CN114736929B - Composition, method and application for producing recombinant baculovirus in insect cells - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly discloses a composition, a method and application for producing recombinant baculovirus in insect cells, wherein the composition comprises a packaging-defective baculovirus plasmid and first rescue recombinant DNA, the packaging-defective baculovirus plasmid lacks CNE sequences and/or NAE sequences in a baculovirus genome, the first rescue recombinant DNA comprises a first insertion sequence and first homology arms positioned at two sides of the first insertion sequence, the first insertion sequence comprises a first functional fragment and a first complement sequence, the first complement sequence is at least one of sequences deleted by the packaging-defective baculovirus plasmid, and the composition can carry out homologous recombination in the insect cells to produce the recombinant baculovirus. The composition provided by the invention can obtain the recombinant baculovirus only by one-step recombination in host insect cells, does not need screening, has no limitation of single recombination site, and has higher purity in preparation of the recombinant baculovirus.

Description

Composition, method and application for producing recombinant baculovirus in insect cells

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a composition, a method and application for producing recombinant baculovirus in insect cells.

Background

Baculoviruses are a class of circular double stranded DNA viruses with envelope encapsulation. At present, many baculovirus strains are studied as Autographa californica (Autographa californica) polynuclear polyhedrosis virus (multiple nuclear polyhedrosis virus, MNPV), abbreviated as AcMNPV. Baculovirus expression systems (BEVS) are eukaryotic expression systems that utilize insect baculovirus vectors to infect host insect cells to produce foreign proteins.

Kitts et al have studied to propose BacPAK baculovirus expression system, which inserts lacZ gene into the position of polyhedra virus gene locus, constructs a replication defective virus linearized by Bsu 36I enzyme digestion by introducing a Bsu 36I enzyme digestion site into orf1629 and orf603 gene locus, and the linearized virus, even if it is unable to produce virus with infectious activity by self-ligation, will rescue recombinant DNA transfer vector and linearized virus co-transfect host insect cells, and the recombinant baculovirus is obtained after homologous recombination. However, recombinant viruses still require plaque screening, which is time-consuming and labor-consuming, since Bsu 36I enzyme cannot cleave viral DNA 100% to linearize it.

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 the system does not require plaque screening, it requires transposition recombination screening in bacteria, which is time and labor consuming as well; and the recombinant baculovirus obtained by the system has an unstable phenomenon in serial passage.

In 2003, zhao et al have proposed a flash-BAC system that inactivates the essential gene orf1629 while introducing a mini-F replicon into the AcMNPV genome, and baculoviruses that inactivate the essential gene orf1629 fail to produce infectious active viruses unless homologous recombination occurs with rescue recombinant DNA transfer vectors. The system combines the advantages of BacPAK and Bac-to-Bac, and can obtain the recombinant baculovirus by only one-step recombination in host insect cells without screening. However, since orf1629 is a trans-element protein associated with replication, viral DNA that has undergone homologous recombination will serve the function of orf1629 and since viral DNA that has not undergone homologous recombination will also be packaged, resulting in the final recombinant baculovirus being impure.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a composition, a method and application for producing recombinant baculovirus in insect cells, and aims to solve the problems that viral DNA which does not undergo homologous recombination is packaged in the existing process of producing recombinant baculovirus by using a flash-BAC system, and the viral DNA which has undergone homologous recombination can provide orf1629 function, so that the recombinant baculovirus is impure.

To achieve the above object, the present invention provides a composition for producing a recombinant baculovirus in an insect cell, comprising a packaging-deficient baculovirus plasmid and a first rescue recombinant DNA, the packaging-deficient baculovirus plasmid lacking a CNE sequence and/or a NAE sequence in the baculovirus genome, the first rescue recombinant DNA comprising a first insertion sequence and a first homology arm flanking the first insertion sequence, the first insertion sequence comprising a first functional fragment and a first complementing sequence, the first complementing sequence being at least one of the sequences deleted by the packaging-deficient baculovirus plasmid, the composition being capable of homologous recombination in an insect cell to produce a recombinant baculovirus.

Preferably, the first functional fragment is a cap gene expression cassette of an AAV and a rep gene expression cassette of an AAV.

Preferably, the first insertion sequence comprises the cap gene expression cassette, the first complement sequence, and the rep gene expression cassette in order from 5 'to 3'.

Preferably, the first insertion sequence comprises the rep gene expression cassette, the first complement sequence and the cap gene expression cassette in order from 5 'to 3'.

Preferably, the first complement sequence is located between the cap gene expression cassette and the rep gene expression cassette, and both ends thereof are close to the start end of the cap gene expression cassette and the start end of the rep gene expression cassette, respectively.

Preferably, the packaging-defective baculovirus plasmid is a recombinant bacmid comprising an AAV ITR core expression element with a foreign gene.

Preferably, the recombinant bacmid is obtained by baculovirus transfer vector mediated transposition of Tn 7.

Preferably, the first functional fragment is an AAV ITR core expression element with a foreign gene.

Preferably, the packaging-defective baculovirus plasmid is a recombinant bacmid comprising an AAV cap gene expression cassette and an AAV rep gene expression cassette.

Preferably, the recombinant bacmid is obtained by baculovirus transfer vector mediated transposition of Tn 7.

Preferably, the first functional fragment is a reporter gene.

Preferably, the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.

Preferably, the composition further comprises a second rescue recombinant DNA, the packaging-deficient baculovirus plasmid lacks a CNE sequence and a NAE sequence in the baculovirus genome, the second rescue recombinant DNA comprising a second insertion sequence and a second homology arm flanking the second insertion sequence, the second insertion sequence comprising a second complement sequence, the first complement sequence being a CNE sequence or a NAE sequence, the second complement sequence being a sequence of the CNE sequence and the NAE sequence different from the first complement sequence.

Preferably, the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and the second insertion sequence further comprises a second functional fragment which is an AAV ITR core expression element with exogenous genes.

According to a further aspect of the present invention there is provided the use of any of the compositions described above for the preparation of recombinant baculovirus and/or recombinant adeno-associated virus in insect cells.

According to another aspect of the invention there is provided an insect cell comprising any of the compositions described above.

According to another aspect of the invention there is provided a method of growing or producing recombinant baculovirus in vitro comprising co-transfecting an insect cell with any of the compositions described above and culturing the insect cell.

According to another aspect of the invention there is provided a method of growing or producing recombinant adeno-associated virus in vitro comprising co-transfecting an insect cell with any of the compositions described above and culturing the insect cell.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

the invention improves on the basis of a flash-BAC system, constructs a packaging-defective baculovirus plasmid, lacks CNE sequences and/or NAE sequences in a baculovirus genome, and cannot normally package baculovirus unless homologous recombination occurs with rescue recombinant DNA. The system can obtain the recombinant baculovirus by only one-step recombination in host insect cells without screening; meanwhile, the insertion sequence can be inserted into any locus of a baculovirus genome, is not limited by the insertion site, can be inserted into a target site according to the requirement, can realize the expression of various exogenous proteins from a single baculovirus, and has higher flexibility. In addition, when the system is used for preparing the recombinant baculovirus, only the virus DNA subjected to homologous recombination is packaged into the recombinant baculovirus, and the virus DNA not subjected to homologous recombination is not packaged, so that the finally obtained recombinant baculovirus is purer.

Drawings

FIG. 1 is a schematic diagram of a first homologous recombination expression cassette of a targeting CNE sequence constructed in example 1 of the present invention.

FIG. 2 is a schematic diagram of a second homologous recombination expression cassette for the targeting NAE sequence constructed in example 1 of the present invention.

FIG. 3 is a schematic diagram of a third homologous recombination expression cassette for the targeting NAE sequence constructed in example 1 of the present invention.

FIG. 4 is a schematic diagram of recombinant DNA fragment Ac96-CNE-GFP with insertion site of orf96 constructed in example 2 of the present invention.

FIG. 5 is a schematic diagram of a recombinant DNA fragment Ac96-NAE-mcherry with an insertion site of orf96 constructed in example 2 of the present invention.

FIG. 6 is a schematic diagram of a recombinant DNA fragment Ac96-Rep-CNE-Cap comprising AAV Cap and Rep expression cassettes constructed in example 2 of the present invention.

FIG. 7 is a schematic diagram of a recombinant DNA fragment Ac96-Rep-NAE-Cap comprising AAV Cap and Rep expression cassettes constructed in example 2 of the present invention.

FIG. 8 is a graph showing green fluorescent plaques produced by co-transfection of insect host cells with a defective baculovirus vector DeltaCNE-Bac and the recombinant DNA fragment Ac96-CNE-GFP according to example 3 of the present invention.

FIG. 9 is a graph showing the effect of expressing green fluorescence after infection of cells with recombinant baculovirus produced by co-transfecting insect host cells with the defective baculovirus vector DeltaCNE-Bac and the recombinant DNA fragment Ac96-CNE-GFP in example 3 of the present invention.

