CN1570121A - Virus vector, cell line, method and production system for high effective production of recombinant adenovirus satellite virus - Google Patents
Virus vector, cell line, method and production system for high effective production of recombinant adenovirus satellite virus Download PDFInfo
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Abstract
The invention discloses a virus vector, cell line and method and its composite production system for high efficiency production of recombinant adenowirus associated virus. The virus vector is recombinant baculovirus vector, and cell line is integrated insect cell line. One or several of the adenowirus associated virus Cap gene, Rep gene, ITR sequence are constructed in or integrated in vector and cell line. Eucaryon promoter, multi-clone sites or target gene are all contained in between ITR sequence. High efficiency recombinant adenowirus associated virus production system is composed of recombinant baculovirus and insect cell line, or integrated insect cell line. The advantages: recombinant baculovirus can reproduce itself and provide largely the protein or genome for producing rAAV, insect cell line is suitable for large scale suspension culture, which reduce the cost, the method is simple, and production efficiency is high.
Description
Technical field
The present invention relates to the production system of virus vector, clone, method and the composition thereof of High-efficient Production recombinant adeno-associated virus, belong to the viral genetic engineering field.
Background technology
In gene therapy research, the rAAV carrier is wide owing to its host range, side effect is little, can mediate characteristics such as the long-time stably express of goal gene obtains widespread use.But the rAAV mass preparation is to limit the principal element of its widespread use in gene therapy, and this can come mass preparation rAAV from the cell that improves individual cells generation number of virus particles and cultivation mass production virus.
In the present production technology, utilize mammalian cell, the output of virus is 100 virions/cell only generally.Behind a large amount of optimization, viral yield can reach 10
4Virion/cell, but the clone of producing usefulness mainly is attached cells such as human embryo kidney (HEK) 293 cells, and these cell large scales are cultivated relatively difficulty, so recombinant virus still is difficult to carry out scale operation.There is the investigator to use baculovirus expression AAV accessory protein in recent years, in insect cell, produces rAAV virus, it is reported to reach 10
5/ cell.Insect cell line (as the Sf9 cell) is fit to suspension culture, can cultivate in a large number in the cell cultures jar, helps the scale operation of rAAV virus.In the insect system that uses, need to use three recombination bacillary viral vectors at present, express Rep, Cap and the ITR of rAAV respectively, its course of infection is still more numerous and diverse.
Summary of the invention
First purpose of the present invention provides the virus vector of High-efficient Production recombinant adeno-associated virus.
Another object of the present invention provides the cell of High-efficient Production recombinant adeno-associated virus.
The 3rd purpose of the present invention provides High-efficient Production recombinant adeno-associated virus method.
The 4th purpose of the present invention provides the production system of High-efficient Production recombinant adeno-associated virus.
For achieving the above object, the present invention is by the following technical solutions:
A kind of virus vector of High-efficient Production recombinant adeno-associated virus, this virus vector is a recombination bacillary viral vector, in this carrier, be built with among Cap gene, Rep gene and this three of ITR sequence of adeno-associated virus one or several, wherein all comprise eukaryotic promoter, multiple clone site or target gene between the ITR sequence.
That baculovirus is commonly used at present is autographa californica nuclear polyhedrosis virus AcMNPV and silkworm baculovirus BmPV, and used in the present invention is autographa californica nuclear polyhedrosis virus AcMNPV.Use baculovirus, relevant albumen that can efficient in insect cell, pro rata expression adeno-associated virus; The ITR sequence provides the packaging signal of adeno-associated virus (AAV), the multiple clone site of integrating promotor and can cloning foreign gene; Rep provides adeno-associated virus (AAV) to duplicate required enzyme; The Cap gene provides viral packing required structural protein.
Be built with the Cap gene of adeno-associated virus in the described recombination bacillary viral vector.
Be built with the Rep gene and the Cap gene of adeno-associated virus in the described recombination bacillary viral vector.
Be built with the ITR sequence of adeno-associated virus in the described recombination bacillary viral vector.
