CN117165629A - Composition for obtaining recombinant baculovirus vector in yeast cells and application - Google Patents
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Abstract
The present invention discloses a composition for obtaining recombinant baculovirus vectors in yeast cells. The composition comprises a baculovirus vector and one or more recombinant fragments; the baculovirus vector comprises a yeast replication element for maintaining replication and inheritance of the baculovirus vector, and one or more target sites corresponding to one or more of the recombinant fragments respectively; the target site is used for linearizing the baculovirus vector under the action of endonuclease so as to generate homologous recombination with the recombination fragment; the recombinant fragment comprises an upstream sequence of a target site, a target gene fragment and a downstream sequence of the target site from a 5 'end to a 3' end, and at least one target gene fragment is provided with an exogenous gene. The invention also discloses application of the composition in producing recombinant baculovirus vectors and producing recombinant baculovirus and recombinant adeno-associated viruses.
Description
Technical Field
The invention belongs to the field of gene recombination, and in particular relates to a composition for obtaining a recombinant baculovirus vector in a yeast cell and application thereof.
Background
Recombinant adeno-associated virus (rAAV) is one of members of the parvoviridae family, and is an icosahedral picornavirus that is unable to autonomously replicate and is free of a membrane. The vector has the characteristics of wide host range, low immunogenicity, high safety, capability of mediating long-term stable expression of exogenous genes in animals and the like, and is one of the vectors with the most application prospect in the current gene therapy field.
Adeno-associated virus (AAV) genomes are linear single stranded DNA molecules having a length of less than about 5,000 nucleotides (nt). The Inverted Terminal Repeat (ITR) flank the single nucleotide coding sequence of the nonstructural replication protein (Rep) and the structural protein (VP). The ITRs are self-complementary and their constitution enables the formation of energy-stable intramolecular double-stranded T-hairpins. These hairpin structures act as origins of viral DNA replication. The Rep gene encodes the Rep proteins Rep78, rep68, rep52, and Rep40.Rep78 and its splice variant Rep68 are translated from mRNA transcribed from the p5 promoter. Rep52 and its splice variant Rep40 are translated from mRNA transcribed from the p19 promoter. The Cap gene encodes VP proteins VP1, VP2, and VP3. These proteins form capsids, synthesized by two spliced mRNAs produced by transcription of the Cap gene from the p40 promoter.
In recent years, AAV has become a preferred viral vector for gene therapy because of its ability to effectively infect cells, integrate into a single chromosomal site in the human genome, and have a low risk of disease to humans. In view of these advantages, recombinant adeno-associated virus (rAAV) is currently used in gene therapy clinical trials for hemophilia b, malignant melanoma, cystic fibrosis, and other diseases.
AAV sequences used to produce AAV in insect cells may be derived from the genome of any AAV serotype. Typically, AAV serotypes have genomic sequences with significant sequence identity at the amino acid and nucleic acid levels, provide a range of genetic functions that are essentially identical, produce virions that are essentially physically and functionally identical, and replicate and assemble by essentially the same mechanisms.
Scientists have developed production systems that exploit the ability of baculoviruses to infect insect cells, i.e., to provide for the integration of Rep, cap and ITR core expression elements into 3 different baculovirus vectors, respectively, by Tn7 recombination, requiring co-infection of insect cells with these 3 recombinant baculovirus vectors to produce rAAV (uarabe et al 2002,Hum.Gene Ther.13:1935-1943; us20030148506; us 20040197895). On this basis, a production system simplified to 2 insect baculoviruses was further produced, i.e. the Rep and Cap expression cassettes were integrated into one insect baculovirus vector (Smith et al 2009, mol. Ther. 17:1888-1896). However, co-infection of two baculoviruses with cells is inefficient, the productivity of each cell cannot be fully utilized, and infection is a random process, so that empty-shell defect rAAV particles without nucleic acid are easy to generate, the preparation process conditions are complicated to optimize, and the quality of rAAVs prepared in different batches is unstable.