FIG. 10 is a graph showing red fluorescence plaques produced in example 3 of the present invention after co-transfection of insect host cells with a defective baculovirus vector DeltaNAE-Bac and a recombinant DNA fragment Ac 96-NAE-mcherry.

FIG. 11 is a graph showing the effect of red fluorescence expression after infection of cells with recombinant baculovirus produced by co-transfection of insect host cells with a defective baculovirus vector ΔNAE-Bac and recombinant DNA fragment Ac96-NAE-mcherry in example 3 of the present invention.

FIGS. 12A-F are schematic representations of recombinant DNA fragments constructed in example 4 of the present invention with insertion sites at orf83, orf126 and orf152, respectively.

FIG. 13 is a graph showing green fluorescence plaques produced in example 4 of the present invention after co-transfection of insect host cells with defective baculovirus vector DeltaCNE-Bac with recombinant DNA fragments Ac83-CNE-GFP, ac126-CNE-GFP and Ac152-CNE-GFP, respectively. FIG. 14 is a graph showing the effect of the recombinant baculovirus of example 4 on expressing green fluorescence after infection of cells by the recombinant baculovirus produced by co-transfecting insect host cells with the defective baculovirus vector DeltaCNE-Bac with the recombinant DNA fragments Ac83-CNE-GFP, ac126-CNE-GFP and Ac152-CNE-GFP, respectively.

FIG. 15 is a graph showing red fluorescence plaques produced in example 4 of the present invention after co-transfection of insect host cells with defective baculovirus vector DeltaNAE-Bac with recombinant DNA fragments Ac83-NAE-mcherry, ac126-NAE-mcherry and Ac152-NAE-mcherry, respectively.

FIG. 16 is a graph showing the effect of the recombinant baculovirus produced in example 4 on expressing red fluorescence after infection of cells with the recombinant DNA fragments Ac83-NAE-mcherry, ac126-NAE-mcherry and Ac152-NAE-mcherry, respectively, after co-transfection of insect host cells with the defective baculovirus vector DeltaNAE-Bac.

FIG. 17 is a Western Blot detection of VP capsid protein and Rep protein expression after co-transfection of insect host cells with defective baculovirus vector DeltaCNE-Bac and recombinant DNA fragment Ac96-Rep-CNE-Cap according to example 5 of the invention.

FIG. 18 is a Western Blot detection of VP capsid protein and Rep protein expression after co-transfection of insect host cells with a defective baculovirus vector DeltaNAE-Bac and a recombinant DNA fragment Ac96-Rep-NAE-Cap according to example 5 of the invention.

FIG. 19 is a schematic diagram of a recombinant DNA fragment Ac83-ITR-NAE containing the AAV core expression element ITR-GOI constructed in example 8 of the present invention.

FIG. 20 is a silver staining test chart of SDS-PAGE of purified recombinant AAV virions after transfection of host cells with three recombinant baculoviruses according to example 9 of the present invention, each showing three capsid proteins VP1/VP2/VP3.

Detailed Description

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.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present invention, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.

As used herein, an expression cassette refers to a nucleic acid construct comprising a coding sequence and a regulatory sequence operably linked when introduced into a host cell, resulting in transcription and/or translation of RNA or polypeptide, respectively. Expression cassette is understood to include a promoter that allows transcription to begin, a gene open reading frame of interest, and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance from the gene of interest that is compatible with expression control.

Cis-acting elements refer to specific DNA sequences in tandem with structural genes, which are binding sites for transcription factors that regulate the precise initiation of gene transcription and transcription efficiency by binding to the transcription factors. Cis-acting elements include promoters, enhancers, regulatory sequences, inducible elements, etc., whose function is to be involved in the regulation of gene expression, do not itself encode any protein, and provide only one site of action.

AAV is a single-stranded DNA virus with a simple genome structure and a full length of about 4.7kb, and contains a rep gene expression cassette, a cap gene expression cassette, and AAV inverted terminal repeats (inverted terminal repeats, ITR) located at both ends of the genome. These are the three elements necessary for packaging AAV viruses. The Cap gene encodes a structural VP capsid protein that comprises three overlapping open reading frames, encoding three types of subunits VP1, VP2, VP3, respectively. The Rep gene encodes four overlapping multifunctional proteins, rep78, rep68, rep52 and Rep40, which are involved in replication and integration of AAV. ITR is a 125 nucleotide palindromic structure at both ends of the genome, which can form a self-complementary inverted T-shaped hairpin structure, and is a cis-acting element required for DNA replication initiation and packaging of recombinant AAV genomes as infectious viral particles. AAV, which is a defective virus, cannot replicate independently in the absence of helper virus, and therefore, AAV is only site-specific for integration into the host cell chromosome, and is thus latent. In the presence of helper virus, increased rep gene expression can rescue AAV genomes integrated in host cell chromosomes, and the AAV DNA is obtained by mass replication, and single-stranded rAAV genomes are packaged into infectious virions under the action of VP capsid proteins.

A conserved non-protein-coding element (CNE) was found in the genome of all sequenced A-type baculoviruses, with a high homology of 154-157bp. It has been reported that an at-rich CNE sequence located in the ac152 region of the noctiluca californica nuclear polyhedrosis virus (Autographa californica multiple nucleopolyhedro-virus, acMNPV) genome is a cis-acting element necessary for virion production.

NAE sequences were originally found to be a necessary element of nucleocapsid assembly found in the genus A, which plays an essential role in nucleocapsid assembly. The native NAE sequence is located in the ac83 gene and its homologous genes in the genus A, and is located at the proximal end thereof (CN 106566829A). ac83 is a core gene associated with baculovirus nucleocapsid assembly, full length 2544bp, encoding 847 amino acids, predicted molecular weight 96.2kDa. Knock-out ac83 does not affect replication of the viral genome, but completely blocks assembly of viral nucleocapsids, and the appearance of large numbers of hollow capsid precursors in the nucleus can be observed under electron microscopy.

The present invention provides a composition for producing a recombinant baculovirus in an insect cell, comprising a packaging-deficient baculovirus plasmid and a first rescue recombinant DNA, the packaging-deficient baculovirus plasmid lacking a CNE sequence and/or a NAE sequence in the baculovirus genome, the first rescue recombinant DNA comprising a first insertion sequence and a first homology arm flanking the first insertion sequence, the first insertion sequence comprising a first functional fragment and a first complementing sequence, the first complementing sequence being at least one of the sequences deleted by the packaging-deficient baculovirus plasmid, the composition being capable of homologous recombination within an insect cell to produce a recombinant baculovirus.

It should be noted that the packaging-defective baculovirus plasmid according to the present invention cannot normally package recombinant baculovirus due to deletion of CNE sequence and/or NAE sequence in baculovirus genome. The CNE sequence may be a CNE sequence that is identical to the wild-type AcMNPV CNE sequence, or may be a CNE sequence from another baculovirus, or an artificial CNE sequence that shares at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or more sequence identity with the wild-type AcMNPV CNE sequence. Likewise, the NAE sequence may be a NAE sequence that is identical to the wild-type AcMNPV NAE sequence, or may be a NAE sequence from another baculovirus, or an artificial NAE sequence that shares at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or more with the wild-type AcMNPV NAE sequence.

Furthermore, when the packaging-defective baculovirus plasmid lacks a CNE sequence in the baculovirus genome, the corresponding first complementing sequence is a CNE sequence; when the packaging-defective baculovirus plasmid lacks a NAE sequence in the baculovirus genome, the corresponding first complementing sequence is a NAE sequence; when the packaging-deficient baculovirus plasmid lacks both CNE sequences and NAE sequences in the baculovirus genome, the corresponding first complementing sequence comprises both CNE sequences and NAE sequences. So that the recombinant baculous particles after homologous recombination in insect cells contain normal CNE and NAE sequences, and recombinant baculovirus can be packaged; when homologous recombination does not occur, CNE or NAE sequences are deleted on the bacmid, so that the recombinant baculovirus cannot be packaged normally. In another embodiment, the deleted sequence can also be made up together with the first rescue recombinant DNA by using a second rescue recombinant DNA comprising a second insertion sequence comprising a second complement sequence, the first complement sequence being a CNE sequence or a NAE sequence, and a second homology arm flanking the second insertion sequence, the second complement sequence being a sequence different from the first complement sequence, of the CNE sequence and the NAE sequence when the defective baculovirus plasmid is packaged and the CNE sequence and the NAE sequence in the baculovirus genome are deleted simultaneously. Notably, the first homology arm and the second homology arm herein are homologous sequences corresponding to different loci on the baculovirus genome, such that the first insertion sequence and the second insertion sequence are capable of insertion into different loci on the baculovirus genome.

The rescue recombinant DNA according to the present invention may be a linear DNA fragment or a baculovirus transfer vector, and is not limited thereto. In addition, the rescue recombinant DNA provided by the invention has no restriction of insertion sites, and when homologous recombination occurs between the rescue recombinant DNA and the packaging-defective baculovirus plasmid, the insertion sequence on the rescue recombinant DNA can be inserted into any gene locus of a baculovirus genome, such as but not limited to Ac18, ac83, ac96, ac126, ac127, ac130 and Ac152.