Be built with the Cap gene and the ITR sequence of adeno-associated virus in the described recombination bacillary viral vector.
Be built with Rep gene, Cap gene and the ITR sequence of adeno-associated virus in the described recombination bacillary viral vector.
A kind of clone of High-efficient Production recombinant adeno-associated virus, this cell is to integrate insect cell line, in the karyomit(e) of this clone, be integrated with among Cap gene, Rep gene and this three of ITR sequence of adeno-associated virus one or several, wherein all comprise eukaryotic promoter, multiple clone site or target gene between the ITR sequence.Insect cell line is the main host cell of baculovirus, cultivates easily, helps improving the production efficiency and the output of virus related to rocombinant adenovirus; At present commonly used have 3 cell strain: Sf9 and Sf21 and a High-Five
TM, all be business-like clone.Can avoid using repeatedly the recombinate shape virus infection insect cell after the relevant sequence of adeno-associated virus (AAV) being incorporated on the karyomit(e) of insect cell.
Be integrated with the Rep gene of adeno-associated virus on the karyomit(e) of described integration insect cell line.
Be integrated with the ITR sequence of adeno-associated virus on the karyomit(e) of described integration insect cell line.
Be integrated with the Rep gene and the ITR sequence of adeno-associated virus on the karyomit(e) of described integration insect cell line.
A kind of method of High-efficient Production recombinant adeno-associated virus is utilized above-mentioned recombination bacillary viral vector infected insect cell system or is utilized above-mentioned recombination bacillary viral vector to infect above-mentioned integration insect cell line, High-efficient Production recombinant adeno-associated virus.Described baculovirus vector is autographa californica nuclear polyhedrosis virus AcMNPV.
The High-efficient Production recombinant adeno-associated virus production system of utilizing aforesaid method to obtain, this system is made up of recombination bacillary viral vector and insect cell line, or is made up of recombination bacillary viral vector and integration insect cell line.Described baculovirus vector is autographa californica nuclear polyhedrosis virus AcMNPV.
Described recombinant adeno-associated virus production system is by insect cell line Sf9 or Sf21 or High-Five
TM, the baculovirus vector that is built with the baculovirus vector of adeno-associated virus Rep gene and Cap gene and is built with adeno-associated virus ITR sequence forms.
Described recombinant adeno-associated virus production system is by insect cell line Sf9 or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Rep gene, Cap gene and ITR sequence form.
Described recombinant adeno-associated virus production system is by the integration insect cell line Sf9 that is integrated with adeno-associated virus Rep gene or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Cap gene and ITR sequence form.
Described recombinant adeno-associated virus production system is by the integration insect cell line Sf9 that is integrated with adeno-associated virus ITR sequence or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Rep gene and Cap gene form.
Advantage of the present invention is: employed recombinant baculovirus can be bred in clone voluntarily, can provide in a large number to produce rAAV desired protein or genome; Insect cell line is fit to suspend and cultivates in a large number, greatly reduces production cost; Use one or two recombinate shape virus infection insect cell line to produce rAAV, more simple and easy to do; This system can improve the efficient of producing rAAV greatly by regulating related gene, proteinic quantity or expression time simultaneously.
The invention will be further described below in conjunction with the drawings and specific embodiments, is not limitation of the invention.
Description of drawings
Fig. 1 is carrier pBac-Cap plasmid construction figure.
Fig. 2 is carrier pBac-RC plasmid construction figure.
Fig. 3 is carrier pBac-ITR plasmid construction figure.
Fig. 4 is carrier pBac-Cap-ITR plasmid construction figure.
Fig. 5 is carrier pBac-Cap-Rep-ITR (pAllinOne) plasmid construction figure.
Fig. 6 is carrier pIB-Rep plasmid construction figure.
Fig. 7 is carrier pIB-Cap-Rep plasmid construction figure.
Fig. 8 is carrier pIB-ITR plasmid construction figure.
Fig. 9 is LacZ detection of expression figure behind rAAV-LacZ infected person embryonic kidney 293 cells.