In 1982, the study of dr.max d.summers, and dr.gale Smith et al proposed a BacPAK baculovirus expression system that constructed a replication defective virus linearized by Bsu 36I cleavage by introducing a Bsu 36I cleavage site at each of the Orf1629 and Orf603 gene sites of AcMNPV, and the linearized virus, due to the deletion of the essential gene Orf1629, failed to produce an infectious virus, co-transfected host insect cells with the rescue recombinant DNA transfer vector, and the linearized virus, and the recombinant baculovirus was obtained after homologous recombination. However, since Bsu361 cannot 100% cleave viral DNA to linearize it, recombinant viruses still require plaque screening, which is time-consuming and labor-consuming.
Subsequently, lee et al have studied to propose a Bac-to-Bac baculovirus expression system that introduces a Bac artificial chromosome element containing mini-F replicon and Tn7 transposition site at the polyhedrin gene site of AcMNPV, allowing replication of baculovirus DNA in bacteria while allowing insertion of foreign gene into viral genome by Tn7 transposition, resulting in recombinant baculovirus. Although this system does not require plaque screening, it requires transposition recombination screening in bacteria, which is also time and labor consuming. And the recombinant baculovirus obtained by the system has an unstable phenomenon in serial passage.
Thus, a baculovirus system (One Bac system) was further developed on this basis, incorporating Rep, cap and ITR core expression elements into a baculovirus vector (CN 103827306A; CN 112553257A). However, the construction of baculovirus systems requires integration of Rep, cap and ITR into one baculovirus vector by one Tn7 transposition and one Red homologous recombination, or two Red homologous recombination, which is time consuming and laborious, and introduces additional resistance gene selection markers for each integration, increasing the safety risk.
Cas9 protein is an endonuclease that, by virtue of base pairing with a target DNA site via a partial sequence of the sgRNA, is able to guide Cas9 binding to this target site and cleavage. Conformational changes occur when Cas9 binds to the target gene site, and the nuclease functional region performs localized cleavage of the reverse strand of the target DNA. The end result of Cas9 mediated DNA cleavage is a double strand break (double strand break, DSB) of the target DNA (about 3-4 nucleotides upstream of PAM sequence). Referred to as Cas9-sgRNA system. This mechanism allows Cas9 proteins the ability to linearize circular genes for gene editing.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides a composition for generating a recombinant baculovirus vector and application thereof, thereby solving the technical problems of time and labor consumption, unstable quality and the like in screening in the prior art.
To achieve the above object, according to one aspect of the present invention, there is provided a composition for obtaining a recombinant baculovirus vector in a yeast cell, comprising a baculovirus vector and one or more recombinant fragments;
wherein the baculovirus vector comprises
Yeast replication elements for maintaining replication and inheritance of said baculovirus vector,
and one or more sites of interest corresponding to one or more of the recombinant fragments, respectively;
the target site is used for linearizing the baculovirus vector under the action of endonuclease so as to generate homologous recombination with the recombination fragment;
the recombinant fragment comprises an upstream sequence of a target site, a target gene fragment and a downstream sequence of the target site from a 5 'end to a 3' end, and at least one target gene fragment is provided with an exogenous gene.
Preferably, the yeast replication elements are CEN and ARS.
Preferably, the exogenous gene is a gene encoding a pharmaceutical polypeptide or protein.
Preferably, the destination site is Ac3, ac29, ac33, ac42, ac49, ac63, ac18, ac83, ac91, ac96, ac126, ac127, ac130 or Ac152.
Preferably, the number of the recombination fragments and the corresponding target sites is plural, and the distance between the adjacent target sites is less than 10k pb.
Preferably, the recombinant expression vector further comprises a parvovirus Cap gene expression cassette, a Rep gene expression cassette and an ITR core expression element which are positioned on the target gene fragment of the recombinant fragment;
the Cap gene expression cassette, the Rep gene expression cassette and the ITR core expression element are not positioned on the same recombinant fragment, and the exogenous gene is positioned in the ITR core expression element.
Preferably, the endonuclease is a Cas9 protein.
According to another aspect of the present invention, there is also provided a method for obtaining a recombinant baculovirus vector using the above composition and a recombinant baculovirus produced using the method.
According to another aspect of the present invention there is also provided a cell comprising the recombinant baculovirus vector described above.