The first functional fragment in the invention can be a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and also can be an AAV ITR core expression element (i.e. ITR-GOI) with exogenous genes, which are used for preparing recombinant adeno-associated viruses. The exogenous Gene may be at least one nucleotide sequence encoding a Gene of Interest (GOI) product, which may be a therapeutic Gene product, and in particular may be a polypeptide, RNA molecule (siRNA) or other Gene product, such as but not limited to lipoprotein esterase, apolipoprotein, cytokine, interleukin or interferon; also useful are reporter proteins that evaluate vector transformation and expression, such as, but not limited to, fluorescent proteins (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyl transferase, beta-galactosidase, beta-glucuronidase, renilla luciferase, firefly luciferase, or alkaline phosphatase. Furthermore, the first functional fragment may also be a reporter gene, such as, but not limited to, a gene sequence expressing a fluorescent protein (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyl transferase, beta-galactosidase, beta-glucuronidase, renilla luciferase, firefly luciferase, or alkaline phosphatase, for evaluation to verify whether homologous recombination has occurred to produce recombinant baculovirus. The first functional fragment may also be any nucleotide sequence encoding a therapeutic gene product, such as, but not limited to, a polypeptide encoding a drug (e.g., an interleukin, etc.) or a viral recombinant subunit protein.

In the embodiment of the present invention, the specific positions and directions of the cap gene expression cassette and the rep gene expression cassette are not limited, and six arrangements of the cap gene expression cassette, the rep gene expression cassette and the first complement sequence may be provided: 1. the cap gene expression cassette, the rep gene expression cassette and the first complement sequence are sequentially arranged from the 5 'end to the 3' end; 2. sequentially arranging a rep gene expression cassette, a cap gene expression cassette and a first complement sequence from a 5 'end to a 3' end; 3. the cap gene expression cassette, the first complement sequence and the rep gene expression cassette are sequentially arranged from the 5 'end to the 3' end; 4. sequentially arranging a rep gene expression cassette, a first complement sequence and a cap gene expression cassette from a 5 'end to a 3' end; 5. sequentially arranging a first complement sequence, a cap gene expression cassette and a rep gene expression cassette from a 5 'end to a 3' end; 6. the first complement sequence, the rep gene expression cassette and the cap gene expression cassette are sequentially arranged from the 5 'end to the 3' end. The cap gene expression cassette and the rep gene expression cassette may be in the same direction or in opposite directions. The order of the cap gene expression cassettes in the recombinant expression cassettes may be either from 5 'to 3' or from 3 'to 5'. Similarly, the sequence of rep gene expression cassettes in the recombinant expression cassette may be either 5 'to 3' or 3 'to 5'.

As a preferred embodiment, the first complement sequence is located between the cap gene expression cassette and the rep gene expression cassette, in particular, the first insert sequence comprises the cap gene expression cassette, CNE sequence and rep gene expression cassette in 5 'to 3' order, or the first insert sequence comprises the cap gene expression cassette, NAE sequence and rep gene expression cassette in 5 'to 3' order, or the first insert sequence comprises the rep gene expression cassette, CNE sequence and cap gene expression cassette in 5 'to 3' order, or the rep gene expression cassette, NAE sequence and cap gene expression cassette in 5 'to 3' order. Further preferably, two ends of the first complement sequence are respectively close to the start end of the cap gene expression cassette and the start end of the rep gene expression cassette, that is, the directions of the cap gene expression cassette and the rep gene expression cassette are opposite, and the start ends of the cap gene expression cassette and the rep gene expression cassette are opposite to each other and face the first complement sequence.

The invention provides an insect cell, which comprises any one of the compositions.

The invention provides a method for growing or producing recombinant baculovirus in vitro, which comprises co-transfecting insect cells with any one of the compositions described above, and culturing the insect cells. And then recovering the recombinant baculovirus.

The invention also provides a method for in vitro growth or production of recombinant adeno-associated virus, which comprises co-transfecting insect cells with the composition and culturing the insect cells to produce recombinant adeno-associated virus. It should be emphasized that the composition required for the preparation of recombinant adeno-associated virus needs to contain the cap gene, rep gene and ITR core expression element of AAV necessary for rAAV production, and therefore, if the first rescue recombinant DNA in the composition contains the cap gene expression cassette of AAV and the rep gene expression cassette of AAV, it is also necessary to insert the AAV ITR core expression element with foreign gene in the packaging-deficient baculovirus plasmid, which can be obtained by baculovirus transfer vector mediated transposition of Tn 7. If the first rescue recombinant DNA in the composition comprises AAV ITR core expression elements with foreign genes, it is also necessary to insert AAV cap gene expression cassettes and AAV rep gene expression cassettes in packaging-deficient baculovirus plasmids, which can be obtained by baculovirus transfer vector mediated Tn7 transposition. In addition, the rAAV can be achieved by two rescue recombinant DNA, and the packaging defective baculovirus plasmid simultaneously lacks a CNE sequence and a NAE sequence in a baculovirus genome, for example, a first rescue recombinant DNA comprises a cap gene expression cassette of AAV, a rep gene expression cassette of AAV and the CNE sequence, and a second rescue recombinant DNA comprises an AAV ITR core expression element with a foreign gene and the NAE sequence.

The following describes the above technical scheme in detail with reference to specific embodiments.

EXAMPLE 1 construction of a bacmid DeltaCNE-Bac lacking CNE sequence, a bacmid DeltaNAE-Bac lacking NAE sequence, and a bacmid DeltaCNE-DeltaNAE-Bac lacking both CNE and NAE sequences

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).

Firstly, constructing a first homologous recombination expression frame (SEQ ID No. 1) of a targeting CNE sequence, wherein the expression frame sequentially comprises a CNE upstream homologous sequence, a chloramphenicol (Chol) resistance gene expression frame and a CNE downstream homologous sequence from 5 'to 3' as shown in FIG. 1; then, the expression frame and the CNE sequence on the bacmid are replaced by utilizing Red recombination technology, so that the bacmid delta CNE-Bac with the CNE sequence deleted is obtained.

Similarly, a second homologous recombination expression cassette (SEQ ID No. 2) of the targeting NAE sequence is firstly constructed, and as shown in FIG. 2, the expression cassette sequentially comprises an NAE upstream homologous sequence, a chloramphenicol (Chol) resistance gene expression cassette and an NAE downstream homologous sequence from 5 'to 3'; then, the expression frame and NAE sequence on the bacmid are replaced by utilizing Red recombination technology, so that bacmid delta NAE-Bac lacking NAE sequence is obtained.

Likewise, a third homologous recombination expression cassette (SEQ ID No. 3) targeting the NAE sequence is first constructed, which expression cassette comprises, in order from 5 'to 3', an NAE upstream homologous sequence, a Gentamicin (GM) resistance gene expression cassette, and an NAE downstream homologous sequence, as shown in FIG. 3; then, the expression frame and NAE sequence on the bacmid delta CNE-Bac are replaced by utilizing Red recombination technology, thereby obtaining the bacmid delta CNE-delta NAE-Bac with CNE and NAE sequences deleted simultaneously.

EXAMPLE 2 construction of recombinant DNA transfer vectors containing CNE or NAE sequences

The various embodiments of the present invention use Green Fluorescent Protein (GFP) or red fluorescent protein (mcherry) as a foreign gene for insertion into the AcMNPV genome. Constructing a first recombinant DNA fragment with an exogenous gene insertion site at an AcMNProof 96 locus: referring to FIG. 4, the recombinant DNA fragment comprises an orf96 upstream homologous sequence, a CNE sequence (SEQ ID No. 4), a GFP expression cassette and an orf96 downstream homologous sequence in sequence from 5 'to 3', and the sequences are connected through artificial direct synthesis or overlap extension PCR amplification to respectively obtain a construct Ac96-CNE-GFP. Similarly, construct Ac96-NAE-mcherry (FIG. 5) containing NAE sequence (SEQ ID No. 5) was constructed. The nucleotide sequences of the construct Ac96-CNE-GFP and the construct Ac96-NAE-mcherry are shown as SEQ ID No.6 and SEQ ID No.7 respectively.

Construction of a second recombinant DNA fragment of AAV rep and cap gene expression cassettes at the AcMNPVorf96 locus at the insertion site: referring to FIG. 6, the recombinant DNA fragment comprises an orf96 upstream homologous sequence, a Rep gene expression cassette (SEQ ID No. 8), a CNE sequence, a Cap gene expression cassette (SEQ ID No. 9) and an orf96 downstream homologous sequence in sequence from 5 'to 3', and the sequences are connected through artificial direct synthesis or overlap extension PCR amplification to respectively obtain a construct Ac96-Rep-CNE-Cap. Similarly, construct Ac96-Rep-NAE-Cap (FIG. 7) containing NAE sequence was constructed. The nucleotide sequences of the construction Ac96-Rep-CNE-Cap and the construction Ac96-Rep-NAE-Cap are respectively shown as SEQ ID No.10 and SEQ ID No. 11.