Embodiment
Embodiment 1, carry and express the structure of the recombinant baculovirus rBac-Cap of AAV cap gene
1, by the synthetic following primer of dna synthesizer (Applied BioSystem 373A):
PCapA：
5’CGGGATCCTGTTAAGA
CGGCTGCCGA
CGGTTATCT
ACC
CGATTGGCTC，
Wherein italic is the BamHI site, and underscore is a catastrophe point.First ATG sports ACG to reduce the expression level of VP1; The ATG of second frame dislocation sports ACG, to eliminate the influence to downstream VP2 and VP3 expression.
PCapB：
5 ' ACAAGC
TTACAGATTACGAGTCAGGTATCTGG, wherein italic is the HindIII site, underscore is a termination codon.
2, be template with plasmid pAAV-RC (Stragagene company product), adopt Roche high-fidelity PCR system to carry out pcr amplification, obtain the Cap gene of AAV-2.Reaction conditions is as follows: template 1ng, and each 100ng of primer P1 and P2, dNTP 0.25mmol/L, reaction buffer 10 microlitres, deionized water to 100 microlitre is mended by enzyme 2.5 units.Adopt U.S. PE company 9600 type PCR instrument carry out the PCR reaction (94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 3 minutes, coamplification 30 circulations.Obtain the 2.3kb dna fragmentation through agarose electrophoresis.
3, each 200ng of the dna fragmentation of above-mentioned acquisition and vector plasmid pFastBacDual (invitrogen company product) carries out enzyme with BamHI+HindIII (being Promega company product) and cuts, reclaiming fragment connects with T4 dna ligase (Promega company product), 16 ℃ after 6 hours, press the described condition transformed into escherichia coli of molecular cloning DH5 α, be coated with ammonia benzyl resistant panel, screen correct plasmid, acquisition can be expressed the structure of the proteic plasmid vector pBac-Cap. of AAV Cap plasmid and see Fig. 1.
4, plasmid pBac-Cap 1ng mixes with 100 μ l competence intestinal bacteria Max Efficiency DH10Bac, ice bath 42 ℃ of hot activations 45 seconds after 30 minutes, ice bath is 2 minutes immediately, add 900 μ l SOC substratum (seeing molecular cloning), 37 ℃ of cultivations are coated with flat board after 1 hour and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 100 μ g/ml BluoGal (Invitrogen company product) and 40 μ g/ml IPTG).37 ℃ of bacterium colonies of cultivating picking white after 48 hours are analyzed.Identifying that correct positive colony is seeded to contains incubated overnight in the above-mentioned antibiotic LB liquid nutrient medium, carries out plasmid extraction with the MidPrep test kit of Invitrogen company, obtains recombination bacillary viral vector DNA.
5, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection, cultivated 72 hours at 27 ℃, collect the supernatant that contains recombinant baculovirus (r Bac-Cap).
Embodiment 2, carry and express AAV cap the structure of recombinant baculovirus rBac-RC of rep gene
The main purpose of present embodiment is to express the Cap gene of AAV simultaneously and duplicate genes involved Rep gene on same Bac carrier.
1, by the synthetic following dna fragmentation of ABI 381 dna synthesizers:
P52A:TCCA CCCGGGATGGAGCTGGTCGGGTGGCTCG, wherein italic is the SmaI site.
The P52B:TGGCAACTAGAAGGCACAGTCGATCAGAGAGAGTGTCCTCGAGCCAAT italic is and BGH polyA signal 5 ' primer complementary sequence.
BGHA:CTCTCTGA TCGACTGTGCCTTCTAGTTGCCA, italic is and the Rep52 complementary sequence.
The BGHB:GGCGTTATCGTTTATTGCCATAGAGCCCACCGCATCC italic is and the IEPromoter complementary sequence.
IE?promoter：
IE1：
GGATGCGGTGGGCTCTATGGCAATAAACGATAACGCCGTTGGTGGCGTGAG
GCATGTAAAAGGTTACATCATTATCTTGTTCATCCGGTTG
IE2：
CAAAAACCAACATGAACGTCTATTTATACCAACCGGATGAACAAGATAATG
ATGTAAC
IE3：
GTATAAATAGACGTTCATGTTGGTTTTTGTTTCAGTTGCAAGTTGGCTGCGG
CGCGCG CAG CACCTTTAGCCATGGCGGGGTTTTACG4GATTG, italic is and the Rep78 complementary sequence.