Preferably, the cell is an insect cell or a yeast cell.
According to another aspect of the present invention, there is also provided a recombinant baculovirus or recombinant adeno-associated virus produced using the above cell.
In general, the above technical solutions conceived by the present invention, compared with the prior art, enable the following beneficial effects to be obtained:
1. the composition can be used for simultaneously inserting target gene fragments into a plurality of target sites through one-time recombination to obtain a recombinant baculovirus vector capable of stably replicating and inheriting in yeast cells; production time and cost are saved;
2. the recombination process does not need to carry out plaque screening or recombination screening in bacteria, so that the time and the labor are saved;
3. in the process of recombining the target gene, no additional resistance gene screening marker is introduced.
Drawings
FIG. 1 is a schematic diagram of the structure of a homologous recombination expression cassette according to example 1 of the present invention;
FIG. 2 is a schematic diagram of the structure of a recombinant fragment GFP-Ac91 in example 1 of the present invention;
FIG. 3 is a schematic diagram of the structure of the recombinant fragment Rep-Cap9-Ac91 in example 2 of the present invention;
FIG. 4 is a schematic diagram of the structure of the recombinant fragment ITR-GOI-Ac96 in example 2 of the present invention;
FIG. 5 is a schematic diagram of the construction process of recombinant baculovirus vector of example 2 of the present invention;
FIG. 6 is a fluorescent microscope observation of insect cells transfected with the recombinant baculovirus vector of example 3 of the present invention.
Detailed Description
A composition for obtaining a recombinant baculovirus vector in a yeast cell is disclosed, comprising a baculovirus vector and one or more recombinant fragments.
Wherein the baculovirus vector comprises a yeast replication element for maintaining replication and inheritance of the baculovirus vector in a yeast cell, the yeast replication element being CEN and ARS;
and one or more sites of interest corresponding to one or more of the recombinant fragments, respectively; the target site is Ac3, ac29, ac33, ac42, ac49, ac63, ac18, ac83, ac91, ac96, ac126, ac127, ac130 or Ac152, etc.
The recombinant fragment comprises an upstream sequence of a target site, a target gene fragment and a downstream sequence of the target site from a 5 'end to a 3' end, and at least one target gene fragment is provided with an exogenous gene. The foreign gene is typically a reporter gene or a gene encoding a pharmaceutical polypeptide or protein, such as lipoprotein esterase, apolipoprotein, cytokine, interleukin, interferon, and the like.
The number of the recombinant fragments is related to the number of the target genes to be inserted, and when only one target gene fragment needs to be inserted, only one recombinant fragment is needed; when a plurality of target gene fragments with different exogenous genes are required to be inserted, or some functional gene fragments are required to be inserted in addition to the exogenous genes, a plurality of recombinant fragments are required; for example, when constructing recombinant adeno-associated viral vectors, at least two recombinant fragments are required.
When the number of the recombination fragments and the corresponding target sites is plural, the distance between adjacent target sites is usually less than 10k pb in order to ensure smooth insertion of the recombination fragments.
On the target gene segment of the recombinant segment, besides the exogenous gene to be expressed, elements of parvovirus Cap gene expression cassette, rep gene expression cassette and ITR core expression element necessary for the expression of adeno-associated virus can be integrated; at this time, the foreign gene is located within the ITR core expression element, and the parvoviral Cap gene expression cassette, rep gene expression cassette, and ITR core expression element are located on different recombinant fragments.