EXAMPLE 3 verification of recombinant baculovirus production by homologous recombination after cotransfection of packaging-defective baculovirus vector and recombinant DNA fragment into insect host cells

In this example, the packaging-deficient baculovirus vectors ΔCNE-Bac and ΔNAE-Bac constructed in example 1 were co-transfected with Sf9 insect host cells, respectively, and after 96 hours of co-transfection, fluorescent microscopy was used to determine whether the host cells were capable of producing green or red fluorescent plaques to determine the production of recombinant baculovirus, with the corresponding recombinant DNA fragments Ac96-CNE-GFP and Ac96-NAE-mcherry constructed in example 2. To further confirm that co-transfection did produce recombinant baculoviruses, sf9 cell culture supernatant was collected 120h after co-transfection, host cells were transferred to suspension culture, and after 72h infection, green fluorescence or red fluorescence luminescence was observed with a fluorescence microscope.

The observation results are shown in fig. 8 to 11: the baculovirus vector delta CNE-Bac and the recombinant DNA fragment Ac96-CNE-GFP co-transfect Sf9 cells for 96 hours to generate green fluorescence plaques (figure 8), after 120 hours of co-transfection, sf9 cell culture supernatant is collected, host cells cultured in a suspension mode are transferred, and a large amount of green fluorescence is generated after 72 hours of infection (figure 9); red fluorescent plaques were produced by co-transfection of Sf9 cells with the baculovirus vector DeltaNAE-Bac and the recombinant DNA fragment Ac96-NAE-mcherry for 96h (FIG. 10), and after 120h of co-transfection, sf9 cell culture supernatants were collected and transferred to suspension-cultured host cells, which produced a large amount of red fluorescence after 72h of infection (FIG. 11). This example shows that the packaging-deficient baculovirus vector delta CNE-Bac or delta NAE-Bac and recombinant DNA fragment co-transfect insect host cells, homologous recombination can occur to produce recombinant baculovirus, and protein expression of exogenous gene can be performed.

Example 4 exogenous genes can be inserted at any locus in the AcMNPV genome, without restriction of the insertion site, and can be inserted at the desired site as desired

First, referring to example 2, recombinant DNA fragments having insertion sites at orf83, orf126 and orf152, respectively, were constructed as shown in FIGS. 12A-F: wherein constructs Ac83-CNE-GFP (FIG. 12A), ac126-CNE-GFP (FIG. 12B) and Ac152-CNE-GFP ((FIG. 12C) are recombinant DNA fragments with CNE complementing sequences at the insertion sites orf83, orf126 and orf152, respectively, constructs Ac83-NAE-mcherry (FIG. 12D), ac126-NAE-mcherry (FIG. 12E) and Ac152-NAE-mcherry (FIG. 12F) are recombinant DNA fragments with NAE complementing sequences at the insertion sites orf83, orf126 and orf152, respectively;

In this example, the packaging-defective baculovirus vectors Δcne-Bac and Δnae-Bac in example 1 were co-transfected with the recombinant DNA fragments in this example into Sf9 insect host cells, respectively, and after 96 hours of co-transfection, the production of recombinant baculovirus was judged by observing whether the host cells were capable of producing green fluorescent plaques or red fluorescent plaques using a fluorescence microscope. To further confirm that co-transfection did produce recombinant baculoviruses, sf9 cell culture supernatant was collected 120h after co-transfection, host cells were transferred to suspension culture, and after 72h infection, green fluorescence or red fluorescence luminescence was observed with a fluorescence microscope.

The observation results are shown in fig. 13 to 16: the baculovirus vector delta CNE-Bac co-transfects Sf9 cells with recombinant DNA fragments Ac83-CNE-GFP, ac126-CNE-GFP and Ac152-CNE-GFP respectively for 96 hours to generate green fluorescence plaques (FIG. 13), after 120 hours of co-transfection, the culture supernatant of Sf9 cells was collected, host cells cultured in suspension were transferred, and a large amount of green fluorescence was generated after 72 hours of infection (FIG. 14); the red fluorescent plaques were produced by co-transfection of the baculovirus vector DeltaNAE-Bac with the recombinant DNA fragments Ac83-NAE-mcherry, ac126-NAE-mcherry and Ac152-NAE-mcherry, respectively, for 96h (FIG. 15), after 120h of co-transfection, the Sf9 cell culture supernatant was collected, and the suspension-cultured host cells were transferred to produce a large amount of red fluorescence after 72h of infection (FIG. 16). This example shows that the foreign gene can be inserted into any locus of AcMNPV genome without restriction of insertion site, and can be inserted into target site as required, and expression of the foreign gene can be performed.

Example 5 obtaining recombinant baculovirus containing Cap and Rep essential for AAV packaging and detecting Cap and Rep expression

Recombinant baculovirus BEV was prepared by co-transfecting Sf9 insect cells with the package-defective baculovirus vector ΔCNE-Bac of example 1 and the recombinant DNA fragment Ac96-Rep-CNE-Cap of example 2, and co-transfecting Sf9 insect cells with the package-defective baculovirus vector ΔNAE-Bac of example 1 and the recombinant DNA fragment Ac96-Rep-NAE-Cap of example 2, respectively. The transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicative BEV resulted in a significant cytopathic effect of Sf9 cells (cytopathic effect, CPE). The culture supernatant of Sf9 cells with CPE was collected and contained a large amount of BEV, namely, the 0 th generation BEV (BEV-P0), while Sf9 cells containing a large amount of rAAV were collected. The prepared BEV-P0 was used to infect suspension-cultured Sf9 cells at a multiplicity of infection (MOI=1), after 72 hours of infection, the cell activity was reduced to below 50%, the cell culture broth was centrifuged at 1000g for 5min, and the culture supernatant and cell pellet were collected, respectively, and the supernatant was labeled as BEV of generation 1 (BEV-P1). Continuing the expansion culture, infecting suspension-cultured Sf9 cells with the prepared BEV-P1 at a multiplicity of infection (MOI=1), decreasing the cell activity to below 50% after 72h of infection, centrifuging 1000g of the cell culture solution for 5min, collecting cell sediment, and performing Western Blot detection on the expression conditions of VP proteins (VP 1, VP2 and VP 3) and Rep proteins (Rep 78 and Rep 52).

The results are shown in fig. 17 and 18: the baculovirus vector delta CNE-Bac and the recombinant DNA fragment Ac96-Rep-CNE-Cap are transfected into Sf9 insect cells to generate recombinant baculovirus, and VP protein and Rep protein are successfully expressed (figure 17); the co-transfection of the baculovirus vector delta NAE-Bac with the recombinant DNA fragment Ac96-Rep-NAE-Cap produced recombinant baculovirus after Sf9 insect cells and successfully expressed VP protein and Rep protein (FIG. 18).

EXAMPLE 6 construction of recombinant baculovirus vectors containing AAV core expression elements (ITR-GOI)

This example method of constructing recombinant AAV bacmid for production of AAV virus in insect cells reference is made to example 1 in the applicant's previous application CN112553257a, comprising the steps of:

(1) Recombinant transfer vectors comprising ITR core elements (ITR-GOI) were constructed. The nucleotide sequence of the ITR-GOI is shown as SEQ ID No. 12. In the embodiment, the GOI in the ITR core element adopts a red fluorescent protein mcherry gene expression cassette, namely, mcherry expression is controlled by a miniEf1a promoter, so that rAAV activity can be conveniently detected, and the ITR and the red fluorescent protein expression cassette are constructed on a transfer vector pFastDual.

(2) Transforming competent cells containing delta CNE-Bac or delta NAE-Bac bacmid by using the recombinant transfer vector constructed in the step (1), and inserting ITR-GOI into Tn7 locus of delta CNE-Bac or delta NAE-Bac bacmid by using Tn7 recombination to finally obtain recombinant baculovirus plasmids with the numbers delta CNE-Bac-Tn7-ITR and delta NAE-Bac-Tn7-ITR respectively, wherein the numbers of the recombinant baculovirus plasmids are delta CNE-Bac-Tn7-ITR and delta NAE-Bac-Tn7-ITR are required for producing rAAV.