P78A：AGCCATGGCGGGGTTTTACGAGATTG
P78B:CGGAATTCCGCTAGCCACCACTGTCTTATTCCTTC, italic is the NheI site
2, be template with plasmid pAAV-RC 1ng, respectively with primer P52A+P52B, P78A+P78B carries out pcr amplification (condition is with embodiment 1), amplifies Rep52 gene and the Rep78 gene of AAV 2; With plasmid pCDNA3.1/Myc-His (Invitrogen company product) 1ng is template, carries out pcr amplification with primer PBHGH1 and PBGH2, obtains the Poly a-signal sequence of BGH; Above-mentioned sequence is carried out agarose electrophoresis, cutting contains the band of dna fragmentation, use the Gel Extraction Kit of QIAGEN to reclaim dna fragmentation, each 5ng of above-mentioned fragment, fragment (the IE1 that adds synthetic IE startup, IE2, IE3) each 5ng is a template with above-mentioned mixing tab section, use P52A and P78B to carry out pcr amplification, obtain to contain Rep52 and BGHpolyA signal thereof, the IE promotor of band deletion mutantion and the dna fragmentation of Rep78 gene as primer.
Above-mentioned fragment reaches and is all cut with the SmaI+NheI enzyme by example one constructed plasmid pBac-Cap, and connects as example 1 usefulness T4 dna ligase, and structure can be expressed AAV Cap albumen and the proteic baculovirus vector of Rep.The building process of plasmid is seen Fig. 2.
3, plasmid pBac-RC 1ng mixes with 100 μ l competence intestinal bacteria Max Efficiency DH10Bac, ice bath 42 ℃ of hot activations 45 seconds after 30 minutes, ice bath is 2 minutes immediately, add 900 μ l SOC substratum (seeing molecular cloning), 37 ℃ of cultivations are coated with flat board after 1 hour and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 100 μ g/ml BluoGal (Invitrogen company product) and 40 μ g/ml IPTG).37 ℃ of bacterium colonies of cultivating picking white after 48 hours are analyzed.Identifying that correct positive colony is seeded to contains incubated overnight in the above-mentioned antibiotic LB liquid nutrient medium, carries out plasmid extraction with the MidPrep test kit of Invitrogen company, obtains recombination bacillary viral vector DNA.
4, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection, cultivated 72 hours at 27 ℃, collect the supernatant that contains recombinant baculovirus (r Bac-RC).
Embodiment 3, carry the structure of the recombinant baculovirus rBac-ITR of AAV ITR sequence
1, each 200ng of vector plasmid pFastBacDual (invitrogen company product) carries out enzyme with Sma I+Stu I (being Promega company product) and cuts, and reclaims big fragment; Cut pAAV-MCS (Stratagene) with Pci I and Sfo I enzyme, the Klenow enzyme is mended flat, electrophoresis reclaims the fragment of carrying ITR, reclaims fragment with two and connects with T4 dna ligase (Promega company product), makes up and carries the baculovirus vector of adeno-associated virus ITR sequence.The building process of plasmid is seen Fig. 3.
2,16 ℃ after 6 hours, press the described condition transformed into escherichia coli of molecular cloning DH5 α, be coated with ammonia benzyl resistant panel, screen correct plasmid, obtain plasmid vector pBac-ITR.
3, plasmid pBac-ITR1ng mixes with 100 μ l competence intestinal bacteria Max Efficiency DH10Bac, ice bath 42 ℃ of hot activations 45 seconds after 30 minutes, ice bath is 2 minutes immediately, add 900 μ l SOC substratum (seeing molecular cloning), 37 ℃ of cultivations are coated with flat board after 1 hour and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 100 μ g/ml BluoGal (Invitrogen company product) and 40 μ g/ml IPTG).37 ℃ of bacterium colonies of cultivating picking white after 48 hours are analyzed.Be seeded to contain in the above-mentioned antibiotic LB liquid nutrient medium and cultivated 24 hours, carry out plasmid extraction, acquisition recombination bacillary viral vector DNA with the MidPrep test kit of Invitrogen company.
4, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection, cultivated 72 hours at 27 ℃, collect the supernatant that contains recombinant baculovirus (r Bac-ITR).Embodiment 4, carry and express the structure of the recombinant baculovirus rBac-Cap-ITR of AAV gene
1, plasmid pBac-Cap cuts with Avr II enzyme, and the Klenow enzyme is mended flat; Pci I and Sfo I enzyme are cut pAAV-MCS, and the Klenow enzyme is mended flat, and electrophoresis reclaims the fragment of carrying ITR, reclaim fragment with two and connect with T4 dna ligase (Promega company product), make up and carry the ITR sequence and express the proteic baculovirus vector of Cap.The building process of plasmid is seen Fig. 4.
2,16 ℃ after 6 hours, press the described condition transformed into escherichia coli of molecular cloning DH5 α, be coated with ammonia benzyl resistant panel, screen correct plasmid, obtain plasmid vector pBac-Cap-ITR.
3, plasmid pBac-Cap-ITR 1ng mixes with 100 μ l competence intestinal bacteria Max Efficiency DH10Bac, ice bath 42 ℃ of hot activations 45 seconds after 30 minutes, ice bath is 2 minutes immediately, add 900 μ l SOC substratum (seeing molecular cloning), 37 ℃ of cultivations are coated with flat board after 1 hour and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 100 μ g/ml BluoGal (Invitrogen company product) and 40 μ g/ml IPTG).37 ℃ of bacterium colonies of cultivating picking white after 48 hours are analyzed.Be seeded to contain in the above-mentioned antibiotic LB liquid nutrient medium and cultivated 24 hours, carry out plasmid extraction, acquisition recombination bacillary viral vector DNA with the MidPrep test kit of Invitrogen company.
4, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection, cultivated 72 hours at 27 ℃, collect the supernatant that contains recombinant baculovirus (r Bac-Cap-ITR).
The structure of embodiment 5, recombinant baculovirus rBac-Cap-Rep-ITR (rBac-AllinOneAAV)
1, plasmid pBac-RC cuts with Avr II enzyme, and the Klenow enzyme is mended flat; PAAV-MCS (LacZ or goal gene insert) cuts with Pci I and Sfo I enzyme, and the Klenow enzyme is mended flat, reclaims the gene fragment of band ITR, and the fragment ligase enzyme of handling with plasmid pBac-RC enzyme is connected.With connecting product transformed into escherichia coli DH5 α, filter out positive recombinant clone, called after pAllinOneAAV, it is standby to extract its plasmid DNA.Plasmid construction such as Fig. 5.
2, plasmid pAllinOneAAV 1ng mixes with 100 μ l competence intestinal bacteria Max Efficiency DH10Bac, ice bath 42 ℃ of hot activations 45 seconds after 30 minutes, ice bath is 2 minutes immediately, add 900 μ l SOC substratum (seeing molecular cloning), 37 ℃ of cultivations are coated with flat board after 1 hour and (contain 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins, 100 μ g/ml BluoGal (Invitrogen company product) and 40 μ g/ml IPTG).37 ℃ of bacterium colonies of cultivating picking white after 48 hours are analyzed.Be seeded to contain in the above-mentioned antibiotic LB liquid nutrient medium and cultivated 24 hours, carry out plasmid extraction, acquisition recombination bacillary viral vector DNA with the MidPrep test kit of Invitrogen company.
3, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection, cultivated 72 hours at 27 ℃, collect the supernatant that contains recombinant baculovirus (r Bac-AllinOneAAV).
