method has the advantages of incapability of amplification, no recombination with an animal cell gene, convenience for large amplification in vitro, high safety and the like.
Description
Technical field
The present invention relates to a kind of method of expression alien gene, relate in particular to and a kind ofly import in zooblast or the animal tissues foreign gene and the method for expressing, belong to field of gene.
Background technology
The treatment that gene therapy is successfully applied to SCID-X1 patient since nineteen ninety has had twenties years so far, and the very large special emphasis of the research of all the time gene therapy is the selection of gene transfer vector.Owing to the work of also expressing in the eukaryotic cell that foreign gene is presented in the body needs virus vector to finish basically, so the choice and application of various virus vector in clinical gene therapy, also be the emphasis of a research in these years, gland relevant viral vector (adeno-associated virus wherein, AAV) be the most successful in the recently research, but also have some problems simultaneously.
At present, the selection problem about gene therapy vector begins to be partial to baculovirus vector both at home and abroad, and wherein, the most frequently used is that baculovirus vector is Autographa californica multiple nucleopolyhedrovirus(AcMNPV); It is the arthropodan large-scale shaft-like enveloped virus of a class single infection, and the circular double stranded DNA genome between the 90-180Kb is packaged in the shaft-like capsid.Because more and more and more and more deep about its research in recent years, in addition its easy-to-operate, be subject to paying much attention to (Cory and Bishop in the expression that is used for various foreign genes and aspect a kind of novel gene therapy vector, 1997, Molecular Biotechnology, 303-313).Set forth difficulty and shortcoming (the Sandig et al. that has some baculoviruss to act in mammalian cell as the transgenosis expression vector in some early stage desk study, 1996, Journal of Molecular Medicine, 205-212), but find that in nearest research baculovirus has the not available characteristics of some carriers that adopt in the past as a kind of novel mammalian cell gene transfer vector, as: recombinant baculovirus makes up convenient, is easy to extensive vitro culture; The foreign gene capacity is large; Do not possess cytotoxicity behind the mammalian cell-infecting, security is good, can not be incorporated in the genome of cell, can not carry out viral dna replication, and it is active etc. that himself promotor does not have in mammalian cell.Yet, the carrier of presenting (gene therapy) as mammiferous foreign gene with AcMNPV exists the difficulty that is very difficult to overcome, be that complement component in the animal blood can deactivation AcMNPV virus particle, make the efficient of foreign gene importing mammalian cell very low, lose thus practical value (Sandig et al., 1996, Hum.Gene Ther.7:1937-1945; Hofmann and Strauss, 1998, Gene Ther.5:531-536).The problems referred to above have limited the range of application of baculovirus AcMNPV in gene therapy, make its range of application substantially be limited to mammalian cell, and can not be used for the application of biont.
Summary of the invention
One of purpose of the present invention provides in a kind of each tissue that foreign gene is imported mammalian cell or biont carries out the silkworm baculovirus carrier that gene is presented;
Two of purpose of the present invention provides a kind of method that makes up the silkworm baculovirus carrier that described gene presents;
Three of purpose of the present invention provide a kind of in zooblast or animal tissues the method for expression alien gene.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Carry out the silkworm baculovirus carrier that gene is presented in a kind of tissue that foreign gene is imported mammalian cell or biont, comprise the expression cassette of skeleton carrier and the restructuring foreign gene to the skeleton carrier;
Described skeleton carrier is silkworm baculovirus BmNPV DNA;
The expression cassette of described foreign gene is comprised of promotor, foreign gene and terminator; Wherein, described promotor is mammalian promoter, described mammalian promoter can be the CMV promotor, human cytomegalic inclusion disease virus early promoter (CMV-IE), people's EF-1-subunit promotor, Rous sarcoma long terminal repeat, people leukosialin gene promoter, mouse glycerol 3-phosphate kinases l (PGKI) gene promoter, human ubiquitin protein (ubiquitin) C gene promoter, the hybrid promoter that is made of avian beta-actin promotor and cmv enhancer sequence or mouse mastoncus virus (MMTV) promotor etc. are preferably CMV promotor (its nucleotides sequence is classified as shown in the SEQ ID No.3) or CAG promotor;
Described terminator is SV40-polyA tail terminator (its nucleotides sequence is classified as shown in the SEQ ID No.4) preferably.
Described foreign gene can be genes involved, cancer suppressor gene, other functional gene or the RNAi etc. that reporter gene (for example, can be green fluorescent protein EGFP gene or luciferase reporter gene (SEQ ID No.2)), antigen gene, treatment inherited disease are used.
The invention provides in a kind of tissue that foreign gene is imported mammalian cell or biont and carry out the silkworm baculovirus Vector construction method that gene is presented, the method comprises:
(1) expression cassette of foreign gene is cloned on the transfer vector, makes up the recombinant transfer vector that obtains containing foreign gene;
(2) the constructed recombinant transfer vector that contains foreign gene and baculovirus cotransfection insect cell are carried out homologous recombination, and get final product.
Wherein, the expression cassette of described foreign gene is comprised of mammalian promoter, foreign gene and terminator; Hybrid promoter or mouse mastoncus viral promotors that described mammalian promoter is CMV promotor, human cytomegalic inclusion disease virus early promoter, people's EF-1-subunit promotor, Rous sarcoma long terminal repeat, people leukosialin gene promoter, mouse glycerol 3-phosphate kinases l gene promoter, human ubiquitin protein C gene promoter, be made of avian beta-actin promotor and cmv enhancer sequence; Described terminator is SV40-polyA tail terminator preferably;
Described transfer vector is the pVL1393 transfer vector preferably;
Described foreign gene can be the genes involved used of reporter gene, antigen gene, treatment inherited disease or cancer suppressor gene etc.; Wherein, preferably green fluorescent protein EGFP gene or luciferase reporter gene of described reporter gene;
Described baculovirus is BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or TeNPV; Be preferably silkworm baculovirus BmNPV.
The present invention further provides a kind of method that foreign gene is imported zooblast or animal tissues and expresses, having comprised: silkworm baculovirus carrier infected insect host or insect cell that the present invention is constructed; Cultivating infected insect host or insect cell increases to the silkworm baculovirus carrier; The product that results and purifying increase.