Under the action of endonucleases such as Cas9 protein and the like, the target site of the baculovirus vector of the composition can be linearized, so that homologous recombination occurs between the target site and a corresponding recombination fragment, and the target gene fragment is integrated into the baculovirus vector to obtain the recombinant baculovirus vector. The recombinant baculovirus vector can be stabilized in yeast cells for replication and stable inheritance, and is used for obtaining recombinant baculovirus or recombinant adeno-associated virus, so as to produce proteins expressed by exogenous genes, such as encoded therapeutic drug polypeptides or proteins and the like.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Example 1 obtaining recombinant baculovirus vector containing Green Fluorescent Protein (GFP) exogenous Gene expression cassette
Construction of baculovirus vector AcMNPV-BAC-YAC containing Yeast replicating element by Step1
Red recombination is a bacterial-level efficient recombination method that can be used to rapidly engineer recombinant baculovirus genomes in E.coli (DH 10 Bac). Red recombination is the homologous recombination of a linear DNA fragment carrying a homology arm introduced into a cell with a specific target sequence of the genome using lambda phage Red recombinase (consisting of 3 proteins of Exo, beta and Gam) to effect replacement of the gene of interest (Doublet et al, 2008,J Microbiol Methods,75 (2): 359 361). At present, many baculovirus strains are studied as Autographa californica (Autographa californica) polynuclear polyhedrosis virus (multiple nuclear polyhedrosis virus, MNPV), abbreviated as AcMNPV. In the embodiment, a Red recombination technology is utilized to recombine a yeast replication element onto a Bac-to-Bac baculovirus expression vector, so as to obtain a baculovirus vector AcMNPV-BAC-YAC containing the yeast replication element.
Constructing a homologous recombination expression frame of a lacZ alpha site of a bac sequence on a targeting plasmid by using Step1.1, wherein the expression frame sequentially comprises an upstream homologous sequence of the lacZ alpha site, a yeast CEN6 centromere and autonomous replication sequence (CEN/ARS), a yeast auxotroph selection marker (HIS 3) expression frame, an escherichia coli chloramphenicol (Chl) resistance gene expression frame and a downstream homologous sequence of the lacZ alpha site from the 5 'end to the 3' end as shown in figure 1; wherein, chloramphenicol resistance gene is used as a screening mark for successfully inserting a homologous recombination expression frame into a Bac-to-Bac baculovirus expression vector in bacteria, and yeast auxotrophy selection mark (HIS 3) is used as a screening mark for successfully recombining a linearized AcMNPV-BAC-YAC vector and a recombination fragment in yeast cells.
Step1.2 inserts the expression cassette into the bacmid at the lacZ alpha site using Red recombination technology, thereby obtaining the baculovirus vector AcMNPV-BAC-YAC containing yeast replication elements.
Construction of recombinant fragment GFP-Ac91 containing GFP Gene by Step2
Firstly, PCR amplification is carried out to obtain a GFP gene expression cassette sequence, then, PCR amplification is carried out in the stem grain to obtain an Ac91 locus upstream homologous sequence Ac91-Hr-up and an Ac91 locus downstream homologous sequence down-Hr-Ac91, and then, the GFP gene expression cassette sequence, the Ac91-Hr-up sequence and the down-Hr-Ac91 sequence are connected in an overlapping PCR mode to obtain a recombinant fragment GFP-Ac91, as shown in figure 2.
Step3 construction of recombinant baculovirus vector AcMNPV-BAC-YAC-GFP containing GFP gene expression cassette
The baculovirus vector AcMNPV-BAC-YAC (gRNA was targeted at Ac91 site: TAATACATAGGTTCGCCCAA AGG) was first linearized with Cas 9. Then, the recombinant fragment GFP-Ac91 is replaced with Ac91 locus sequence on the baculovirus vector by a yeast transformation coupling homologous recombination (TAR) method, so that the recombinant baculovirus vector AcMNPV-BAC-YAC-GFP containing GFP gene expression cassette is obtained.
Example 2
Step1 construction of a Yeast replication element-containing baculovirus vector AcMNPV-BAC-YAC in the same procedure as in example 1
Step2 construction of recombinant fragment Rep-Cap9-Ac91 containing AAV9 Cap gene and AAV2 Rep gene
Step2.1 PCR-amplified from AAV genome to obtain Cap gene sequence of AAV9 and Rep gene sequence of AAV2, and PCR-amplified from the bacmid to obtain Ac91-Hr-up homologous sequence of Ac91 locus and down-Hr-Ac91 homologous sequence of Ac91 locus.
Step2.2 recombinant fragment Rep-Cap9-Ac91 is obtained by ligating the Cap gene sequence, the Rep gene sequence, the Ac91-Hr-up sequence, the down-Hr-Ac91 sequence and the corresponding promoter sequences by means of overlap PCR, as shown in FIG. 3.