Embodiment 6, and the proteic Sf9 clone of stably express Rep makes up
1, pIB/V5-his carrier (Invitrogen company) is cut with Hind III enzyme, and the Klenow enzyme is cut with Xba I enzyme after mending and putting down again, reclaims big fragment; PBac-RC cuts back to close the Rep gene with Sma I and Nhe I enzyme, with the carrier dna ligase of above-mentioned processing.With connecting product transformed into escherichia coli DH5 α, filter out positive recombinant clone, called after pIB-Rep, it is standby to extract its plasmid DNA.Plasmid construction such as Fig. 6.
2, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), and cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection.After 48 hours, collecting cell also is diluted to 1 * 10 27 ℃ of cultivations
4Cells/ml; Be inoculated on 96 well culture plates with nonresistant substratum doubling dilution then, screen resistance clone (concrete operations are according to the specification sheets of Invitrogen company) with the resistance substratum that contains proper concn after the incubated overnight.Obtain the proteic Sf9 clone of stably express AAV-Rep at last, called after Sf9-Rep.
The proteic Sf9 clone of embodiment 7, stably express AAV-Cap-Rep makes up
1, pIB/V5-his carrier BspH I, the Klenow enzyme is mended flat, reclaims big fragment; The pBac-Cap plasmid is cut with Avr II+Bst1107I enzyme, and the Klenow enzyme reclaims the Cap gene, with the carrier dna ligase of above-mentioned processing after mending and putting down.With connecting product transformed into escherichia coli DH5 α, filter out positive recombinant clone, called after pIB-Cap, it is standby to extract its plasmid DNA.
2, the pIB-Cap-K plasmid is cut with Hind III enzyme, and after the Klenow enzyme was mended and put down, XbaI enzyme cutting reclaimed big fragment; The Rep52-Rep78 pcr amplification product cuts back to close big fragment with Sma I+Nhe I enzyme, is connected with dna ligase with the pIB-Cap-K plasmid vector of previous processed.With connecting product transformed into escherichia coli DH5 α, filter out positive recombinant clone, called after pIB-Cap-Rep, it is standby to extract its plasmid DNA.Plasmid construction such as Fig. 7.
3, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), and cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection.After 48 hours, collecting cell also is diluted to 1 * 10 27 ℃ of cultivations
4Cells/ml; Be inoculated on 96 well culture plates with nonresistant substratum doubling dilution then, (screen resistance clone (concrete operations are according to the specification sheets of Invitrogen company) with the resistance substratum that contains proper concn after the incubated overnight.Obtain the proteic Sf9 clone of stably express AAV-Cap-Rep at last, called after Sf9-Cap-Rep.
Embodiment 8, stable Sf9 clone of carrying AAV-ITR make up
1, the pIB/V5-his carrier is cut with EcoR V enzyme; Pci I and Sfo I enzyme are cut pAAV-MCS, the Klenow enzyme is mended flat, electrophoresis reclaims the fragment of carrying ITR, to reclaim fragment is connected with T4 dna ligase (Promega company product) with carrier, 16 ℃ after 6 hours, press the described condition transformed into escherichia coli of molecular cloning DH5 α, be coated with ammonia benzyl resistant panel, screen correct plasmid, obtain plasmid vector pIB-ITR.It is standby to extract its plasmid DNA.Plasmid construction such as Fig. 8.
2, above-mentioned plasmid DNA is with Cellfectin reagent (Invitrogen company) transfection Sf9 cell (being undertaken by the test kit specification sheets), and cell cultures is in Grace ' s insect cell substratum (Invitrogen company) after the transfection.After 48 hours, collecting cell also is diluted to 1 * 10 27 ℃ of cultivations
4Cells/ml; Be inoculated on 96 well culture plates with nonresistant substratum doubling dilution then, screen resistance clone (concrete operations are according to the specification sheets of Invitrogen company) with the resistance substratum that contains proper concn after the incubated overnight.The Sf9 clone that AAV-ITR is surely carried in acquisition at last, called after Sf9-ITR.
