CN114703203B - Baculovirus vector and its use - Google Patents

Abstract

The present application relates to a method for preparing a linear double-stranded DNA (neDNA) expression vector without ends by using an insect cell-baculovirus system, wherein the neDNA vector contains AAV inverted terminal repeats (ITRs) and a gene expression cassette. The insect cell-baculovirus system of the present application has a high yield for preparing neDNA, with an average yield increase of 2-3 times. Compared with other Bac-reps, after three consecutive baculovirus passages, the expression stability of Rep protein (Rep78) is better.

Description

Baculovirus vector and its use

Technical Field

The present application relates to the field of biomedicine, and specifically to a baculovirus vector and its use.

Background technique

Gene therapy is a new treatment method that achieves the purpose of treating diseases by regulating the biological process of gene expression at the nucleic acid level. An ideal gene therapy vector must have the characteristics of high delivery efficiency, high safety, scalable production, and low cost. Recombinant adeno-associated virus (rAAV) vectors are efficient and safe and are currently the main gene delivery vectors used for gene therapy of genetic diseases. rAAV still faces the problems of difficulty in production and amplification and high production cost. Non-viral vector gene therapy based on DNA therapy is expected to solve this problem. In 2013, Robert Kotin et al. used the insect cell baculovirus expression system to prepare a linear double-stranded no-end DNA (neDNA), the two ends of which were closed by the inverted terminal repeats (ITRs) of the AAV genome. Robert Kotin's preparation method was directly transformed from the AAV baculovirus production system developed by his laboratory in 2002, and the yield was not optimized for neDNA; at the same time, when the recombinant baculovirus Bac-Rep expresses Rep protein, homologous recombination occurs between Rep78 and Rep52, and the system is unstable.

Therefore, there is still a need to overcome the above-mentioned serious limitations of large-scale (commercial) production of neDNA in insect cells. It is therefore an object of the present invention to provide means and methods for stable and high-yield (large-scale) production of neDNA in insect cells.

Summary of the invention

The purpose of this application is to design an efficient insect cell baculovirus expression system and a method for preparing neDNA, optimize the Rep protein expression vector, and improve the stability of Rep protein expression, thereby improving the yield of neDNA and the stability of the production system. The features of the present invention include: 1) using a strong promoter (p10 promoter) to increase the expression of Rep78; 2) optimizing the sequence codons of Rep52 to avoid homologous recombination between Rep78 and Rep52 sequences. Effects of the present invention: 1) The yield is increased by 2-3 times compared to Robert Kotin's preparation method; 2) The Rep protein baculovirus expression vector is still stable after 3 generations.

In one aspect, the present application provides an isolated nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:12.

In certain embodiments, the isolated nucleic acid molecule encodes adeno-associated virus (AAV) Rep52 protein.

On the other hand, the present application provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the adeno-associated virus (AAV) Rep78 protein and a nucleotide sequence encoding the Rep52 protein, wherein the nucleic acid molecule encoding the Rep52 protein comprises the nucleotide sequence shown in SEQ ID NO:12.

In certain embodiments, the nucleotide sequence encoding the Rep78 protein is wild type.

In certain embodiments, the nucleic acid molecule encoding the Rep78 protein comprises the nucleotide sequence shown in SEQ ID NO:11.

In certain embodiments, it further comprises a first promoter for initiating transcription of the nucleotide sequence encoding the AAV Rep78 protein and a second promoter for initiating transcription of the nucleic acid molecule encoding the AAV Rep52 protein, wherein the first promoter is the same as or different from the second promoter.

In certain embodiments, the first promoter and the second promoter comprise insect cell promoters.

In certain embodiments, the first promoter comprises a strong promoter.

In certain embodiments, the first promoter has the same or higher transcription initiation ability as the second promoter.

In certain embodiments, the first promoter and the second promoter are each independently selected from the group consisting of: p10 promoter, Polyhedrin (polh) promoter and IE1 promoter.

In certain embodiments, the transcription directions of the first promoter and the second promoter are the same or opposite.