Described insect host is preferably silkworm, wild silkworm, Semen Ricini silkworm, wild silkworm, Philosamia cynthia, tussah, yamama, wild giant silkworm, autographa california, tea geometrid, cabbage army worm, autumn mythimna separata, cabbage looper, armyworm, bollworm, the cotton mythimna separata of the U.S., oriental tobacco budworm, cigarette beetle, oriental armyworm or gypsymoth; Described insect cell is bombyx mori cell, wild silkworm cell, Semen Ricini silkworm cell, wild silkworm cell, Philosamia cynthia cell, tussah cell, yamama cell, wild giant silkworm cell, autographa california cell, tea geometrid cell, cabbage army worm cell, Spodoptera frugiperda cell, cabbage looper cell, armyworm cell, bolworm cell, the cotton mythimna separata cell of the U.S., oriental tobacco budworm cell, cigarette beetle cell, oriental armyworm cell or gypsymoth cell preferably;
Described infection is the silkworm baculovirus carrier to be eaten to infect insect larvae or the pupal cell in 1-5 age or seen through epidermis by mouth infect 1-5 insect larvae or the pupal cell in age; Preferably, described infection is with silkworm baculovirus carrier infected silkworm cell or with silkworm baculovirus carrier percutaneous puncture-inoculation 1-5 silkworm larva or the pupal cell in age, collects afterwards the silkworm larva that contains recombinant baculovirus or body fluid or the tissue homogenate of pupa in 3-6 days infecting; Wherein, described pupal cell is preferably 1-2 days early stage tender pupa.
The skeleton carrier of presenting foreign gene DNA is silkworm baculovirus BmNPV, and this promotor of presenting the skeleton carrier front is the promotor that silkworm expression is used, and can not express in mammalian cell; For this reason, the present invention is with between the mammalian promoter and terminator SV40 of exogenous gene cloning to the transfer vector, make up the recombinant transfer vector that is used for foreign gene transfer, then obtained recombinant baculovirus by carrying out homologous recombination with silkworm baculovirus BmNPV cotransfection insect cell; The recombinant baculovirus that obtains can import foreign gene and carry out gene in each tissue of mammalian cell (African green monkey kidney cell vero cell and human neuroblastoma clone SH-SY5Y) or biont and present.For the ease of detecting, the present invention makes up respectively as foreign gene with green fluorescent protein EGFP gene and luciferase reporter gene respectively and has obtained silkworm baculovirus and be delivery carrier, in zooblast (Vero cell, SH-SY5Y cell) or animal tissues's (tissue of mouse tissue, chicken), present and express experiment respectively, whether detect the expression of green fluorescence and luciferase in zooblast or the animal tissues, and measure carrier copy number in the cell unit wherein with the efficient of presenting of definite this carrier with quantitative fluorescent PCR.Experimental result shows with what BmNPV made up presents skeleton carrier and can well foreign gene be imported in the zooblast (Vero cell, SH-SY5Y cell) and express, and (in mouse, the chicken) also has obvious effect in each vital tissue organ of animal individual such as brain, skin, liver, lung etc. equally.
Therefore, adopt the present invention with silkworm baculovirus BmNPV DNA as framework construction can the tissue that foreign gene is imported mammalian cell or biont in carry out the silkworm baculovirus carrier that gene is presented, can not only finely be applied to the presenting or importing of foreign gene of various mammalian cells, also can be advantageously applied to Mammals (people's) dna vaccination (reporter gene is replaced with antigen gene), the treatment of inherited disease (reporter gene is replaced with the genes involved that the treatment inherited disease is used), the treatment of cancer (getting final product with replacement reporter genes such as antioncogenes), drug targets research, interaction of genes, humanized exogenous protein expression, the expression of the little RNA that uses as RNAi and the purposes such as synthetic.Because BmNPV can be by the institute's deactivations such as complement in the animal blood, therefore the envelope protein that exposes such as GP64 gene etc. can be substituted into other baculovirus or other virus, also can reach same effect.In like manner also can find to have in other baculovirus can not be by the baculovirus of the institute's deactivations such as complement in the animal blood, and perhaps the building mode by hybrid virus obtains the similar function of this kind.
Thus, the constructed silkworm baculovirus carrier of the present invention can be applied to the purposes of the aspects such as expression alien gene, preparation dna vaccination, gene therapy, drug targets research or interaction of genes in zooblast or tissue.
The invention provides a kind of gene therapy medicament or dna vaccination, described gene therapy medicament or dna vaccination comprise silkworm baculovirus carrier and the pharmaceutically acceptable auxiliary material that the present invention is constructed.
The present invention further provides a kind of oral preparations of presenting foreign gene, and described oral preparations includes gene, and to present silkworm baculovirus carrier of the present invention and the final concentration of significant quantity be the lipid acid of 25-35%.
Description of drawings
Fig. 1 pEGFP-N3 carrier synoptic diagram.
Fig. 2 pVL1393 schematic representation intention.
Fig. 3 mouse tissue Green fluorescence intensity contrast (tissue slice); A: lung's green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); B; Spleen green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); C: kidney green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); D: brain green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group).
Fig. 4 chicken is organized Green fluorescence intensity contrast (tissue smear); A: lung's green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); B; Spleen green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); C: kidney green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); D: brain green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); E: lymph green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group); F: fabricius bursa green fluorescence observation in the 11st day (left side: fill with and feed group, in: injection group, the right side: control group).
Luciferase contrast in Fig. 5 different tissues of mice.
Luciferase contrast in Fig. 6 chicken different tissues.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of 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 make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Experiment material
1. the structure of bacterial strain, virus strain and carrier e. coli strains: Bmbacmid and BmNPV is with reference to related documents (application for a patent for invention publication number: CN102286534A; Denomination of invention: express insect bio-reactor and construction process and the application of many foreign genes); Transfer vector pVL1393 is available from Invitrogen company; Bombyx mori cell BmN, Bm-5 and Sf-21 cell are available from Invitrogen company; African green monkey kidney cell (Vero cell) and human neuroblastoma clone (SH-SY5Y) are preserved the ATCC of mechanism available from cell and the microorganism of the U.S.; Intestinal bacteria (Top10, DH10B) and pGL-3 carrier are available from Promega company; PEGFP-N3 carrier (cpg biotech, CPC012); PMD-18T carrier and pMD-18T-simple carrier are available from TaKaRa company.