The production of embodiment 9, reorganization AAV virion
1, material: r Bac-RC, r Bac-ITR and High-Five
TMCell
2, method:
Use Grace ' s substratum, 10% foetal calf serum is cultivated High-Five under 27-28 ℃ of condition
TM(Invitrogen company) insect cell, when growing to 80% individual layer, with the recombinant baculovirus (rBac-RC and r Bac-ITR) that makes up with 2 M.O.I coinfection insect cells, 27 ℃ of cultivations after 72 hours, collecting cell and supernatant.By the freeze thawing fragmentation, the centrifugal removal cell debris of 12000g, it is 10% to final concentration that supernatant adds PEG8000, the centrifugal acquisition virion precipitation of 12000g, through cesium chloride density gradient centrifugation, find in density to be that the isolating virion of 1.37g/ml is a reorganization AAV particle.According to tiring of the AAV of Stratagene company titration step measurements reorganization AAV, the result shows that every cell can reach 2 * 10
4Virion.
The production of embodiment 10, reorganization AAV virion
1, material: r Bac-AllinOneAAV and Sf21 cell
2, method:
Cultivate the Sf21 insect cell, under suitable stand density condition, with recombinant baculovirus (rBac-AllinOneAAV) infected insect cell that makes up, 27 ℃ cultivate 72 hours after, collecting cell and supernatant.By the freeze thawing fragmentation, the centrifugal removal cell debris of 12000g, it is 10% to final concentration that supernatant adds PEG8000, the centrifugal acquisition virion precipitation of 12000g, through cesium chloride density gradient centrifugation, find in density to be that the isolating virion of 1.37-1.4g/ml is a reorganization AAV particle.According to tiring of the AAV of Stratagene company titration step measurements reorganization AAV, the result shows that every cell can reach 4 * 10
4Virion.
The production of embodiment 11, reorganization AAV virion
1, material: r Bac-Cap-ITR and Sf9-Rep cell
2, method:
R Bac-Cap and r Bac-ITR coinfection Sf9-Rep insect cell line are produced the rAAV virus vector, and concrete operations are with embodiment 10.The result shows that every cell can reach 105 virions.
The production of embodiment 12, reorganization AAV virion
1, material: rBac-RC, r Bac-Cap, r Bac-Rep and Sf9-ITR cell
2, method:
R Bac-Rep and r Bac-Cap coinfection Sf9-ITR insect cell line are produced the rAAV virus vector, and concrete operations are with embodiment 10.
R Bac-RC infects the Sf9-ITR insect cell line and produces the rAAV virus vector, and concrete operations are with embodiment 10.The result shows that every cell can reach 4 * 10
4Virion.
AAV infected person embryonic kidney 293 cells that carry beta-galactosidase gene of embodiment 13, insect cell production
All insert the LacZ gene in the above-described embodiments on the multiple clone site between the ITR as reporter gene, because the rAAV that produces all carries the LacZ gene.Detect LacZ expression of gene situation and can react the rAAV titre of our production and the efficient of production system.
Be used in the rAAV-LacZ particle of producing in the insect cell, infect 293 cells of cultivating, after 24 hours, take a picture, the results are shown in Figure 9 with X-gal dyeing.
The result shows that rAAV-LacZ infected person embryonic kidney 293 cells that baculovirus produces can detect the expression of LacZ.
Claims (19)
1, a kind of virus vector of High-efficient Production recombinant adeno-associated virus, it is characterized in that: this virus vector is a recombination bacillary viral vector, in this carrier, be built with among Cap gene, Rep gene and this three of ITR sequence of adeno-associated virus one or several, wherein all comprise eukaryotic promoter, multiple clone site or target gene between the ITR sequence.
2, a kind of clone of High-efficient Production recombinant adeno-associated virus, it is characterized in that: this cell is to integrate insect cell line, in the karyomit(e) of this clone, be integrated with among Cap gene, Rep gene and this three of ITR sequence of adeno-associated virus one or several, wherein all comprise eukaryotic promoter, multiple clone site or target gene between the ITR sequence.
3, a kind of method of High-efficient Production recombinant adeno-associated virus, utilize claim 1 described recombination bacillary viral vector infected insect cell system or utilize the described recombination bacillary viral vector of claim 1 to infect the described integration insect cell line of claim 2, High-efficient Production recombinant adeno-associated virus.