In certain embodiments, the first promoter is operably linked to the nucleotide sequence encoding the Rep78 protein, and the second promoter is operably linked to the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are the same, it sequentially comprises the first promoter, a nucleotide sequence encoding the Rep78 protein, the second promoter, and a nucleotide sequence encoding the Rep52 protein.

In certain embodiments, the nucleotide sequence encoding the Rep78 protein and the nucleotide sequence encoding the Rep52 protein further comprise a nucleotide sequence encoding polyA (pA) downstream thereof.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are the same, it sequentially comprises the first promoter, a nucleotide sequence encoding the Rep78 protein, a first pA, a second promoter, a nucleotide sequence encoding the Rep52 protein, and a second pA.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are opposite, they sequentially comprise a nucleotide sequence encoding Rep78, a first promoter, a second promoter, and a nucleotide sequence encoding Rep52, wherein the first promoter initiates the transcription of the nucleotide sequence encoding the Rep78 protein, and the second promoter initiates the transcription of the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, the 5' end of the first promoter is directly or indirectly connected to the 5' end of the second promoter.

In certain embodiments, the 3’ end of the first promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep78.

In certain embodiments, it further comprises a first pA, wherein the 3' end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly connected to the 5' end of the first pA.

In certain embodiments, the 3’ end of the second promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep52.

In certain embodiments, it further comprises a second pA, wherein the 3' end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly connected to the 5' end of the second pA.

In certain embodiments, the pA is selected from any one of SV40 polyA and HSV TK polyA.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8.

On the other hand, the present application provides an isolated nucleic acid molecule, which comprises, in sequence, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a transcriptional promoter of a nucleotide sequence encoding a Rep78 protein and the first pA, and the second promoter is a transcriptional promoter of a nucleotide sequence encoding a Rep52 protein and the second polyA, wherein the sequences of the nucleotide sequence encoding the Rep52 protein and/or the nucleotide sequence encoding the Rep78 protein are codon-optimized to avoid homologous recombination, the first promoter and the second promoter comprise insect cell promoters, and the first promoter is a strong promoter.

In certain embodiments, the first promoter comprises p10 promoter, polh promoter or IE1 promoter.

In certain embodiments, the p10 promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

In certain embodiments, the second promoter comprises a p10 promoter, a polh promoter or an IE1 promoter.

In certain embodiments, the polh promoter comprises the nucleotide sequence shown in SEQ ID NO:10.

In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described in the present application.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises a baculovirus vector.

In certain embodiments, the vector comprises a pFastBac vector.

In certain embodiments, the vector comprises the nucleotide sequence shown in SEQ ID NO:14.

In another aspect, the present application provides a cell comprising the isolated nucleic acid molecule described herein or the vector described herein.

In certain embodiments, the cell comprises an insect cell.

In certain embodiments, the cell comprises a Spodoptera frugiperda (Sf9) cell.

On the other hand, the present application provides a baculovirus expression system, which comprises a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a target gene, wherein the first baculovirus vector is the baculovirus vector described in the present application.

In certain embodiments, from the 5' end to the 3' end, the nucleic acid sequence encoding the target gene comprises, in sequence, a first parvovirus inverted terminal repeat sequence (ITR), the target gene, and a second ITR.

In certain embodiments, at least one promoter is further included between the first ITR and the target gene.

In certain embodiments, at least one eukaryotic promoter is further included between the first ITR and the target gene.

In certain embodiments, at least one mammalian cell promoter is further included between the first ITR and the target gene.

In certain embodiments, a mammalian cell promoter and an insect cell promoter are further included between the first ITR and the target gene.

In certain embodiments, the mammalian cell promoter includes a general promoter and a tissue-specific promoter.

In certain embodiments, the ubiquitous promoter comprises a CMV, SV40, EF1a, CAG or UBC promoter.

In certain embodiments, the tissue-specific promoter comprises ALB, hAAT, TBG, TTR, GFAP, MHCK7 or hSyn promoter.

In certain embodiments, the mammalian cell promoter comprises a CMV promoter.

In certain embodiments, the insect cell promoter comprises the p10 promoter.

In certain embodiments, the promoter comprises CMV and p10 promoters.