Construction of recombinant fragment ITR-GOI-Ac96 containing ITR core element by Step2.3
To facilitate detection of AAV packaging rates, GOI in the ITR core element was tested using GFP gene expression cassettes as exogenous genes. The ITR core element containing GFP expression cassette was ligated with the Ac96-Hr-up sequence upstream of the Ac96 locus and the down-Hr-Ac96 sequence downstream of the Ac96 locus by overlap PCR to obtain recombinant fragment ITR-GOI-Ac96, as shown in FIG. 4.
Step3 construction of recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR containing AAV viral packaging element
Step3.1 linearizes the baculovirus vector AcMNPV-BAC-YAC with Cas9 at the Ac91 site and Ac96 site (sgRNA target at Ac91 site:
TAATACATAGGTTCGCCCAAAGG; sgrnas target at Ac96 locus: TCACACGCTCGTTCTCGATGTGG).
Step3.2A linearized baculovirus vector obtained in Step3.1 was co-transformed with recombinant fragments Rep-Cap9-Ac91 and ITR-GOI-Ac96 into yeast cells by a yeast transformation-coupled homologous recombination (TAR) method, rep-Cap9-Ac91 was recombined with the baculovirus vector at the Ac91 locus, ITR-GOI-Ac96 was recombined with the baculovirus vector at the Ac96 locus, and thus a recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR containing AAV packaging elements was obtained, and the overall procedure of example 2 was as shown in FIG. 5.
EXAMPLE 3 preparation of recombinant baculovirus vector AcMNPV-BAC-YAC-GFP transfected sf9 cells
Step1 is used for initially extracting saccharomyces cerevisiae (VL 6-48) genome containing recombinant baculovirus vector AcMNPV-BAC-YAC-GFP
The method comprises the following specific steps: (1) culturing the recombinant baculovirus vector obtained in the example 1 in yeast cells, screening, then picking and monoclonal culturing in SD-HIS culture medium at 28 ℃ overnight with shaking for 14-18h, and then culturing in 1:10 amplification until the OD600 is about 1.5; (2) 50ml of the culture medium was centrifuged at 1860g for 5min, and the supernatant was discarded, followed by 50ml of ddH 2 O is resuspended, 1860g is centrifuged for 5min, and the supernatant is discarded; (3) with 5ml SPE solution (1M sorbitol, 0.01M sodium phosphate solution (2.08 g/l Na) 2 HPO 4 ·7H 2 O,0.32g/l NaH 2 PO 4 ·H 2 O),0.01M Na 2 EDTA (pH 7.5)) resuspended cells; (4) adding 40 μl wall breaking enzyme (Yeasen), 10 μl beta-mercaptoethanol, processing at 30deg.C for 90min, centrifuging at 1294g at 4deg.C for 5min, and discarding supernatant; (5) the cells were resuspended in 250. Mu.l of 1M sorbitol, 3ml of lysate (0.05M Tris-HCl,0.02M EDTA,1% SDS (pH 12.8)) was added and treated at 37℃for 30min; (6) adding 5ml of phenol chloroform isoamyl alcohol, gently reversing for 20 times, centrifuging for 20min with 4194g, and extracting the upper layer solution; (7) adding 1/10 volume of 3M sodium humate and equal volume of isopropanol, gently inverting for several times, centrifuging at 4194g at 4deg.C for 30min, and discarding the supernatant; (8) rinsing with 70% ethanol twice, and air drying to remove liquid.
Step2 refined extraction of AcMNPV-BAC-YAC-GFP Saccharomyces cerevisiae (VL 6-48) genome containing recombinant baculovirus vector
Treatment with omega Bac/Yac extraction kit. Finally, the yeast genome containing the recombinant baculovirus vector AcMNPV-BAC-YAC-GFP is obtained.
Finally, transferring DH10B electrically, taking 20 mu l of the extracted yeast genome, uniformly mixing with 100 mu l of DH10B in a competent mode, performing electric shock on a 1.6KV/mm pulse field in a 1mm electric rotating cup, adding LB culture medium, performing shaking incubation at 37 ℃ for 90min, then coating the mixture in a LB+Kana+Chl plate, and performing overnight culture at 37 ℃. The single clone was picked up and cultured overnight with shaking in LB+Kana+Chl medium at 37℃and then the recombinant baculovirus vector AcMNPV-BAC-YAC-GFP was extracted with omega Bac/Yac extraction kit.