2. enzyme and reagent: restriction enzyme, ligase enzyme are Promega company product.
3. biochemical reagents: IPTG, X-Gal, SDS are Sigma company product.Lipofectin, low melting point agarose LMP, PCR test kit, T
4Dna ligase, RNA enzyme, Proteinase K, cell culture medium TC-100, foetal calf serum and other reagent are purchased from Invitrogen company, and luciferase test kit Luciferase Assay System is available from Promega.
4. substratum: Escherichia coli culture medium is LB(1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0); The insect cell substratum is TC-100; The DMEM substratum is available from Sigma company and Invitrogen company.
5. primer:
The primer in table 1 experiment
1. enhanced green fluorescence protein (EGFP) gene cloning and gene thereof are presented the structure of skeleton carrier
Because presenting to express, the EGFP gene needs special mammalian promoter and terminator in mammalian cell, the present invention selects CMV promotor and SV40 terminator (also can increase again insulator to increase expression efficiency), the present invention chooses the pEGFP-N3 vector plasmid as the source of EGFP gene after analyzing relatively, simultaneously use very easily its upstream CMV promotor and downstream SV40-polyA, its synoptic diagram is seen Fig. 1; The nucleotides sequence of the expression cassette that is comprised of CMV promotor, EGFP gene and downstream SV40-polyA is classified as shown in the SEQ ID No.1.
The EGFP upstream region of gene is CMV promotor and polyclone restriction enzyme site MCS in the carrier, and the downstream is SV40 sequence and polyA tailer sequence, this experiment will with sequence be these several parts of CMV promotor, EGFP gene, SV40 sequence and polyA tail.MCS sequence wherein is that the characteristics of isocaudarner cut away by Bgl II and BamH I, then increase from the CMV sequence to one section sequence the polyA tail, and be connected on the pVL1393 transfer vector by BamH I and EcoR I restriction enzyme site, thereby obtain and to carry out the baculovirus vector that gene is presented and expressed at mammalian cell in order to recombinate with the Bmbacmid homologous recombination.
1.1. about the preparation of solution and substratum with reference to related documents or reference book (Joseph et al., the molecular cloning experiment guide third edition, 2002; Ao Sibai et al., fine works molecular biology guide, 1998).
Solution I: 50mmol/L glucose, 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA.
Solution II: 0.2mol/L NaOH, 1%SDS (now with the current).
Solution III: 100mL system, 5mol/L Potassium ethanoate 80mL, glacial acetic acid 12mL, ddH
2O 8mL.
TER solution: pancreas RNAse (RNAse A) is dissolved among 10mM Tris-HCl, the 15mMNaCl, and the storage liquid-20 that is made into 10mg/mL is ℃ frozen, uses 1 * TE buf to be diluted to 4 ℃ of preservations of working fluid of 20 μ g/mL again.
PPt Buffer: Virahol 22mL; 5mol/mL KAc 1mL; DdH
2O 2mL.
1.2 the excision of MCS sequence in the pEGFP-N3 plasmid
1.2.1 pEGFP-N3 plasmid enzyme restriction
It is as follows that enzyme is cut system:
37 ℃ of reactions will add the 3M sodium-acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes after 2.5 hours in the above-mentioned system, the centrifugal supernatant that removes of 12000rpm changes Buffer D into, added 1 μ L EcoR I and moisturizing to 50 μ L and placed 37 ℃ of reactions 2 hours.
1.2.2 enzyme is cut product and is reclaimed
Enzyme is cut the glass milk method recovery of product and is carried out (Joseph et al., the molecular cloning experiment guide third edition, 2002 according to document or the described method of biology tool book; Ao Sibai et al., fine works molecular biology guide, 1998).
1.2.3 reclaiming product connects
Because need behind the used plasmid enzyme restriction from connecting, linked system is as follows:
PEGFP-N3 plasmid 4 μ L
T
4Dna ligase 0.5 μ L
10×Buffer 0.5μL
16 ℃ of reactions are spent the night.
Transform the TOP10 competent cell 1.2.4 connect product
1.2.4.1 a small amount of of competent cell or a large amount of preparation are carried out (Joseph et al., the molecular cloning experiment guide third edition, 2002) according to document or the described method of molecular biosciences reference book
Transform (Joseph et al., the molecular cloning experiment guide third edition, 2002 1.2.4.2 connect product; Ao Sibai et al., fine works molecular biology guide, 1998).
(1) plasmid DNA 20～100ng or connect mixture 3 μ L and be added in the competent cell of the above-mentioned preparation of 100 μ L, mixing gently, ice bath 30min;
(2) carry out heat-shocked (42 ℃ of insulation 90s), put rapidly 1～2min on ice, add 1mL and be incubated to 37 ℃ LB substratum, 37 ℃ of shaking culture 1h;
(3) slightly centrifugal, go resuspended precipitation behind the part supernatant, coat several and contain on the corresponding antibiotic LB flat board.Be inverted overnight incubation for 37 ℃.
1.2.5 the extraction of purpose plasmid is identified
1.2.5.1 the extraction of purpose plasmid
(1) a plurality of single colony transformation of picking are inoculated in the LB substratum that 4mL contains 80 μ g/mLAmp respectively, and 37 ℃ of shaking culture are spent the night;
(2) extraction of common plasmid DNA
A. get above-mentioned bacterium liquid 1.5mL in the Eppendorf pipe, the centrifugal 5min of 5,000g collects thalline, abandons supernatant;
B. suspend with 150 μ LSol I and precipitate, place 5min on ice;
C. add 300 μ LSol II and 50 μ L chloroforms, reverse gently ice bath 5min behind the mixing;
D. add 450 μ LSol III, ice bath 5-10min behind the violent mixing;
E.4 ℃ 12, the centrifugal 10min of 000g gets supernatant, adds 450 μ L Virahols, places 20min in-20 ℃ behind the mixing;
F.12, the centrifugal 10min of 000g abandons supernatant, and precipitation is dissolved among the 250 μ LTER (TE that contains 20 μ g/mLRNaseA), 37 ℃ of digestion 20min; Add 350 μ LPPt deposit B uffer, the rearmounted 4 ℃ of 20min of mixing;
G.12, the centrifugal 10min of 000g abandons supernatant, and 70% ethanol is washed once, the vacuum-drying precipitation, and with 40 μ L0.1 * TE (pH8.0) dissolving ,-20 ℃ save backup.