On the other hand, the present application provides an insect cell containing a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein, and the second nucleotide sequence encodes a nucleotide sequence of a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:11, and the second nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:12.

In certain embodiments, the first and second nucleotide sequences are part of a nucleic acid construct, wherein the first and second nucleotide sequences are each operably linked to an expression control sequence for insect cell expression.

In certain embodiments, the insect cell further comprises: a third nucleic acid sequence comprising at least one parvovirus inverted terminal repeat nucleotide sequence (ITR).

In certain embodiments, the third nucleotide sequence further contains at least one nucleotide sequence encoding a target gene.

In certain embodiments, the third nucleotide sequence contains two parvovirus ITR nucleotide sequences, and the at least one nucleotide sequence encoding a gene of interest is located between the two parvovirus ITR nucleotide sequences.

In certain embodiments, the parvovirus comprises an adeno-associated virus.

In certain embodiments, the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression.

In certain embodiments, the nucleic acid construct is an insect cell compatible vector.

In certain embodiments, the nucleic acid construct is a baculovirus vector.

In certain embodiments, it comprises a baculovirus vector as described herein.

On the other hand, the present application provides the use of the baculovirus expression system described in the present application or the insect cell described in the present application in preparing a target nucleic acid molecule.

In certain embodiments, the target nucleic acid molecule is a linear DNA molecule (neDNA) with covalently blocked ends.

On the other hand, the present application provides a method for preparing a target nucleic acid molecule, comprising culturing the insect cells described in the present application.

In certain embodiments, the preparation method comprises:

1) Providing the baculovirus expression system described in the present application;

2) inserting the target gene sequence into the second baculovirus vector;

3) co-transfecting the first baculovirus vector and the second baculovirus vector into insect cells;

4) growing the insect cells under conditions that allow replication and release of the DNA containing the gene of interest;

5) Collect the target nucleic acid molecules.

In certain embodiments, the method further provides for isolating the nucleic acid molecule of interest.

In another aspect, the present application provides a kit comprising the isolated nucleic acid molecule described in the present application, the baculovirus expression system described in the present application and/or the insect cell described in the present application.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

FIG. 1A shows a plasmid map of pFastBac-ITR-EGFP described in this application.

FIG. 1B shows the plasmid map of pFastBac-p10Rep described in this application.

FIG. 2 shows a schematic diagram of transcription of the p10Rep, RepWT, inRep and CORep genes described in the present application.

Figure 3 shows the results of Western Blotting analysis of Rep protein expression and stability; wherein, Ctr: uninfected Sf9 cells; 1: Sf9 cells infected with BacV-RepWT; 2: Sf9 cells infected with BacV-inRep; 3: Sf9 cells infected with BacV-CORep; 4: Sf9 cells infected with BacV-p10Rep.

Figure 4 shows the identification results of the neDNA-ITR-EGFP gene expression vector driven by different Rep proteins described in the present application; wherein, M: DNA Marker; 1-4: electrophoresis patterns of the neDNA-ITR-EGFP gene expression vector driven by RepWT, inRep, CORep and p10Rep, respectively.

Figures 5A-5B show the results of enzyme digestion identification of the neDNA-ITR-EGFP gene expression vector described in the present application.

Figure 6 shows the fluorescence expression graph of RepWT, inRep, CORep and p10Rep described in the present application driving the neDNA-ITR-EGFP gene expression vector and transfecting HEK293 cells, which was taken under a fluorescence microscope 72 hours after transfection.

FIG. 7 shows the results of luciferase expression in C57BL/6 mice by nanolipid particle delivery of neDNA-ITR-FLuc.

FIG8 shows the sequence comparison of Rep52 before and after optimization described in the present application (Query: Rep52WT, Sbjct: Rep52-CO).