Example 7 preparation of AAV recombinant baculovirus containing AAV packaging essential elements Cap, rep and ITR-GOI Using DeltaCNE-Bac-Tn 7-ITR or DeltaNAE-Bac-Tn 7-ITR defective bacmid

The recombinant bacmid Δcne-Bac-Tn7-ITR prepared in example 6 and the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in example 2, and the recombinant bacmid Δnae-Bac-Tn7-ITR prepared in example 6 and the recombinant DNA fragment Ac97-Rep-NAE-Cap prepared in example 2 were respectively co-transfected into host insect cell lines and cultured to obtain AAV recombinant baculoviruses numbered Δcne-Bac-AAV and Δnae-Bac-AAV, respectively, as follows:

and (3) extracting the recombinant bacmid and transfer vector DNA to co-transfect Sf9 insect cells, and preparing recombinant baculovirus BEV and rAAV. The successful production of BEV by Sf9 insect cells after co-transfection, and further infection with a large number of replicative BEVs resulted in significant cytopathic effects (CPE) of Sf9 cells. The culture supernatant of Sf9 cells with CPE was collected and contained a large amount of BEV, namely, the 0 th generation BEV (BEV-P0), while Sf9 cells containing a large amount of rAAV were collected. The prepared BEV-P0 was used to infect suspension-cultured Sf9 cells at a multiplicity of infection (MOI=1), after 72 hours of infection, the cell activity was reduced to below 50%, the cell culture broth was centrifuged at 1000g for 5min, the culture supernatant and cell pellet were collected, the supernatant was designated as BEV 1 generation (BEV-P1), and the cells were designated as rAAV packaged with BEV-P0.

Example 8 preparation of AAV recombinant baculovirus containing AAV packaging essential elements Cap, rep and ITR-GOI Using DeltaCNE-DeltaNAE-Bac defective bacmid

First, a recombinant DNA fragment of AAV ITR core expression element (ITR-GOI) was constructed at the AcMNPV orf83 locus: referring to FIG. 19, the recombinant DNA fragment comprises an orf83 upstream homologous sequence, an ITR-GOI sequence, a NAE sequence and an orf83 downstream homologous sequence in sequence from 5 'to 3', and the sequences are connected by artificial direct synthesis or overlap extension PCR amplification to obtain a construct Ac83-ITR-NAE.

Then, the defective bacmid ΔCNE- ΔNAE-Bac prepared in example 1 was co-transfected with the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in example 2, and the recombinant DNA fragment Ac83-ITR-NAE prepared in this example was cultured to obtain AAV recombinant baculovirus, which was numbered ΔCNE- ΔNAE-Bac-AAV, for specific procedures as in example 7.

Example 9 purification of recombinant AAV virions and detection of their packaging Rate

The recombinant baculoviruses ΔCNE-Bac-AAV, ΔNAE-Bac-AAV and ΔCNE- ΔNAE-Bac-AAV of examples 7 and 8 were further expanded according to the procedure of example 5 until rAAV was packaged by infecting suspension-cultured Sf9 cells with BEV-P2 at a multiplicity of infection (MOI=1) in a package volume of 300mL to 400mL. Monitoring cell activity after 3 days of infection, centrifuging to obtain cell sediment and supernatant, purifying the cell sediment and supernatant, repeatedly freezing and thawing the cells for 3 times, centrifuging at 5000rpm for 10min, collecting supernatant, adding nuclease (Benzonase) into the supernatant, treating in water bath at 37deg.C for 60min, and centrifuging at 5000rpm for 10min. The collected cell lysates and the collected supernatant PEG were precipitated and purified by iodixanol density gradient centrifugation after resuspension (see Aslanidi et al 2009, proc. Natl. Acad. Sci. USA, 206:5059-5064). The final purified finished virus was resuspended in 80. Mu.L to 190. Mu.L PBS and 10. Mu.L of purified finished virus was run on SDS-PAGE gels and silver stained.

SDS-PAGE gel is shown in FIG. 20: wherein, lane 1 shows three capsid proteins VP1/VP2/VP3 from silver staining detection patterns of SDS-PAGE of purified recombinant AAV virions after infection of host cells with recombinant baculovirus delta CNE-Bac-AAV; lane 2 shows a silver staining detection pattern of SDS-PAGE of purified recombinant AAV virions after infection of host cells with recombinant baculovirus delta NAE-Bac-AAV, showing three capsid proteins VP1/VP2/VP3; lane 3 is a silver staining assay of SDS-PAGE of purified recombinant AAV virions after infection of host cells with recombinant baculovirus ΔCNE- ΔNAE-Bac-AAV, showing three capsid proteins VP1/VP2/VP3.

The present example also uses Q-PCR to detect the packaging rate of the harvested rAAV, and uses a pair of primers (Q-ITR-F: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA) that target the ITR sequence. The test results are shown in Table 1.

TABLE 1 packing fraction detection results of rAAV virions produced by recombinant baculovirus

Recombinant baculovirus numbering	Cell packing rate (VG/cell)
		△CNE-Bac-AAV	4.83E+05
△NAE-Bac-AAV	5.26E+05
		△CNE-△NAE-Bac-AAV	4.85E+05

This example shows that the defective baculovirus vector and recombinant DNA fragment provided by the invention can be directly recombined in insect host cells to prepare AAV recombinant baculovirus containing AAV packaging essential elements Cap, rep and ITR-GOI, and AAV virions can be successfully produced.

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.

SEQUENCE LISTING

<110> Rui Zhi medical science and technology (Wuhan) Co., ltd

<120> a composition, method and use for producing recombinant baculovirus in insect cells