1.2.5.2 the enzyme of plasmid is cut evaluation
Two restriction enzyme sites of the EcoR I of the company of choosing and HindIII carry out double digestion, can be accredited as the plasmid that cuts away middle MCS if find behind the electrophoresis not have to cut fully.
Identification system is as follows:
37 ℃ of enzymes are cut 1-2h, add Marker as the reference standard, detect enzyme with agarose gel electrophoresis and cut the result.
If the buffer difference of two kinds of enzymes is then used first single endonuclease digestion 1-2h, add the dehydrated alcohol precipitation DNA of the 3M sodium-acetate of 1/10 volume and 2 times of volumes, remove supernatant and carry out again second enzyme and cut.Obtain getting rid of the pEGFP-N3 vector plasmid of CMS after identifying correctly.
1.3 the purpose fragment sequence is connected in the pVL1393 carrier
1.3.1 the amplification of purpose fragment sequence
With CMVSV40-F and two pairs of primers of CMVSV40-R the pEGFP-N3 plasmid that obtains in 1.2 is carried out pcr amplification and obtain the CMV-EGFP-SV40-polyA sequence.
The PCR system is as follows:
The PCR response procedures is as follows:
1,95 ℃ of denaturation 5min
2,95 ℃ of sex change 1min
3,58 ℃ of annealing 1min
4,72 ℃ are extended 1.5min
5, repeating step 2-429 circulation
6,72 ℃ are extended 5min
1.3.2 PCR product electrophoresis reclaims and is connected on the pMD-18T carrier
With above-mentioned PCR product electrophoresis and downcut fragment corresponding to purpose length with Marker contrast in 1% sepharose, and reclaim DNA in the glue, the same 1.2.2 of method with glass milk method.
To reclaim product and be connected on the pMD-18T carrier, reaction system is as follows:
PCR product 3.5 μ L
PMD-18T carrier 0.5 μ L
Solution I 4μL
16 ℃ of reaction 30min.
1.3.3 connect conversion and the recombinant screen of product
Above-mentioned connection product is transformed intestinal bacteria Top10 competent cell and incubated overnight, and method is with 1.2.The preliminary screening method of recon is the disrupt red cell Rapid identification
Plasmid after the restructuring and original plasmid molecule amount can detect recon (Beuken, et al., Biotechniques, 72:3827-3836,1998) with this method when certain difference is arranged.
(1) a plurality of single colony transformation of picking are inoculated in the LB substratum that 4mL contains 80 μ g/mLAmp respectively, and 37 ℃ of shaking culture are spent the night;
(2) get 300～500 μ L bacterium liquid in the Eppendorf pipe, the centrifugal 10sec of 12,000g abandons supernatant, adds 30 μ L damping fluids (6% sucrose, 0.1% tetrabromophenol sulfonphthalein), adds 20 μ L phenol/chloroforms (1:1) again, and fully thalline is upspring in vibration;
The centrifugal 5min of (3) 12,000g 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)
1.3.4 the extraction of recon plasmid and order-checking
The recombinant plasmid of above-mentioned preliminary evaluation is extracted, and method is chosen 3 samples and is carried out sequencing reaction with 1.2, analyzes sequencing result and obtains right-on plasmid pMD18T-EGFP.
1.3.5pMD18T-EGFP the enzyme of plasmid and pVL1393 carrier is cut
The synoptic diagram that contains the pVL1393 carrier of ORF1629 homology arm is seen Fig. 2.Its main element is as follows:
The upstream homology arm is Recombination sequence (ORF603+): bases 1-3997, the polyhedron upstream promoter is Polyhedrin promoter:bases 3998-4092, polyhedrosis gene is Polyhedrin gene:bases 4093-4738, multiple clone site is Multiple cloning site:bases 4128-4179, the downstream homology arm is Recombination sequence (ORF1629+): bases 4738-7002, the intestinal bacteria replication origin is ColE1 origin:bases 8029-7356, and the resistance screening gene is ammonia benzyl mycin resistant gene Ampicillin resistance gene:bases 8965-8177.
PMD18T-EGFP plasmid and pVL1393 carrier are carried out BamH I and EcoR I double digestion simultaneously, and electrophoresis reclaims the fragment of 1.5kb size in the pMD18T-EGFP plasmid enzyme restriction product, and pVL1393 carrier enzyme is cut 65 ℃ of deactivation endonuclease activities of product.
1.3.6EGFP connect the pVL393 carrier
Linked system and transformation and selection strategy be with described in 1.2, filters out recon and enzyme and cut order-checking obtain the recombinating transfer vector pVL1393-EGFP of CMV-EGFP-SV40-polyA.
1.4EGFP gene recombination obtains the delivery carrier that is of EGFP gene to the BmNPV DNA
With the pVL1393-EGFP plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell, because the pVL1393 plasmid that BmNPVDNA carries out homology arm is recombinated, the gene without being the baculovirus skeleton after the restructuring in the polyhedron plaque of the cell culture that obtains is delivery carrier.
Inoculate about 0.5-1 * 10
6The Bm-N cell is in 15cm
2In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary with serum free medium, add again the 1mL serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-EGFP plasmid DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that the 4-6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%, sealing, cultivate 4-5d for 27 ℃, after floating to cell, collect supernatant liquor and be used for screening and the expression that restructuring is delivery carrier.
1.5 gene is presented screening, the purification and amplification of skeleton carrier (recombinant virus) Bm-EGFP
Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, with the cotransfection supernatant liquor by 10
-3-10
-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, 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.Microscopically is chosen without polyhedron recombinant virus plaque, get the recombinant virus spot with the Tip choicest in the super clean bench, be dissolved in the 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out the pcr amplification analysis, the virus of finding 33 spots all is the recombinant virus Bm-EGFP that contains the EGFP gene, the viral supernatant liquor that can amplify the purpose fragment spreads spot and carries out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, about 5d is after cell floats afterwards in infection, and 4 ℃ of preservations are in order to infect.