Detailed ways

The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Definition of Terms

The viruses of the Parvoviridae family are small DNA animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvoviridae that infects vertebrates and the Densovirinae that infects insects. The members of the Parvoviridae subfamily are referred to herein as Parvovirus and include the genus Dependovirus. As can be inferred from their genus name, the members of the Dependovirus are unique because they usually require co-infection with helper viruses such as adenovirus or herpes virus to produce infection in cell culture. The Dependovirus genus includes AAV, which is very common in humans and other primates, and several serotypes have been isolated from various tissue samples. Serotypes 2, 3, 5 and 6 were found in human cells, and AAV serotypes 1, 4 and 7-11 in non-human primate samples. Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996) describes additional information about parvoviruses and other members of the Parvoviridae family. It will be appreciated that the present invention is not limited to AAV but is equally applicable to other parvoviruses.

The genome of AAV is a linear single-stranded DNA molecule with a length of less than about 5000 nucleotides (nt). Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for nonstructural replication (rep) proteins and structural proteins (VP). The VP proteins (VP1, VP2 and VP3) constitute the capsid. The 145nt at the end are self-complementary and ordered so as to form an energetically stable intramolecular double strand forming a T-shaped hairpin. The function of these hairpin structures is the starting point for viral DNA replication and is used as a primer for the cellular DNA polymerase complex. After wtAAV infects mammalian cells, the Rep genes (i.e., Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and the two expressed Rep proteins both play a role in the replication of the viral genome. The splicing event in the Rep ORF actually leads to the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52 and Rep40). However, unspliced mRNA encoding Rep78 and Rep52 is sufficient to produce AAV vectors in mammalian cells. Rep78 and Rep52 proteins are also sufficient to produce AAV vectors in insect cells.

In this application, the term "AAV vector" or "rAAV vector" generally refers to a vector comprising one or more polynucleotide sequences of interest, a gene of interest or a "transgene" flanked by parvoviral or AAV inverted terminal repeats (ITRs).

In this application, the term "operably linked" refers to the connection of polynucleotide (or polypeptide) elements in a functional relationship. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably linked". For example, if a transcription regulatory sequence affects the transcription of a coding sequence, it is operably linked to the coding sequence. Operably linked means that the linked DNA sequences are generally continuous and, when it is necessary to connect two protein coding regions, the linked DNA sequences are continuous and in reading frame.

In the present application, the term "expression control sequence" generally refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. When an expression control sequence controls and regulates the transcription and/or translation of a nucleotide sequence, the expression control sequence is "operably linked" to the nucleotide sequence. Therefore, an expression control sequence may include a promoter, an enhancer, an internal ribosome entry site (IRES), a transcription terminator, a start codon before a protein-coding gene, an intron splicing signal, and a stop codon. The term "expression control sequence" is intended to include, at a minimum, a sequence that exists to affect expression, and may also include other advantageous components. For example, a leader sequence and a fusion partner sequence are expression control sequences. The term may also include a nucleic acid sequence design that removes unwanted possible start codons from the sequence both within and outside the frame. It may also include a nucleic acid sequence design that removes unwanted possible splicing sites. It includes a sequence that directs the addition of a polyA tail, a string of adenine residues at the 3' end of the mRNA, or a polyadenylation sequence (pA). It can also be designed to increase the stability of the mRNA. It is known that there are expression control sequences such as promoters that affect the stability of transcription and translation, as well as sequences that enable translation, such as Kozak sequences, in insect cells. Expression control sequences have the property of regulating the nucleotide sequence to which they are operably linked, thereby reducing or increasing the level of expression.

In the present application, term " promoter (promoter) " or " transcriptional regulatory sequence " generally refers to such a nucleic acid fragment, described nucleic acid fragment has the effect of controlling the transcription of one or more coding sequences, it is located at the transcription direction upstream of the transcription start site of described coding sequence, and can carry out structural recognition by the DNA dependent RNA polymerase binding site, transcription start site and any other DNA sequence that exist, described other DNA sequence includes but is not limited to transcription factor binding site, repressor and activator protein binding site and any other nucleotide sequence that can directly or indirectly play the effect of regulating promoter transcription amount known to those skilled in the art. " Constitutive " promoter is a kind of promoter that has activity in most tissues under most physiological and developmental conditions. " Inducible " promoter is for example, by using chemical inducer to carry out the promoter of physiological or developmental regulation. " Tissue-specific " promoter is only active in specific tissue or cell type.