Step3 the obtained recombinant baculovirus vector DNA is used for transfecting Sf9 insect cells to prepare the recombinant baculovirus.
It was verified that the transfected Sf9 insect cells successfully produced BEV and that further infection with a large number of replicative and proliferating BEVs resulted in significant cytopathic effects (CPE) of Sf9 cells. And the transferred sf9 cells were observed with a fluorescence microscope with green fluorescence, as shown in fig. 6.
EXAMPLE 4 extraction of recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR transfected sf9 cells, preparation of recombinant baculovirus and packaging of AAV viral particles
Step1 is used for initially extracting saccharomyces cerevisiae (VL 6-48) genome containing recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR by wall breaking enzyme
The method comprises the following specific steps: (1) culturing the artificial chromosome of the candida glabrata obtained in the example 2 in yeast cells, screening, picking up a monoclonal, culturing in SD-HIS culture medium at 28 ℃ overnight under shaking for 14-18h, and then culturing in a 1:10 amplification manner until the OD600 is about 1.5; (2) 50ml of the culture medium was centrifuged at 1860g for 5min, and the supernatant was discarded, followed by 50ml of ddH 2 O is resuspended, 1860g is centrifuged for 5min, and the supernatant is discarded; (3) with 5ml SPE solution (1M sorbitol, 0.01M sodium phosphate solution (2.08 g/l Na) 2 HPO 4 ·7H 2 O,0.32g/l NaH 2 PO 4 ·H 2 O),0.01M Na 2 EDTA (pH 7.5)) resuspended cells; (4) adding 40 μl wall breaking enzyme (Yeasen), 10 μl beta-mercaptoethanol, processing at 30deg.C for 90min, centrifuging at 1294g at 4deg.C for 5min, and discarding supernatant; (5) the cells were resuspended with 250. Mu.l of 1M sorbitol and added3ml of lysate (0.05M Tris-HCl,0.02M EDTA,1% SDS (pH 12.8)), treated at 37℃for 30min; (6) adding 5ml of phenol chloroform isoamyl alcohol, gently reversing for 20 times, centrifuging for 20min with 4194g, and extracting the upper layer solution; (7) adding 1/10 volume of 3M sodium humate and equal volume of isopropanol, gently inverting for several times, centrifuging at 4194g at 4deg.C for 30min, and discarding the supernatant; (8) rinsing with 70% ethanol twice, and air drying to remove liquid.
Step2 refined the Saccharomyces cerevisiae (VL 6-48) genome containing the recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR. Treatment with omega Bac/Yac extraction kit. The final DNA concentration 1089 ng/. Mu.l, a260/a280=2.04, a260/a230=2.25.
Mu.l of the extracted yeast genome was taken, mixed with 100. Mu.l of DH10B competent, shocked in a 1mm electric cup with a pulse field of 1.6KV/mm, added with LB medium, incubated with shaking at 37℃for 90min, then plated on LB+Kana+Chl plates, and incubated overnight at 37 ℃. The single clone was picked up and cultured overnight at 37℃in LB+Kana+Chl medium, and then the recombinant baculovirus vector AcMNPV-BAC-YAC-RC-ITR was extracted using omega Bac/Yac extraction kit. A total of 5 samples were taken to test DNA concentrations as shown in table 1.
TABLE 1
Step3 transfects Sf9 insect cells with the obtained recombinant bacmid DNA to prepare recombinant baculovirus and package AAV viral particles.
The transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicative BEV resulted in significant cytopathic effects (CPE) of Sf9 cells. The culture supernatant of Sf9 cells, which had undergone CPE, was collected and contained a large amount of BEV, namely, BEV generation 0 (BEV P0). The prepared BEV P0 was infected with Sf9 cells cultured in suspension at a multiplicity of infection (moi=1), after 72 hours of infection, the cell activity was reduced to 50% or less, 1000g of the cell culture broth was centrifuged for 5min, and the culture supernatant was collected and labeled as BEV generation 1 (BEV P1).