4, the High-efficient Production recombinant adeno-associated virus production system that obtains of method according to claim 3, it is characterized in that: this system is made up of recombination bacillary viral vector and insect cell line, or is made up of recombination bacillary viral vector and integration insect cell line.
5, the virus vector of High-efficient Production recombinant adeno-associated virus according to claim 1 is characterized in that: the Cap gene that is built with adeno-associated virus in the described recombination bacillary viral vector.
6, the virus vector of High-efficient Production recombinant adeno-associated virus according to claim 1 is characterized in that: the Rep gene and the Cap gene that are built with adeno-associated virus in the described recombination bacillary viral vector.
7, the virus vector of High-efficient Production recombinant adeno-associated virus according to claim 1 is characterized in that: the ITR sequence that is built with adeno-associated virus in the described recombination bacillary viral vector.
8, the virus vector of High-efficient Production recombinant adeno-associated virus according to claim 1 is characterized in that: the Cap gene and the ITR sequence that are built with adeno-associated virus in the described recombination bacillary viral vector.
9, the virus vector of High-efficient Production recombinant adeno-associated virus according to claim 1 is characterized in that: the Rep gene, Cap gene and the ITR sequence that are built with adeno-associated virus in the described recombination bacillary viral vector.
10, the clone of High-efficient Production recombinant adeno-associated virus according to claim 2 is characterized in that: the Rep gene that is integrated with adeno-associated virus on the karyomit(e) of described integration insect cell line.
11, the clone of High-efficient Production recombinant adeno-associated virus according to claim 2 is characterized in that: the ITR sequence that is integrated with adeno-associated virus on the karyomit(e) of described integration insect cell line.
12, the clone of High-efficient Production recombinant adeno-associated virus according to claim 2 is characterized in that: the Rep gene and the ITR sequence that are integrated with adeno-associated virus on the karyomit(e) of described integration insect cell line.
13, High-efficient Production recombinant adeno-associated virus production system according to claim 4, it is characterized in that: described recombinant adeno-associated virus production system is by insect cell line Sf9 or Sf21 or High-Five
TM, the baculovirus vector that is built with the baculovirus vector of adeno-associated virus Rep gene and Cap gene and is built with adeno-associated virus ITR sequence forms.
14, High-efficient Production recombinant adeno-associated virus production system according to claim 4, it is characterized in that: described recombinant adeno-associated virus production system is by insect cell line Sf9 or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Rep gene, Cap gene and ITR sequence form.
15, High-efficient Production recombinant adeno-associated virus production system according to claim 4, it is characterized in that: described recombinant adeno-associated virus production system is by the integration insect cell line Sf9 that is integrated with adeno-associated virus Rep gene or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Cap gene and ITR sequence form.
16, High-efficient Production recombinant adeno-associated virus production system according to claim 4, it is characterized in that: described recombinant adeno-associated virus production system is by the integration insect cell line Sf9 that is integrated with adeno-associated virus ITR sequence or Sf21 or High-Five
TM, and the baculovirus vector that is built with adeno-associated virus Rep gene and Cap gene form.
17, according to claim 1 or 5 or 6 or 7 or the virus vector of 8 or 9 described High-efficient Production recombinant adeno-associated virus, it is characterized in that: described baculovirus vector is autographa californica nuclear polyhedrosis virus AcMNPV.
18, the method for High-efficient Production recombinant adeno-associated virus according to claim 3 is characterized in that: described described baculovirus vector is autographa californica nuclear polyhedrosis virus AcMNPV.
19, according to claim 4 or 13 or 14 or 15 or 16 described High-efficient Production recombinant adeno-associated virus production systems, it is characterized in that: described baculovirus vector is autographa californica nuclear polyhedrosis virus AcMNPV.
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| CN 03150374 CN1570121A (en) | 2003-07-25 | 2003-07-25 | Virus vector, cell line, method and production system for high effective production of recombinant adenovirus satellite virus |
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