In this application, the term "strong promoter" generally refers to a promoter with a high affinity for RNA polymerase, which can guide the synthesis of a large amount of mRNA, that is, a promoter that can efficiently initiate DNA transcription. Mammalian strong promoters include CMV promoter, CAG promoter, EF1a promoter, SV40 promoter, etc. Insect cell strong promoters include p10 promoter, polh promoter, IE1 promoter, etc. Take the p10 promoter in the insect cell-baculovirus expression system in the specification as an example: the p10 promoter (p10 promoter) is the p10 promoter expressed in the middle and late stages of Autographa californica nuclear polyhedrosis virus (AcMNPV), and is a sequence that can drive the high expression of the target gene in insect cells.

In this application, the term "linear double-stranded no end DNA (neDNA)" generally refers to a linear, double-stranded, closed-end DNA vector, both ends of which are closed by the inverted terminal repeats (ITRs) of the AAV genome. The neDNA vector has covalent closure and is therefore resistant to nucleases (such as exonuclease I or exonuclease III). neDNA can have a variety of configurations, such as: monomers, dimers, trimers and polymers.

In this application, the term "vector" or "construct" is generally a nucleic acid molecule (generally DNA or RNA) used to transfer a passenger nucleic acid sequence (i.e., DNA or RNA) to a host cell. Three common types of vectors include plasmids, bacteriophages, and viruses. The vector is preferably a virus. Vectors containing promoters and polynucleotides that can be operably connected to cloning sites therein are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from suppliers such as Stratagene (LaJolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it is necessary to remove, add, or change the 5' and/or 3' non-translated portions of the clone to remove additional, possibly inappropriate variable translation initiation codons or other sequences that can interfere or reduce expression at the transcription or translation level.

In this application, the term "viral vector" generally refers to a vector that contains some or all of the following viral genes, control sequences, and viral packaging sequences.

In this application, "parvoviral vector" may be defined as a recombinantly produced parvovirus or parvoviral particle comprising a polynucleotide to be delivered to a host cell (in vivo, ex vivo or in vitro). Examples of parvoviral vectors include, for example, adeno-associated virus vectors.

In this application, the term "baculovirus insect cell expression system" or "BEVS" (Baculovirus expression vector system) generally refers to a eukaryotic expression system for expressing foreign proteins. The most widely used baculovirus expression system is the split virus of Autographa california multiply enveloped nuclear polyhedrosisvirus (AcMNPV), referred to as baculovirus. The characteristics of this type of vector are: the baculovirus expression system uses all protein modification, processing and transport systems present in higher eukaryotic cells, and belongs to a eukaryotic expression system; AcMNPV is a non-helper virus and can be suitable for large-scale proliferation in suspended insect cells without any auxiliary factors, so it is convenient to express recombinant proteins in large quantities; this expression system makes the expression product in a soluble state; the baculovirus gene is very large (130kb), which is suitable for cloning large fragments of foreign genes. Baculovirus does not infect vertebrates and viral promoters are inactive in mammalian cells.

In this application, the term "Bacmid" or "bacmid" generally refers to a baculovirus recombinant DNA that can shuttle between insect cells and E. coli. The name is derived from Baculovirus and plasmid.

In the present application, the term "substantially identical", "substantially identical" or "substantially similar" or "substantially similar" generally refers to when two peptide sequences or two nucleotide sequences are optimally aligned, for example, by the GAP or BESTFIT program using default parameters, they share at least a certain percentage of sequence identity, as defined in other parts of the specification. GAP uses the Needleman and Wunsch full sequence alignment algorithm to align two full-length sequences and maximize the number of matches and minimize the number of gaps. The GAP default parameters are generally used, with a gap creation penalty of 50 (nucleotides)/8 (proteins) and a gap extension penalty of 3 (nucleotides)/2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and the default scoring matrix used for proteins is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). When an RNA sequence is considered to be substantially similar to a DNA sequence or to have a certain degree of sequence identity, it is clear that thymine (T) in the DNA sequence is equivalent to uracil (U) in the RNA sequence. The percentage of sequence identity can be determined by sequence alignment and scoring using a computer program. Alternatively, similarity or identity percentages can also be determined by searching databases such as FASTA, BLAST, etc.