The expansion culture was continued as described above until the number of infections with BEV-P2 seed viruses was measured (MOI=1) And (3) infecting the suspension-cultured Sf9 cells to package AAV, wherein the packaging volume is 300-400 mL. Cell activity was monitored 3 days after infection, after activity was less than 50%, cell cultures were harvested and 500. Mu.l of the harvested mixture (mixture of cells and supernatant) was placed in a 1.5ml EP tube and freeze-thawed repeatedly 4 times in a liquid nitrogen and 37℃water bath. 0.1. Mu.l of Benzonase was added to 500. Mu.l of the mixture, and the mixture was homogenized in a 37℃water bath for 1h and then in a 95℃water bath for 10min.2500g was centrifuged for 10min and the supernatant was collected in a fresh 1.5ml EP tube. 200. Mu.l of the supernatant was added to 10. Mu.l of 10% SDS solution to give a final concentration of 0.5% SDS. A further 1.2. Mu.l of Proteinase K (working concentration 112. Mu.g/ml) was added, the final volume being about 210. Mu.l, and the water bath was 55℃for 1h. After brief centrifugation, a 95℃water bath was used for 10min. After mixing, 5. Mu.l of the sample was removed therefrom and 145. Mu.l of ddH was added thereto 2 O makes the final volume 150. Mu.l; after mixing, 10. Mu.l of the sample was taken out and 90. Mu.l of ddH was added 2 O was set to a final volume of 100. Mu.l, and 2. Mu.l was taken as the Q-PCR template to measure the titer of the virus sample.
Verification of experimental results
The titer of BEV and AAV packaging titer in example 4 were determined by the method of fluorescent quantitative PCR (qPCR), the titer units being expressed in VG/mL (VG, viruses genome). Baculovirus titers were determined using the GP64 gene (QGP 64F: ATTGGCAATCTCATGAACAA and QGP 64R: CTTCTTTGTAGATGCTGTTG); AAV packaging titers were determined using a pair of primers targeting the ITR sequence (Q-ITR-F: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA). The results of the 5 samples in table 1 of corresponding example 4 are shown in table 2, respectively.
TABLE 2 baculovirus and AAV packaging titres
It can be seen that the recombinant baculovirus vector obtained in example 2 transfects cells and recombinant baculovirus is produced in the cells and AAV viral particles are packaged.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A composition for obtaining a recombinant baculovirus vector in a yeast cell, comprising a baculovirus vector and one or more recombinant fragments;
the baculovirus vector comprises
Yeast replication elements for maintaining replication and inheritance of said baculovirus vector,
and one or more sites of interest corresponding to one or more of the recombinant fragments, respectively;
the target site is used for linearizing the baculovirus vector under the action of endonuclease so as to generate homologous recombination with the recombination fragment;
the recombinant fragment comprises an upstream sequence of a target site, a target gene fragment and a downstream sequence of the target site from a 5 'end to a 3' end, and at least one target gene fragment is provided with an exogenous gene.
2. The composition of claim 1, wherein the yeast replication elements are CEN and ARS.
3. The composition of claim 1, wherein the exogenous gene is a gene encoding a pharmaceutical polypeptide or protein.
4. The composition of claim 1, further comprising a parvoviral Cap gene expression cassette, a Rep gene expression cassette, and an ITR core expression element located on the gene fragment of interest of the recombinant fragment;
the number of the recombination fragments and the corresponding target sites is multiple, the Cap gene expression cassette, the Rep gene expression cassette and the ITR core expression element are not positioned on the same recombination fragment, and the exogenous gene is positioned in the ITR core expression element.
5. The composition of claim 1, wherein the endonuclease is a Cas9 protein.
6. A method of obtaining a recombinant baculovirus vector using the composition of any one of claims 1-5.
7. A recombinant baculovirus vector produced using the method of claim 6.
8. A cell comprising the recombinant baculovirus vector of claim 7.
9. The cell of claim 8, wherein the cell is an insect cell or a yeast cell.
10. A recombinant baculovirus or recombinant adeno-associated virus produced using the cell of claim 8 or 9.
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