1.6 gene is presented the amplification of skeleton carrier (recombinant virus) Bm-EGFP in silkworm
Above-mentioned 33 recombinant virus Bm-EGFP nutrient solutions are pressed 10
5The pfu/ head is injected five ages childrens silkworm, after the silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, and the silkworm baculovirus that obtains containing the EGFP gene is delivery carrier.2 restructuring BmNPV presenting the EGFP gene in Vero cell, SH-SY5Y cell and Mammals (mouse, chicken).
There is the BmNPV virus of EGFP gene to inoculate respectively Vero cell and SH-SY5Y cell restructuring, injection or oral to mouse and chicken, after for some time in the observation of cell, whether have fluorescence and fluorescence power to come its situation of presenting to the EGFP gene of principium identification in each tissue of mouse and chicken, then adopt the method for absolute quantitation PCR to measure and calculate to recombinate in the cell and in each tissue of mouse the copy number of the BmNPV that the EGFP gene is arranged.
2.1BmNPV presenting the EGFP gene in Vero cell, SH-SY5Y cell
2.1.1 inoculation
The cell conditioned medium liquid that contains virus particle of collecting in 1.5 is inoculated into 15cm
2In the Vero cell in the culturing bottle, inoculum size is 100 μ L, and the virus of the about 10PFU of each cell repeats 5 groups, the cell that the experiment contrast group infects for the BmNPV that does not contain EGFP, and the blank group is that normal cell is not done the infection processing.
2.1.2 the observation of green fluorescence
The cell of above-mentioned inoculation is continued to cultivate, and Continuous Observation, beginning to observe at the 4th day has green fluorescence to occur in the experimental group, collect the cell culture that one bottle of observation has green fluorescence continuous 5 day every day, shows that EGFP can continuous expression in Vero cell, SH-SY5Y cell.
Have the copy number working method that is delivery carrier (Bm-EGFP) of EGFP gene as follows 2.1.3 absolute quantitation PCR detects restructuring:
A, the cell culture of above-mentioned collection is centrifugal, collecting cell removes supernatant, adds the polyhedron lysate (0.1MNa that contains 1%NP-40
2CO
3, 0.15M NaCl, pH10.4) and dissolving, by equal-volume phenol, chloroform extraction genomic dna and quantitative, carry out qPCR with it as template and analyze after the ultrasonication.
B, RT-PCR reaction system are mixed various compositions: SYBR Green Realtime PCR Master Mix 10 μ L, Primer 1(10mM) 0.5 μ L, Primer 2(10mM) 0.5 μ L, dna profiling 1 μ L, use ddH again
2O is supplemented to final volume 20 μ L.
C, quantitative fluorescent PCR program are as follows: 95 ℃ of 3min; 95 ℃ of 15sec, 60 ℃ of 15sec, 72 ℃ of 30sec, 40 circulations, by Chromo 4 Detector, MJ Research quantitative real time PCR Instrument detects.
The making of D, typical curve: carry out quantitative fluorescent PCR according to EGFP gene design primer (EGFP-F and EGFP-R) among the Bm-EGFP, the quantity of virus genom DNA in the host cell actin gene (design of primers is as actin-vero-F and actin-vero-R, see the primer tabulation) homogenization, EGFP and actin pass through the pcr amplification rear clone to the pGEM-T carrier, measure restructuring T carrier concn and carry out 10 times of gradient dilutions, each dilute sample is carried out qPCR 3 times with EGFP and actin special primer respectively, generate typical curve.Extract DNA to every group and carry out qPCR 3 times with EGFP and actin special primer.
Detected result is shown as the DNA copy that contains 4-6 the EGFP gene of having an appointment in each cell.
2.2BmNPV presenting the EGFP gene in mouse
2.2.1 sample preparation and to injected in mice or oral
The silkworm blood sample of collecting in 1.6 press after the concentration dilution of 1:4 and the ultrasonication centrifugal with PBS, get supernatant liquor for injection treatment; With mixing with lipid acid (final concentration 30%) and propolis (final concentration is 10%) behind the silkworm blood sample preliminary purification of collecting in 1.6, after ultrasonic emulsification, obtain oral preparations, with this oral preparations filling hello mouse.
The injection experimental group: respectively 10 mouse are carried out tail vein injection, injection volume is 200 μ L supernatant liquors/only, about 10
9PFU; Fill with to feed experimental group: 10 mouse of another group are carried out oral filling feed, fill with the amount of feeding and be 200 μ L oral preparations/only; Other gets, and 20 mouse carry out tail vein injection with the BmNPV that does not contain the EGFP gene respectively and processing is fed in oral filling, normally raise as the experiment contrast group; 10 mouse are left intact as the blank group in addition.The normal raising.
2.2.2 the observation of fluorescence and result in the mouse tissue
Two experimental group (injection experimental group and filling hello experimental group) are being injected or are being filled with and fed processing rear the 5th day by mouse, 11 days, 17 days, in every group, choose respectively two in 21 days and put to death and take out respectively its brain, heart, lung, liver, spleen, kidney, muscle, small intestine, several tissues such as reproductive system, organizing it respectively, freezing microtome section prepares the fixing rear direct compression of 5 μ m thickness section, at the lung of fluorescence microscopy Microscopic observation discovery two experimental group, kidney, fluorescence and control group obvious difference in the spleen, can be judged as and express the EGFP that external source is arranged, variant demonstration EGFP was expressing at the 5th day, 11 days the most obvious, expression amount reduced in 17 days, and the fundamental sum control group did not have difference in 21 days; Fundamental sum blank group does not have difference in other tissue.In 2 experiment contrast groups the section after and the blank group substantially do not have difference (Fig. 3).
2.2.3 restructuring has the mensuration that is the delivery carrier copy number of EGFP in the tissue
Measuring method is with described in the 2.1.3, the mouse tissue confidential reference items are elected β-actin as, the PCR primer is that actin-mou-F and actin-mou-R(see primer tabulation), do together the copy number that is delivery carrier in the quantitative fluorescent PCR drawing standard curve detection tissue with the selected gene fragment of EGFP.The result is shown as above-mentioned observation to be had and is that the delivery carrier copy number is basically identical to be each cell 1-2 copy number in the different tissues of fluorescence.