In this application, the term "comprise" and its variations such as "comprises" and "comprising" generally refer to including the stated integers or steps or groups of integers or steps, but not excluding any other integers or steps or groups of integers or steps. The term "comprises" used herein may also be replaced by the term "containing" or "including", or the term used herein may sometimes be replaced by the term "having".

It must be noted that, as used in this specification and the appended claims, the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a/an" and the terms "one or more" and "at least one" may be used interchangeably herein.

DETAILED DESCRIPTION OF THE INVENTION

Isolated nucleic acid molecules

In one aspect, the present application provides an isolated nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:12.

In certain embodiments, the isolated nucleic acid molecule encodes adeno-associated virus (AAV) Rep52 protein.

The codons of the Rep52 sequence of the present application are optimized, and after optimization, homologous recombination between Rep78 and Rep52 is avoided. Codon optimization can be performed based on the codon usage of the fall armyworm organism found in the codon usage database (see, for example, http://www.kazusa.or.jp/codon/), or can be performed manually by extracting Sf9 insect cell transcriptome sequencing data from the NCBI database and extracting parameters such as the codon preference adaptation index. Suitable computer programs for codon optimization are available to those skilled in the art (see, for example, Anders Fuglsang, 2003, Protein Expression and Purification 31: 247-249; Jayaraj et al., 2005, Nucl. Acids Res. 33 (9): 3011-3016; Chin et al., 2014 Bioinformatics 30 (15) 2210-2212 and on the Internet). Alternatively, the optimization can be performed manually using the same codon usage database. To avoid homologous recombination, candidate sequences with nucleotide sequences with the same number of consecutive bases less than or equal to 30 and homology less than or equal to 85% were selected for subsequent experimental verification, and finally the Rep52 codon optimized sequence was obtained.

The nucleic acid sequence of Rep52-WT is shown in SEQ ID NO: 13, the nucleic acid sequence of Rep52-CO is shown in SEQ ID NO: 12, and the comparison between Rep52-WT and Rep52-CO before and after codon optimization is shown in FIG8 .

On the other hand, the present application provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the adeno-associated virus (AAV) Rep78 protein and a nucleotide sequence encoding the Rep52 protein, wherein the nucleic acid molecule encoding the Rep52 protein comprises the nucleotide sequence shown in SEQ ID NO:12.

In certain embodiments, the nucleotide sequence encoding the Rep78 protein is wild type.

In certain embodiments, the nucleic acid molecule encoding the Rep78 protein comprises the nucleotide sequence shown in SEQ ID NO:11.

In certain embodiments, it further comprises a first promoter for initiating transcription of the nucleotide sequence encoding the AAV Rep78 protein and a second promoter for initiating transcription of the nucleic acid molecule encoding the AAV Rep52 protein, wherein the first promoter is the same as or different from the second promoter.

In certain embodiments, the first promoter has the same or higher transcription initiation ability as the second promoter.

In certain embodiments, the first promoter comprises the p10 promoter.

In certain embodiments, the first promoter comprises the full-length p10 promoter.

In certain embodiments, the first promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

In certain embodiments, the second promoter comprises a Polyhedrin (polh) promoter.

In certain embodiments, the second promoter comprises the nucleotide sequence shown in SEQ ID NO:10.

In certain embodiments, the transcription directions of the first promoter and the second promoter are the same or opposite.

In certain embodiments, the first promoter is operably linked to the nucleotide sequence encoding the Rep78 protein, and the second promoter is operably linked to the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are the same, it sequentially comprises the first promoter, a nucleotide sequence encoding the Rep78 protein, the second promoter, and a nucleotide sequence encoding the Rep52 protein.

In certain embodiments, the nucleotide sequence encoding the Rep78 protein and the nucleotide sequence encoding the Rep52 protein further comprise a nucleotide sequence encoding polyA (pA) downstream thereof.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are the same, it sequentially comprises the first promoter, a nucleotide sequence encoding the Rep78 protein, a first pA, a second promoter, a nucleotide sequence encoding the Rep52 protein, and a second pA.