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 1590

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 1

tgctcctgac gccgctcctg acggcgatgg ctgcgactgc ttgaagacgg ctggctgcga 60

ctgcttgaag acggctgggc ttcgggagat gttgtaaagt tgatgcggcg acggctgaga 120

gacagcctgt ggcggcggct gctgctggga gtggcggcgt tgatttggcg actcatggct 180

gggctggtag gatactgttc actaggctgt gaggcttgaa ctgtgcttac gagtagaacg 240

gcagctgtat ttatactgtt tatcagtact gcacgactga taagacaata gtggtggggg 300

aacttgccag gcaaaaatga gaagttccta tactttctag agaataggaa cttcatttaa 360

atggcgcgcc ttacgccccg ccctgccact catcgcagta ctgttgtatt cattaagcat 420

ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc 480

accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc 540

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 600

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 660

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 720

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 780

accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat caggcgggca 840

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 900

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 960

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 1020

ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa tacgcccggt 1080

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1140

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1200

tctgcgaagt gatcttccgt cacaggtagg cgcgccgaag ttcctatact ttctagagaa 1260

taggaacttc tcattttaaa tttatcatat cacaggctgc agtttctgtt atctgtcccc 1320

cactcaggcg tgcagctata aaagcaggca ctcaccaact cgtaagcaca gttcgttgtg 1380

aagtgaacac ggagagcctg ccaataagca aaatgccaag ggacaccaac aatcgccacc 1440

ggtctacgcc atatgaacgt cctacgcttg aagatctccg cagacagttg caagacaatt 1500

tggacagcat aaaccgccga gacagaatgc aagaagaaca agaagaaaac ctgcgctatc 1560

aagtgcgtag aaggcagcgt caaaaccagc 1590

<210> 2

<211> 1277

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 2

cacccgacgt aataggctac gtgtcggata ttatgcaaaa cacttatatt gtaacgtggt 60

tcaacaccgt cgacctttcc acctatcacg aaagcgtgca tgatgaccgg attgaaattt 120

ttgatttctt aaatcaaaaa tttcaacctg ttgatcgaat cgtacacgat cgcgttagag 180

caaatgatga aaatcccaac gaagttccta tactttctag agaataggaa cttcatttaa 240

atggcgcgcc ttacgccccg ccctgccact catcgcagta ctgttgtatt cattaagcat 300

ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc 360

accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc 420

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 480

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 540

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 600

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 660

accagctcac cgtctttcat tgccatacgt aattccggat gagcattcat caggcgggca 720

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 780

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 840

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 900

ttctccattt tagcttcctt agctcctgaa aatctcgaca actcaaaaaa tacgcccggt 960

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1020

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1080

tctgcgaagt gatcttccgt cacaggtagg cgcgccgaag ttcctatact ttctagagaa 1140

taggaacttc gcacgtcaaa aacggccaat acatggcgtg tcccgaagaa ttgtacgata 1200

acaacgaatt taaatgtaac atagaatcgg ataaattata ctatttggat aatttacaag 1260

aagattccat tgtataa 1277

<210> 3

<211> 1528

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette homologous recombinant

<400> 3

atgatgtctg gcgtaatgtt gctcatgctt gccatttttc ttataatagc gtttacttta 60

atgtatttgg caatttattt tgaatttgac gaaacgactt tcaccaagcg gctccaagtg 120

atgactgaat atgtgaagcg caccaacgca gacgaaccca cacccgacgt aataggctac 180

gtgtcggata ttatgcaaaa cacttatatt gtaacgtggt tcaacaccgt cgacctttcc 240

acctatcacg aaagcgtgca tgatgaccgg attgaaattt ttgatttctt aaatcaaaaa 300

tttcaacctg ttgatcgaat cgtacacgat cgcgttagag caaatgatga aaatcccaac 360

ataacttcgt ataatttata ctatacgaag ttatatttaa atttaggtgg cggtacttgg 420

gtcgatatca aagtgcatca cttcttcccg tatgcccaac tttgtataga gagccactgc 480

gggatcgtca ccgtaatctg cttgcacgta gatcacataa gcaccaagcg cgttggcctc 540

atgcttgagg agattgatga gcgcggtggc aatgccctgc ctccggtgct cgccggagac 600

tgcgagatca tagatataga tctcactacg cggctgctca aacctgggca gaacgtaagc 660

cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga atgtcttact 720

acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg tggctacgtc 780

tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt ggtcagggcc 840

gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt tagggcgact 900

gccctgctgc gtaacatcgt tgctgctgcg taacatttta gcttccttag ctcctgaaaa 960

tctcgacaac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga 1020

acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt 1080

atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtaggcg 1140

cgccataact tcgtataatt tatactatac gaagttatgc acgtcaaaaa cggccaatac 1200

atggcgtgtc ccgaagaatt gtacgataac aacgaattta aatgtaacat agaatcggat 1260

aaattatact atttggataa tttacaagaa gattccattg tataaacatt ttatgtcgaa 1320

aacaaatgac atcattccgg atcatgattt acgcgtagaa ttctacttgt aaagcaagtt 1380

aaaataagcc gtgtgcaaaa atgacatcag acaaatgaca tcatctacct atcatgatca 1440

tgttaataat catgttttaa aatgacatca gcttatgact aataattgat cgtgcgttac 1500

aagtagaatt ctactcgtaa agcgagtt 1528

<210> 4

<211> 156

<212> DNA

<213> Artificial Sequence

<220>

<223> CNE

<400> 4

acttttttgt aatgcaaaaa agttgatagt gtagtagtat attgggagcg tatcgtacag 60

tgtagactat tctaataaaa tagtctacga tttgtagaga ttgtactgta tatggagtgt 120

caggcaaaag tgaacttttt tgcattgcaa aaaaat 156

<210> 5

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<223> NAE

<400> 5

cattttcagc gacgtatatt gacaaatata ctacagtcgg acgtttgtgc cgacctatat 60

actacacttt accaaaaata tactacacta aactctaaat atactacaac tccacttcaa 120

tataaccaca ctctcgtaaa acggcccaaa aatatcgaaa tatatggggc aaatacacgt 180

ttaaaaaacg ctacgattcc 200

<210> 6

<211> 2254

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-CNE-GFP

<400> 6

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg cctaggctca agcagtgatc agatccagac 480

atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 540

tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 600

caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 660

gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagta 720

cttctcgaca agcttggatc cgcgcccgat ggtgggacgg tatgaataat ccggaatatt 780

tataggtttt tttattacaa aactgttacg aaaacagtaa aatacttatt tatttgcgag 840

atggttatca ttttaattat ctccatgatc tattaatatt ccggaatttt tttgcaatgc 900

aaaaaagttc acttttgcct gacactccat atacagtaca atctctacaa atcgtagact 960

attttattag aatagtctac actgtacgat acgctcccaa tatactacta cactatcaac 1020

ttttttgcat tacaaaaaag tgtatacgga cctttaattc aacccaacac aatatattat 1080

agttaaataa gaattattat caaatcattt gtatattaat taaaatacta tactgtaaat 1140

tacattttat ttacaatcac tcgacgaaga cttgatcacc cgggatggtg agcaagggcg 1200

aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1260

acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 1320

agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1380

cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1440

agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 1500

actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 1560

tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 1620

acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 1680

tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 1740

acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 1800

ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 1860

ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaggtacc gggagatggg 1920

ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa 1980

taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcatgtggt cgaccaacag 2040

attcagtttt caatactcga cattatcaat tatttgattt acaatggcta cgtggatttg 2100

ttggccgaat aacgcgtata tagacgcttg tacgttcatc gtagtaatca ttttaataca 2160

tttgattgaa ctaaacatac atctgcaatg ggtgaaagag tcactaaatt ttgcaatgga 2220

aaacggcgat aaagaagaca gcgacaatga atag 2254

<210> 7

<211> 2289

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-NAE-mcherry

<400> 7

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg cctaggctca agcagtgatc agatccagac 480

atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 540

tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 600

caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 660

gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagta 720

cttctcgaca agcttggatc cgcgcccgat ggtgggacgg tatgaataat ccggaatatt 780

tataggtttt tttattacaa aactgttacg aaaacagtaa aatacttatt tatttgcgag 840

atggttatca ttttaattat ctccatgatc tattaatatt ccggacattt tcagcgacgt 900

atattgacaa atatactaca gtcggacgtt tgtgccgacc tatatactac actttaccaa 960

aaatatacta cactaaactc taaatatact acaactccac ttcaatataa ccacactctc 1020

gtaaaacggc ccaaaaatat cgaaatatat ggggcaaata cacgtttaaa aaacgctacg 1080

attccgtata cggaccttta attcaaccca acacaatata ttatagttaa ataagaatta 1140

ttatcaaatc atttgtatat taattaaaat actatactgt aaattacatt ttatttacaa 1200

tcactcgacg aagacttgat cacccgggat ggtgagcaag ggcgaggagg ataacatggc 1260

catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga 1320

gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct 1380

gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat 1440

gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc 1500

cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 1560

cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg 1620

caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 1680

ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa 1740

gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc 1800

cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga 1860

ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat 1920

ggacgagctg tacaagtaag gtaccgggag atgggggagg ctaactgaaa cacggaagga 1980

gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg 2040

gtgttgggtc gtttgttcat gtggtcgacc aacagattca gttttcaata ctcgacatta 2100

tcaattattt gatttacaat ggctacgtgg atttgttggc cgaataacgc gtatatagac 2160

gcttgtacgt tcatcgtagt aatcatttta atacatttga ttgaactaaa catacatctg 2220

caatgggtga aagagtcact aaattttgca atggaaaacg gcgataaaga agacagcgac 2280

aatgaatag 2289

<210> 8

<211> 1866

<212> DNA

<213> Artificial Sequence

<220>

<223> Rep gene

<400> 8

ctggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg agtgggagtt gccgccagat 120

tctgacttgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

cgcgactttc tgacggagtg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240

caatttgaga agggagagag ctacttccac ttacacgtgc tcgtggaaac caccggggtg 300

aaatccttag ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaacggc 420

gccggaggcg ggaacaaggt ggtggacgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatttagaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgacgcgc cggtgatcag atcaaaaact 660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 840

cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1800

ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 1860

caataa 1866

<210> 9

<211> 2211

<212> DNA

<213> Artificial Sequence

<220>

<223> Cap gene

<400> 9

ctggctgccg acggttatct acccgattgg ctcgaggaca accttagtga aggaattcgc 60

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740

gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800

atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860

aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920

aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980

gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040

gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100

tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160

tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211

<210> 10

<211> 5623

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-Rep-CNE-Cap

<400> 10

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg gaacaaacga cccaacaccc gtgcgtttta 480

ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt 540

tcagttagcc tcccccatct cccggtacct tattgttcaa agatgcagtc atccaaatcc 600

acattgacca gatcgcaggc agtgcaagcg tctggcacct ttcccatgat atgatgaatg 660

tagcacagtt tctgatacgc ctttttgacg acagaaacgg gttgagattc tgacacggga 720

aagcactcta aacagtcttt ctgtccgtga gtgaagcaga tatttgaatt ctgattcatt 780

ctctcgcatt gtctgcaggg aaacagcatc agattcatgc ccacgtgacg agaacatttg 840

ttttggtacc tgtctgcgta gttgatcgaa gcttccgcgt ctgacgtcga tggctgcgca 900

actgactcgc gcacccgttt gggctcactt atatctgcgt cactgggggc gggtcttttc 960

ttggctccac cctttttgac gtagaattca tgctccacct caaccacgtg atcctttgcc 1020

caccggaaaa agtctttgac ttcctgcttg gtgaccttcc caaagtcatg atccagacgg 1080

cgggtgagtt caaatttgaa catccggtct tgcaacggct gctggtgttc gaaggtcgtt 1140

gagttcccgt caatcacggc gcacatgttg gtgttggagg tgacgatcac gggagtcggg 1200

tctatctggg ccgaggactt gcatttctgg tccacgcgca ccttgcttcc tccgagaatg 1260

gctttggccg actccacgac cttggcggtc atcttcccct cctcccacca gatcaccatc 1320

ttgtcgacac agtcgttgaa gggaaagttc tcattggtcc agtttacgca cccgtagaag 1380

ggcacagtgt gggctatggc ctccgcgatg ttggtcttcc cggtagttgc aggcccaaac 1440

agccagatgg tgttcctctt gccgaacttt ttcgtggccc atcccagaaa gacggaagcc 1500

gcatattggg gatcgtaccc gtttagttcc aaaattttat aaatccgatt gctggaaatg 1560

tcctccacgg gctgctggcc caccaggtag tcgggggcgg ttttagtcag gctcataatc 1620

tttcccgcat tgtccaaggc agccttgatt tgggaccgcg agttggaggc cgcattgaag 1680

gagatgtatg aggcctggtc ctcctggatc cactgcttct ccgaggtaat ccccttgtcc 1740

acgagccacc cgaccagctc catgtacctg gctgaagttt ttgatctgat caccggcgcg 1800

tcagaattgg gattctgatt ctctttgttc tgctcctgcg tctgcgacac gtgcgtcaga 1860

tgctgcgcca ccaaccgttt acgctccgtg agattcaaac aggcgcttaa atactgttct 1920

aaattagtcc acgcccactg gagctcaggc tgggttttgg ggagcaagta attggggatg 1980

tagcactcgt ccaccacctt gttcccgcct ccggcgccgt ttctggtctt tgtgaccgcg 2040

aaccagtttg gcaaagtcgg ctcgatcccg cggtaaattc tctgaatcag tttttcgcga 2100

atctgactca ggaaacgtcc caaaactaag gatttcaccc cggtggtttc cacgagcacg 2160

tgtaagtgga agtagctctc tcccttctca aattgcacaa agaaaagggc ctccggggcc 2220

ttactcacac ggcgccactc cgtcagaaag tcgcgctgca gcttctcggc cacggtcagg 2280

ggtgcctgct caatcagatt cagatccaag tcagaatctg gcggcaactc ccactccttc 2340

tcggccaccc agttcacaaa gctgtcagaa atgccgggca gatgcccgtc aaggtcgctg 2400

gggaccttaa tcacaatctc gtaaaacccc gccagggcgg ccccgggtga tcaagtcttc 2460

gtcgagtgat tgtaaataaa atgtaattta cagtatagta ttttaattaa tatacaaatg 2520

atttgataat aattcttatt taactataat atattgtgtt gggttgaatt aaaggtccgt 2580

atacactttt ttgtaatgca aaaaagttga tagtgtagta gtatattggg agcgtatcgt 2640

acagtgtaga ctattctaat aaaatagtct acgatttgta gagattgtac tgtatatgga 2700

gtgtcaggca aaagtgaact tttttgcatt gcaaaaaaat tccggaatat taatagatca 2760

tggagataat taaaatgata accatctcgc aaataaataa gtattttact gttttcgtaa 2820

cagttttgta ataaaaaaac ctataaatat tccggattat tcataccgtc ccaccatcgg 2880

gcgcggatcc gccgccctgg ctgccgacgg ttatctaccc gattggctcg aggacaacct 2940

tagtgaagga attcgcgagt ggtgggcttt gaaacctgga gcccctcaac ccaaggcaaa 3000

tcaacaacat caagacaacg ctcgaggtct tgtgcttccg ggttacaaat accttggacc 3060

cggcaacgga ctcgacaagg gggagccggt caacgcagca gacgcggcgg ccctcgagca 3120

cgacaaggcc tacgaccagc agctcaaggc cggagacaac ccgtacctca agtacaacca 3180

cgccgacgcc gagttccagg agcggctcaa agaagatacg tcttttgggg gcaacctcgg 3240

gcgagcagtc ttccaggcca aaaagaggct tcttgaacct cttggtctgg ttgaggaagc 3300

ggctaagacg gctcctggaa agaagaggcc tgtagagcag tctcctcagg aaccggactc 3360

ctccgcgggt attggcaaat cgggtgcaca gcccgctaaa aagagactca atttcggtca 3420

gactggcgac acagagtcag tcccagaccc tcaaccaatc ggagaacctc ccgcagcccc 3480

ctcaggtgtg ggatctctta caatggcttc aggtggtggc gcaccagtgg cagacaataa 3540

cgaaggtgcc gatggagtgg gtagttcctc gggaaattgg cattgcgatt cccaatggct 3600

gggggacaga gtcatcacca ccagcacccg aacctgggcc ctgcccacct acaacaatca 3660

cctctacaag caaatctcca acagcacatc tggaggatct tcaaatgaca acgcctactt 3720

cggctacagc accccctggg ggtattttga cttcaacaga ttccactgcc acttctcacc 3780

acgtgactgg cagcgactca tcaacaacaa ctggggattc cggcctaagc gactcaactt 3840

caagctcttc aacattcagg tcaaagaggt tacggacaac aatggagtca agaccatcgc 3900

caataacctt accagcacgg tccaggtctt cacggactca gactatcagc tcccgtacgt 3960

gctcgggtcg gctcacgagg gctgcctccc gccgttccca gcggacgttt tcatgattcc 4020

tcagtacggg tatctgacgc ttaatgatgg aagccaggcc gtgggtcgtt cgtcctttta 4080

ctgcctggaa tatttcccgt cgcaaatgct aagaacgggt aacaacttcc agttcagcta 4140

cgagtttgag aacgtacctt tccatagcag ctacgctcac agccaaagcc tggaccgact 4200

aatgaatcca ctcatcgacc aatacttgta ctatctctca aagactatta acggttctgg 4260

acagaatcaa caaacgctaa aattcagtgt ggccggaccc agcaacatgg ctgtccaggg 4320

aagaaactac atacctggac ccagctaccg acaacaacgt gtctcaacca ctgtgactca 4380

aaacaacaac agcgaatttg cttggcctgg agcttcttct tgggctctca atggacgtaa 4440

tagcttgatg aatcctggac ctgctatggc cagccacaaa gaaggagagg accgtttctt 4500

tcctttgtct ggatctttaa tttttggcaa acaaggaact ggaagagaca acgtggatgc 4560

ggacaaagtc atgataacca acgaagaaga aattaaaact actaacccgg tagcaacgga 4620

gtcctatgga caagtggcca caaaccacca gagtgcccaa gcacaggcgc agaccggctg 4680

ggttcaaaac caaggaatac ttccgggtat ggtttggcag gacagagatg tgtacctgca 4740

aggacccatt tgggccaaaa ttcctcacac ggacggcaac tttcaccctt ctccgctgat 4800

gggagggttt ggaatgaagc acccgcctcc tcagatcctc atcaaaaaca cacctgtacc 4860

tgcggatcct ccaacggcct tcaacaagga caagctgaac tctttcatca cccagtattc 4920

tactggccaa gtcagcgtgg agatcgagtg ggagctgcag aaggaaaaca gcaagcgctg 4980

gaacccggag atccagtaca cttccaacta ttacaagtct aataatgttg aatttgctgt 5040

taatactgaa ggtgtatata gtgaaccccg ccccattggc accagatacc tgactcgtaa 5100

tctgtaaaag cttgtcgaga agtactagag gatcataatc agccatacca catttgtaga 5160

ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa 5220

tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag 5280

catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 5340

actcatcaat gtatcttatc atgtctggat ctgatcactg cttgagccta ggatgtggtc 5400

gaccaacaga ttcagttttc aatactcgac attatcaatt atttgattta caatggctac 5460

gtggatttgt tggccgaata acgcgtatat agacgcttgt acgttcatcg tagtaatcat 5520

tttaatacat ttgattgaac taaacataca tctgcaatgg gtgaaagagt cactaaattt 5580

tgcaatggaa aacggcgata aagaagacag cgacaatgaa tag 5623

<210> 11

<211> 5667

<212> DNA

<213> Artificial Sequence

<220>

<223> Ac96-Rep-NAE-Cap

<400> 11

tcaaaaaaaa ttgtaaaatg ttgtcaatca tgttggctat cgtgtttgta cttttcgtgt 60

taatttattt aataatttcg atcaaaaatc accatccatt cttacataga atagaaacgc 120

taatacaaga tttcaacaac acattgttgt ttggcgcgta tgtacagatt tacgatttaa 180

gcacgcccgc ccgcaccgaa cgattgttta ttattgcgcc cgaaaatgtg gtgttgtata 240

attttaacaa aacgctctat tattacttgg actcggcgaa cgtgttttgt cccaacgagt 300

ttagcgtgac cacgttcacg caatccacta ttaaaacgat caacgagacg ggaatatatg 360

ccaccgcatg cacgccggtc agcagcttga cgctaattga acattttgca acattaaaaa 420

ataacgtgcc cgatcacacg ctcgttctcg gaacaaacga cccaacaccc gtgcgtttta 480

ttctgtcttt ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt 540

tcagttagcc tcccccatct cccggtacct tattgttcaa agatgcagtc atccaaatcc 600

acattgacca gatcgcaggc agtgcaagcg tctggcacct ttcccatgat atgatgaatg 660

tagcacagtt tctgatacgc ctttttgacg acagaaacgg gttgagattc tgacacggga 720

aagcactcta aacagtcttt ctgtccgtga gtgaagcaga tatttgaatt ctgattcatt 780

ctctcgcatt gtctgcaggg aaacagcatc agattcatgc ccacgtgacg agaacatttg 840

ttttggtacc tgtctgcgta gttgatcgaa gcttccgcgt ctgacgtcga tggctgcgca 900

actgactcgc gcacccgttt gggctcactt atatctgcgt cactgggggc gggtcttttc 960

ttggctccac cctttttgac gtagaattca tgctccacct caaccacgtg atcctttgcc 1020

caccggaaaa agtctttgac ttcctgcttg gtgaccttcc caaagtcatg atccagacgg 1080

cgggtgagtt caaatttgaa catccggtct tgcaacggct gctggtgttc gaaggtcgtt 1140

gagttcccgt caatcacggc gcacatgttg gtgttggagg tgacgatcac gggagtcggg 1200

tctatctggg ccgaggactt gcatttctgg tccacgcgca ccttgcttcc tccgagaatg 1260

gctttggccg actccacgac cttggcggtc atcttcccct cctcccacca gatcaccatc 1320

ttgtcgacac agtcgttgaa gggaaagttc tcattggtcc agtttacgca cccgtagaag 1380

ggcacagtgt gggctatggc ctccgcgatg ttggtcttcc cggtagttgc aggcccaaac 1440

agccagatgg tgttcctctt gccgaacttt ttcgtggccc atcccagaaa gacggaagcc 1500

gcatattggg gatcgtaccc gtttagttcc aaaattttat aaatccgatt gctggaaatg 1560

tcctccacgg gctgctggcc caccaggtag tcgggggcgg ttttagtcag gctcataatc 1620

tttcccgcat tgtccaaggc agccttgatt tgggaccgcg agttggaggc cgcattgaag 1680

gagatgtatg aggcctggtc ctcctggatc cactgcttct ccgaggtaat ccccttgtcc 1740

acgagccacc cgaccagctc catgtacctg gctgaagttt ttgatctgat caccggcgcg 1800

tcagaattgg gattctgatt ctctttgttc tgctcctgcg tctgcgacac gtgcgtcaga 1860

tgctgcgcca ccaaccgttt acgctccgtg agattcaaac aggcgcttaa atactgttct 1920

aaattagtcc acgcccactg gagctcaggc tgggttttgg ggagcaagta attggggatg 1980

tagcactcgt ccaccacctt gttcccgcct ccggcgccgt ttctggtctt tgtgaccgcg 2040

aaccagtttg gcaaagtcgg ctcgatcccg cggtaaattc tctgaatcag tttttcgcga 2100

atctgactca ggaaacgtcc caaaactaag gatttcaccc cggtggtttc cacgagcacg 2160

tgtaagtgga agtagctctc tcccttctca aattgcacaa agaaaagggc ctccggggcc 2220

ttactcacac ggcgccactc cgtcagaaag tcgcgctgca gcttctcggc cacggtcagg 2280

ggtgcctgct caatcagatt cagatccaag tcagaatctg gcggcaactc ccactccttc 2340

tcggccaccc agttcacaaa gctgtcagaa atgccgggca gatgcccgtc aaggtcgctg 2400

gggaccttaa tcacaatctc gtaaaacccc gccagggcgg ccccgggtga tcaagtcttc 2460

gtcgagtgat tgtaaataaa atgtaattta cagtatagta ttttaattaa tatacaaatg 2520

atttgataat aattcttatt taactataat atattgtgtt gggttgaatt aaaggtccgt 2580

ataccatttt cagcgacgta tattgacaaa tatactacag tcggacgttt gtgccgacct 2640

atatactaca ctttaccaaa aatatactac actaaactct aaatatacta caactccact 2700

tcaatataac cacactctcg taaaacggcc caaaaatatc gaaatatatg gggcaaatac 2760

acgtttaaaa aacgctacga ttcctccgga atattaatag atcatggaga taattaaaat 2820

gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt tgtaataaaa 2880

aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg atccgccgcc 2940

ctggctgccg acggttatct acccgattgg ctcgaggaca accttagtga aggaattcgc 3000

gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 3060

aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 3120

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 3180

cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 3240

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 3300

gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 3360

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 3420

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 3480

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 3540

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 3600

gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 3660

accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 3720

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 3780

tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 3840

ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 3900

caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 3960

acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 4020

gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 4080

acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 4140

ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 4200

cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 4260

gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 4320

ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 4380

ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 4440

tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 4500

ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 4560

ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 4620

accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 4680

gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 4740

atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 4800

aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 4860

aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 4920

gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 4980

gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 5040

tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 5100

tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta aaagcttgtc 5160

gagaagtact agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 5220

aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 5280

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 5340

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 5400

tatcatgtct ggatctgatc actgcttgag cctaggatgt ggtcgaccaa cagattcagt 5460

tttcaatact cgacattatc aattatttga tttacaatgg ctacgtggat ttgttggccg 5520

aataacgcgt atatagacgc ttgtacgttc atcgtagtaa tcattttaat acatttgatt 5580

gaactaaaca tacatctgca atgggtgaaa gagtcactaa attttgcaat ggaaaacggc 5640

gataaagaag acagcgacaa tgaatag 5667

<210> 12

<211> 2407

<212> DNA

<213> Artificial Sequence

<220>

<223> ITR-GOI

<400> 12

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgccgccc gtcagtgggc agagcgcaca 180

tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga 240

aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 300

ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 360

tttgccgcca gaacacgcgt aagggatccg ccaccatggt gagcaagggc gaggaggata 420

acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg 480

gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 540

ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc 600

agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 660

agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 720

tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 780

tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 840

aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga 900

ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca 960

agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc 1020

acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg 1080

gcggcatgga cgagctgtac aagtaagaat tcgatatcaa gcttatcgat aatcaacctc 1140

tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 1200

tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 1260

ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 1320

tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 1380

ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 1440

cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 1500

acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 1560

ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 1620

accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 1680

ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg 1740

agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt 1800

tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga 1860

ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt 1920

tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc 1980

acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc 2040

tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg 2100

gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat 2160

ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc 2220

tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg 2280

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 2340

gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 2400

ctgcagg 2407

Claims (13)

1. A composition for producing recombinant baculovirus in an insect cell, characterized by: a recombinant DNA comprising a packaging-deficient baculovirus plasmid and a first rescue DNA, the packaging-deficient baculovirus plasmid lacking a CNE sequence or a NAE sequence in the baculovirus genome, the first rescue recombinant DNA comprising a first insertion sequence comprising a first functional fragment and a first complementing sequence, and a first homology arm flanking the first insertion sequence, the first complementing sequence being a sequence deleted from the packaging-deficient baculovirus plasmid, the composition being capable of undergoing homologous recombination in insect cells to produce a recombinant baculovirus;

the first functional fragment is an AAV ITR core expression element with an exogenous gene, a reporter gene or a nucleotide sequence encoding a therapeutic gene product; or the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV.

2. The composition of claim 1, wherein: the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and the first insertion sequence comprises the cap gene expression cassette, the first complement sequence and the rep gene expression cassette in the order from 5 'to 3'.

3. The composition of claim 2, wherein: the first complement sequence is positioned between the cap gene expression cassette and the rep gene expression cassette, and the two ends of the first complement sequence are respectively close to the starting end of the cap gene expression cassette and the starting end of the rep gene expression cassette.

4. The composition of claim 1, wherein: the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, and the packaging-defective baculovirus plasmid is a recombinant bacmid containing an AAV ITR core expression element with exogenous genes.

5. The composition of claim 4, wherein: the recombinant bacmid is obtained through Tn7 transposition mediated by baculovirus transfer vectors.

6. The composition of claim 1, wherein: the first functional fragment is an AAV ITR core expression element with an exogenous gene, and the packaging-defective baculovirus plasmid is a recombinant bacmid containing an AAV cap gene expression cassette and an AAV rep gene expression cassette.

7. The composition of claim 6, wherein: the recombinant bacmid is obtained through Tn7 transposition mediated by baculovirus transfer vectors.

8. The composition of claim 1, wherein: the recombinant plasmid further comprises a second rescue recombinant DNA, the CNE sequence and the NAE sequence in the baculovirus genome of the packaging-defective baculovirus plasmid are deleted, the second rescue recombinant DNA comprises a second insertion sequence and second homology arms positioned at two sides of the second insertion sequence, the second insertion sequence comprises a second complement sequence, the first complement sequence is the CNE sequence or the NAE sequence, and the second complement sequence is a sequence different from the first complement sequence in the CNE sequence and the NAE sequence.

9. The composition of claim 8, wherein: the first functional fragment is a cap gene expression cassette of AAV and a rep gene expression cassette of AAV, the second insertion sequence also comprises a second functional fragment, and the second functional fragment is an AAV ITR core expression element with exogenous genes.

10. Use of a composition according to any one of claims 1-9 for the preparation of recombinant baculovirus or recombinant adeno-associated virus in insect cells.

11. An insect cell, characterized in that: a composition comprising any of claims 1-9.

12. A method of growing or producing a recombinant baculovirus in vitro, comprising: comprising co-transfecting the composition of any one of claims 1-9 into an insect cell and culturing the insect cell.

13. A method of growing or producing a recombinant adeno-associated virus in vitro, comprising: comprising co-transfecting an insect cell with the composition of any one of claims 4, 5, 6, 7 and 9, and culturing the insect cell.

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