2.3BmNPV presenting the EGFP gene in chicken
The silkworm blood sample of collecting in 1.6 press after the concentration dilution of 1:4 and the ultrasonication centrifugal with PBS, get supernatant liquor for injection treatment; With mixing with lipid acid (final concentration 30%) and propolis (final concentration is 10%) behind the silkworm blood sample preliminary purification of collecting in 1.6, after ultrasonic emulsification, obtain oral preparations, with this oral preparations filling hello mouse.
The processing mode of chicken is with described in 2.2, choose 30 chick that just hatched, fill with to feed 200 μ L oral preparations/only for 10, intravenous injection 200 μ L silkworm blood supernatant liquors under 10 wings/only, 10 as the blank group, observes that variant demonstration EGFP was expressing at the 5th day, and 11-13 days the most obvious, expression amount reduced in 20 days, and the fundamental sum control group did not have difference in 25 days; Fluorescence and control group obvious difference in the lung of two experimental group, kidney, spleen, lymph, the fabricius bursa, fundamental sum blank group does not have difference (Fig. 4) in other tissue.Chicken organizes confidential reference items to elect β-actin as in the quantitative fluorescent PCR, the PCR primer is that actin-chi-F and actin-chi-R(see primer tabulation), the result is shown as above-mentioned observation to be had and is that the delivery carrier copy number is basically identical to be each cell 1-2 copy number in the different tissues of fluorescence.
Clone and the gene of embodiment 2 luciferase reporter genes (luciferase) are presented
The structure that the clone of 1 luciferase reporter gene (luciferase) and gene thereof are presented skeleton carrier
The same with the EGFP gene among the embodiment 1, (derive from the pGL3-Basic plasmid with the luciferase gene, available from promega company, gene order is seen SEQ ID No.2) reporter gene presented as gene expresses in mammalian cell, and its gene upstream and downstream still is respectively CMV promotor (gene order is seen SEQID No.3) and SV40-polyA tail (sequence is seen SEQID No.4); Sequential analysis according to MCS in the pVL1393 carrier, adopt CMV clone primer upstream EcoR V, downstream BamH I, SV40-polyA clone primer upstream Not I-Pst I, downstream Bgl II, middle luciferase gene carry out enzyme with upstream BamH I, downstream Pst I restriction enzyme site and cut the connection structure.
1.1 luciferase gene, CMV promotor, SV40-polyA clone respectively and are connected on the pMD18T carrier
1.1.1 luciferase gene clone
With primer Luc-F and Luc-R the pGL3-Basic carrier is carried out pcr amplification, upstream primer is introduced BamH I restriction enzyme site, downstream primer is introduced Pst I restriction enzyme site, the PCR product is reclaimed and is connected on the pMD-18T carrier, selecting positive colony checks order, obtain the pMD-18T-Luc plasmid, method is with embodiment 1.
1.1.2 the clone of CMV promotor
With primer CMV-F and CMV-R the pEGFP-N3 carrier is carried out pcr amplification and obtain the CMV promotor, upstream primer is introduced EcoR V restriction enzyme site, downstream primer is introduced BamH I restriction enzyme site, the PCR product is reclaimed and is connected on the pMD-18T carrier, selecting positive colony checks order, obtain the pMD-18T-CMV plasmid, method is with embodiment 1.
1.1.3 the clone of SV40-polyA
With primer SV40-F and SV40-R the pEGFP-N3 carrier is carried out pcr amplification and obtain the SV40-polyA sequence, upstream primer is introduced Not I and Pst I restriction enzyme site, downstream primer is introduced Bgl II restriction enzyme site, the PCR product is reclaimed and is connected on the pMD-18T carrier, selecting positive colony checks order, obtain the pMD-18T-SV40 plasmid, method is with embodiment 1.
1.2 CMV promotor, SV40-polyA sequence and Luciferase gene are connected on the pVL-1393 carrier successively
1.2.1 the CMV promotor is connected on the pVL-1393 carrier
Plasmid pMD-18T-CMV and the transfer vector pVL-1393 that will contain the CMV promotor carry out EcoR V and BamHI double digestion, the pMD-18T-CMV enzyme is cut the product electrophoresis and is reclaimed about 600 fragment, be connected on the pVL-1393 carrier, detect the correct rear plasmid that extracts and obtain the pVL-CMV plasmid vector.
1.2.2 the SV40-polyA sequence is connected on the pVL-CMV carrier
PMD-18T-SV40 plasmid and recombinant transfer vector pVL-CMV are carried out respectively Pst I and Bgl II double digestion, the pMD-18T-SV40 enzyme is cut product and is reclaimed about 250 fragment, be connected on the pVL-CMV carrier, detect the correct rear plasmid that extracts and obtain the pVL-CMV-SV40 plasmid vector.
1.2.3 the Luciferase gene is connected on the pVL-CMV-SV40 carrier
PMD-18T-Luc plasmid and pVL-CMV-SV40 carrier are carried out respectively BamH I and Pst I double digestion, the pMD-18T-Luc enzyme is cut the fragment about product recovery 1.6k, be connected on the pVL-CMV-SV40 carrier, detect the correct rear plasmid that extracts and obtain the pVL-CMV-SV40-Luc plasmid vector.
Obtain its restructuring on the BmNPV genomic dna 1.3 luciferase reporter gene is recombinated and present skeleton carrier
With the pVL-CMV-SV40-Luc plasmid with BmNPV DNA with liposome embedded cotransfection bombyx mori cell, because the pVL1393 plasmid that BmNPV DNA carries out homology arm is recombinated, the gene without being the baculovirus skeleton after the restructuring in the polyhedron plaque of the cell culture that obtains is delivery carrier.