In certain embodiments, when the transcription directions of the first promoter and the second promoter are opposite, they sequentially comprise a nucleotide sequence encoding Rep78, a first promoter, a second promoter, and a nucleotide sequence encoding Rep52, wherein the first promoter initiates the transcription of the nucleotide sequence encoding the Rep78 protein, and the second promoter initiates the transcription of the nucleotide sequence encoding the Rep52 protein.

In certain embodiments, the 5' end of the first promoter is directly or indirectly connected to the 5' end of the second promoter.

In certain embodiments, the 3’ end of the first promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep78.

In certain embodiments, it further comprises a first pA, wherein the 3' end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly connected to the 5' end of the first pA.

In certain embodiments, the 3’ end of the second promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep52.

In certain embodiments, it further comprises a second pA, wherein the 3' end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly connected to the 5' end of the second pA.

In certain embodiments, the pA is selected from any one of SV40 polyA and HSV TK polyA.

In certain embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8.

On the other hand, the present application provides an isolated nucleic acid molecule, which comprises, in sequence, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a transcriptional promoter of a nucleotide sequence encoding a Rep78 protein and the first pA, and the second promoter is a transcriptional promoter of a nucleotide sequence encoding a Rep52 protein and the second polyA, wherein the sequences of the nucleotide sequence encoding the Rep52 protein and/or the nucleotide sequence encoding the Rep78 protein are codon-optimized to avoid homologous recombination, and the first promoter has the same or higher transcriptional initiation ability than the second promoter.

Compared to the second promoter, a first promoter having the same or higher transcription initiation ability can be defined as follows: The strength of the promoter can be determined by the expression obtained under the conditions used in the method of the present application.

In certain embodiments, the first promoter or the second promoter is selected from the group consisting of the polh promoter, the p10 promoter, the basic protein promoter, an inducible promoter or the IE1 promoter, or any other late or very late baculovirus gene promoter.

In one embodiment, the first promoter is a p10 promoter and the second promoter is a polh promoter. In another embodiment, the first promoter in the nucleic acid construct of the present invention is a polh promoter and the second promoter is an IE1 promoter. In another embodiment, the first promoter in the nucleic acid construct of the present invention is a p10 promoter and the second promoter is an IE1 promoter. In another embodiment, the first promoter in the nucleic acid construct of the present invention is a polh promoter and the second promoter is a polh promoter.

In certain embodiments, the first promoter comprises the p10 promoter.

In certain embodiments, the first promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

In certain embodiments, the second promoter comprises a Polyhedrin promoter.

In certain embodiments, the second promoter comprises the nucleotide sequence shown in SEQ ID NO:10.

Vectors and expression systems

In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described in the present application.

In certain embodiments, the vector is suitable for expression and/or replication in insect cells.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises a baculovirus vector, for example, the vector may be a pFastBac vector.

In some embodiments, the vector comprises the nucleotide sequence shown in SEQ ID NO:14 or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO:14.

In another aspect, the present application provides a cell comprising the isolated nucleic acid molecule described in the present application or the vector described in the present application.

In certain embodiments, the cell comprises an insect cell. For example, the cell may be an Sf9 cell.

On the other hand, the present application provides a baculovirus expression system, which comprises a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a target gene, wherein the first baculovirus vector is the baculovirus vector described in the present application.

In certain embodiments, from the 5' end to the 3' end, the nucleic acid sequence encoding the target gene comprises, in sequence, a first parvovirus inverted terminal repeat sequence (ITR), the target gene, and a second ITR.

In certain embodiments, the gene of interest comprises at least one nucleotide sequence encoding a gene product of interest expressed in mammalian cells.

In certain embodiments, at least one promoter is included between the first ITR and the target gene.

In certain embodiments, at least one eukaryotic promoter is included between the first ITR and the target gene.

In certain embodiments, at least one mammalian cell promoter is included between the first ITR and the target gene.