Inoculate about 0.5-1 * 10
6The Bm-N cell is in 15cm
2In the culturing bottle, adherent culture spend the night (being that cell density is about 80%).Remove the substratum that contains FBS, wash the cell secondary with serum free medium, add again the 1mL serum free medium.The DNA1 μ g that in 50 μ L reaction systems, adds above-mentioned BmNPV, pVL1393-EGFP plasmid DNA 2ug, liposome 5uL mixes, and add water and supply volume, mixing gently, 27 ℃ of incubation 15min dropwise add in the culturing bottle, and the limit edged shakes up.Cultivate the serum free medium that the 4-6h hypsokinesis removes to contain transfection liquid for 27 ℃, add the 4.5mL serum free medium and add the FBS of 500 μ L, making its final concentration is 10%, sealing, cultivate 4 ~ 5d for 27 ℃, after floating to cell, collect supernatant liquor and be used for screening and the expression that restructuring is delivery carrier.
1.5 screening, the purification and amplification of skeleton carrier BmNPV:Bm-Luc are presented in restructuring
Inoculate an amount of Bm cell (plate bottom area 80%) in the little dish of 35mm, cultivate 16h to cell attachment for 27 ℃, suck substratum, with the cotransfection supernatant liquor by 10
-3-10
-5After doing suitably dilution, get the 1mL diluent and be added in the attached cell, be evenly distributed.27 ℃ are infected 1h, 2% low melting point sepharose is melted in 60 ℃ of water-baths, 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.Microscopically is chosen without polyhedron recombinant virus plaque, get the recombinant virus spot with the Tip choicest in the super clean bench, be dissolved in the 400 μ L TC-100 substratum, placing 4h for 4 ℃ discharges virus particle, get 100 μ L virus liquids and infect cell in 24 orifice plates, behind 27 ℃ of cultivation 3d, get 150uL cells infected supernatant and prepare fast virus genom DNA by alkaline denaturation, carry out the pcr amplification analysis, the virus of finding 25 spots all is the recombinant virus Bm-Luc that contains the luciferase gene, the viral supernatant liquor that can amplify the purpose fragment spreads spot and carries out the screening first time, the final pure recombinant virus that obtains to contain goal gene.Get respectively the Bm-5 attached cell of the pure recombinant virus infection normal growth of 100pL, about 5d is after cell floats afterwards in infection, and 4 ℃ of preservations are in order to infect and amplification.
1.6 gene is presented the expression of skeleton carrier (recombinant virus) Bm-Luc in silkworm
Above-mentioned 33 recombinant virus Bm-Luc nutrient solutions are pressed 10
5The pfu/ head is injected children silkworm in five ages, after the silkworm morbidity, cuts foot, collects silkworm blood, and-20 ℃ frozen, increases in silkworm, obtains containing mammalian promoter the luciferase gene that starts and the silkworm baculovirus that contains the SV40 terminator and is delivery carrier.
2 BmNPV are presenting the Luciferase gene in Vero cell, SH-SY5Y cell and Mammals.
2.1 the gene to luciferase of BmNPV in vero cell and SH-SY5Y cell presented
2.1.1 inoculation
The supernatant that will contain after the Bm cell culture fragmentation of Bm-Luc is inoculated into respectively in Vero cell, the SH-SY5Y cell culture, and infects simultaneously the BmNPV that does not contain Luc and cultivate one group of blank, and method is with embodiment 1.
2.1.2 photon is measured
The culture of above-mentioned two kinds of cells was begun experimental group is begun to detect its photon intensity method following (luciferase cell cultures lysate lysis buffer wherein and luciferase detection substrate are the luciferase detection system test kit available from Promega company) in 24 hours in cultivation respectively:
Be dissolved in after cell culture is centrifugal among an amount of Lysis buffer, get 5 μ L and join in the 15 μ L substrates, put at once the photon instrument and detect, record data repeat 3 times, average, and contrast the expression that determines whether luciferase with the blank group.
Detected result is presented at beginning in the 4th day, luciferase reporter gene begins to express, and design primer Luc1-F and luc1-R do absolute quantitative PCR with actin in the vero cell of choosing among the embodiment 1 and the SH-SY5Y cell to calculate its copy number be each cell 4-6 copy number.
2.2 the gene to luciferase of restructuring BmNPV:Bm-luc in the mouse tissue organ presented
2.2.1 Mice Inoculated
The silkworm blood sample of collecting in 1.6 press after the concentration dilution of 1:4 and the ultrasonication centrifugal with PBS, get supernatant liquor for injection treatment; With mixing with lipid acid (final concentration 30%) and propolis (final concentration is 10%) behind the silkworm blood sample preliminary purification of collecting in 1.6, obtain oral preparations through ultrasonic emulsification, with this oral preparations filling hello mouse.
The injection experimental group: respectively 10 mouse are carried out tail vein injection, injection volume is 200 μ L supernatant liquors/only, about 109PFU; Fill with to feed experimental group: 10 mouse of another group are carried out oral filling feed, fill with the amount of feeding and be 200 μ L oral preparations/only; Other gets 20 mouse and carries out tail vein injection and oral filling hello processing with the BmNPV that does not contain the EGFP gene respectively, and normal the raising as tail vein injection experiment contrast group and oral filling fed processing experiment contrast group; 10 mouse are left intact as the blank group in addition.The normal raising.
2.2.2 detect the expression of luciferase in tissue
In every group, choose respectively equally two at the 5th day, 11 days, 17 days, 21 days and put to death and take out respectively its brain, heart, lung, liver, spleen, kidney, muscle, small intestine, the several tissues of reproductive system, each tissue is suspended among the lysis buffer of 10 times of volumes ultrasonication with the liquid nitrogen processing and after grinding.Detect photon with the luciferase substrate reactions and determine that whether it express.
Detected result shows that photon intensity in several tissues such as beginning to fill with the brain of feeding experimental group and intravenous injection experimental group, lung, liver, spleen, kidney on the 5th day all exceeds 2-3 doubly than the background of blank group, substantially can't detect in other tissue, and photon intensity is almost identical with the blank group in the time of the 21st day, illustrate that the 5th day beginning silkworm baculovirus carrier has been presented to luciferase gene in the above-mentioned tissue and expression, the expression of luciferase weakens in the time of the 21st day.Photon mensuration intensity quite is background level (Fig. 5) in other three control groups (tail vein injection experiment contrast group, oral filling are fed and processed experiment contrast group, blank group).
Above-mentioned several organizing extracted respectively full genome and detected the wherein absolute copy number of silkworm baculovirus carrier with the method among the embodiment 1, and the result shows that copy number in the different tissues is substantially at the 1-2copies/ cell.
2.3 the gene to luciferase of restructuring BmNPV:Bm-luc in the chicken histoorgan presented
Choosing the chick of new hatching processes, method is with 2.2, the result shows that photon intensity in several tissues such as beginning to fill with the brain of feeding experimental group and intravenous injection experimental group, lung, liver, spleen, kidney, lymph, the fabricius bursa on the 5th day all exceeds 3-4 doubly (Fig. 6) than the background of blank group, and copy number is substantially at the 1-2copies/ cell.
Sequence table
＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences
＜120〉method of expression alien gene in zooblast or animal tissues
<130> DQXL--0078
<160> 4
<170> PatentIn version 3.5
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atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
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agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 840
gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 900
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gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac 1380
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taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 1500
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gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt 360
tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420
aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga 480
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540
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tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg 660
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aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg 900
attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct 960
aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat 1020
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140
acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt 1200
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260
ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct 1320
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cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500
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gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
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ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
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<210> 4
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tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 60
tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 120
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Claims (10)
1. foreign gene is imported in the tissue of mammalian cell or biont and carry out the silkworm baculovirus carrier that gene is presented, it is characterized in that: the expression cassette that comprises skeleton carrier and the restructuring foreign gene to the skeleton carrier; Described skeleton carrier is silkworm baculovirus BmNPV DNA; The expression cassette of described foreign gene is comprised of mammalian promoter, foreign gene and terminator.
2. according to silkworm baculovirus carrier claimed in claim 1, it is characterized in that: hybrid promoter or mouse mastoncus viral promotors that described mammalian promoter is CMV promotor, human cytomegalic inclusion disease virus early promoter, people's EF-1-subunit promotor, Rous sarcoma long terminal repeat, people leukosialin gene promoter, mouse glycerol 3-phosphate kinases l gene promoter, human ubiquitin protein C gene promoter, be made of avian beta-actin promotor and cmv enhancer sequence; Described terminator is SV40-polyA tail terminator.
3. according to silkworm baculovirus carrier claimed in claim 1, it is characterized in that: described foreign gene is genes involved, cancer suppressor gene or the RNAi that reporter gene, antigen gene, treatment inherited disease are used; Wherein, described reporter gene is green fluorescence protein gene or luciferase reporter gene.
4. method that makes up any one described silkworm baculovirus carrier of claim 1-3, the method comprises:
(1) expression cassette of foreign gene is cloned on the transfer vector, makes up the recombinant transfer vector that obtains containing foreign gene; The expression cassette of described foreign gene is comprised of mammalian promoter, foreign gene and terminator; (2) with the constructed recombinant transfer vector that contains foreign gene and baculovirus cotransfection insect cell, carry out homologous recombination, and get final product.
5. it is characterized in that in accordance with the method for claim 4:
Described foreign gene is the genes involved used of reporter gene, antigen gene, treatment inherited disease, cancer suppressor gene, or RNAi; Wherein, preferably green fluorescent protein EGFP gene or luciferase reporter gene of described reporter gene;
Described transfer vector is the pVL1393 transfer vector;
Hybrid promoter or mouse mastoncus viral promotors that described mammalian promoter is CMV promotor, human cytomegalic inclusion disease virus early promoter, people's EF-1-subunit promotor, Rous sarcoma long terminal repeat, people leukosialin gene promoter, mouse glycerol 3-phosphate kinases l gene promoter, human ubiquitin protein C gene promoter, be made of avian beta-actin promotor and cmv enhancer sequence; Described terminator is SV40-polyA tail terminator preferably;
Described baculovirus is BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or TeNPV; Be preferably silkworm baculovirus BmNPV.
Described insect cell is bombyx mori cell.
6. the method that foreign gene is imported zooblast or animal individual tissue and expresses comprises: with the described silkworm baculovirus carrier of claim 1-3 infected insect host or insect cell; Cultivating infected insect host or insect cell increases to the silkworm baculovirus carrier; The product that results and purifying increase.
7. it is characterized in that in accordance with the method for claim 6: described insect host is silkworm, wild silkworm, Semen Ricini silkworm, wild silkworm, Philosamia cynthia, tussah, yamama, wild giant silkworm, autographa california, tea geometrid, cabbage army worm, autumn mythimna separata, cabbage looper, armyworm, bollworm, the cotton mythimna separata of the U.S., oriental tobacco budworm, cigarette beetle, oriental armyworm or gypsymoth; Described insect cell is bombyx mori cell, wild silkworm cell, Semen Ricini silkworm cell, wild silkworm cell, Philosamia cynthia cell, tussah cell, yamama cell, wild giant silkworm cell, autographa california cell, tea geometrid cell, cabbage army worm cell, Spodoptera frugiperda cell, cabbage looper cell, armyworm cell, bolworm cell, the cotton mythimna separata cell of the U.S., oriental tobacco budworm cell, cigarette beetle cell, oriental armyworm cell or gypsymoth cell;
Described infection is the silkworm baculovirus carrier to be eaten to infect insect larvae or the pupal cell in 1-5 age or seen through epidermis by mouth infect 1-5 insect larvae or the pupal cell in age; Preferably, described infection is with silkworm baculovirus carrier infected silkworm cell or with silkworm baculovirus carrier percutaneous puncture-inoculation 1-5 silkworm larva or the pupal cell in age, collects afterwards the silkworm larva that contains recombinant baculovirus or body fluid or the tissue homogenate of pupa in 3-6 days infecting; Wherein, described pupal cell is preferably 1-2 days early stage tender pupa.
8. the purposes of the described silkworm baculovirus carrier of claim 1-3 in preparation gene therapy medicament or dna vaccination.
9. a gene therapy medicament or dna vaccination is characterized in that, comprising: the described silkworm baculovirus carrier of claim 1-3 and pharmaceutically acceptable auxiliary material.
10. an oral preparations of presenting foreign gene is characterized in that: comprise that it is the lipid acid of 25-35% that gene is presented silkworm baculovirus carrier and the final concentration of the described purifying of claim 1-3 of significant quantity.
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