In some embodiments, at least one nucleotide sequence of the target gene product expressed in mammalian cells is operably connected to at least one mammalian cell compatible expression control sequence such as a promoter. Many such promoters are known in the art. The constitutive promoter widely expressed in many kinds of cells can be used, for example the CMV promoter. In other embodiments, the promoter is inducible, tissue-specific, cell type-specific or cell cycle-specific. For example, for liver-specific expression, the promoter can be selected from α1-antitrypsin promoter, thyroid hormone binding globulin promoter, albumin promoter, LPS (thyroxine binding globulin) promoter, HCR-ApoCII hybrid promoter, HCR-hAAT hybrid promoter and apolipoprotein E promoter. Other examples include the E2F promoter for tumor-selective, particularly neuronal tumor-selective, expression (Parr et al., 1997, Nat. Med. 3: 1145-9) or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et al., 1997, J Exp Med; 185: 2101-10).

In certain embodiments, a mammalian cell promoter and an insect cell promoter are included between the first ITR and the target gene.

In certain embodiments, the mammalian cell promoter comprises a CMV promoter.

In certain embodiments, the insect cell promoter comprises the p10 promoter.

In certain embodiments, the promoter comprises CMV and p10 promoters.

Insect cells

On the other hand, the present application provides an insect cell containing a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein, and the second nucleotide sequence encodes a nucleotide sequence of a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:11, or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO:11, and the second nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:12, or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO:12.

For example, the cell line used can be from Spodopterafrugiperda, Drosophila cell line, or mosquito cell line, such as Aedes albopictu derived cell line. Preferred insect cells or cell lines are cells from insect species susceptible to baculovirus infection, including, for example, Se301, SeIZD2109, SeUCRU Sf9, Sf900+, Sf21, BT1-TN-5B1-4, MG-1, Tn368, HzAml, Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and (US 6,103,526; Protein Sciences Corp., CT, USA).

In certain embodiments, the first and second nucleotide sequences are part of a nucleic acid construct, wherein the first and second nucleotide sequences are each operably linked to an expression control sequence for insect cell expression.

In certain embodiments, the insect cell further comprises: a third nucleic acid sequence comprising at least one parvovirus inverted terminal repeat (ITR) nucleotide sequence.

In this application, the term "parvovirus ITR" is generally understood to mean a palindromic sequence containing mostly complementary, symmetrically arranged sequences also called "A" and "C" regions. The function of the ITR is a replication origin - a site that has a "cis" effect in replication, that is, it serves as a recognition site for trans-acting replication proteins such as Rep78 (or Rep68), which recognize the palindrome and specific sequences inside the palindrome. An exception to the symmetry of the ITR sequence is the "D" region of the ITR. It is unique (no complementary sequence within one ITR). The cleavage of the single-stranded DNA occurs at the junction between the A region and the D region. It is the region where new DNA synthesis starts. The D region is usually located on one side of the palindrome and provides directionality for the nucleic acid replication step. Parvoviruses that replicate in mammalian cells usually contain two ITR sequences. However, an ITR can be designed so that the binding sites are symmetrically distributed on the two strands of the A region and the D region, one on each side of the palindrome. Thus, on a double-stranded circular DNA template (such as a plasmid), Rep78 or Rep68-assisted nucleic acid replication is performed in both directions, and a single ITR sequence is sufficient for parvoviral replication of the circular vector. Thus, one ITR nucleotide sequence can be used in the context of the present invention. However, preferably, two or other even numbers of regular ITRs are used. Most preferably, two ITR sequences are used. In one embodiment, the parvoviral ITR is an AAV ITR.

In certain embodiments, the third nucleotide sequence further contains at least one nucleotide sequence encoding a target gene.

In certain embodiments, the third nucleotide sequence contains two parvovirus ITR nucleotide sequences, and the at least one nucleotide sequence encoding a gene of interest is located between the two parvovirus ITR nucleotide sequences.

In certain embodiments, the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression.

In certain embodiments, the nucleic acid construct is an insect cell compatible vector.

In the present application, the term "insect cell compatible vector" is generally understood to be a nucleic acid molecule capable of productive transformation or transfection of insects or insect cells. Examples of biological vectors include plasmids, linear nucleic acid molecules and recombinant viruses. Any vector can be used as long as it is compatible with insect cells. The vector can be integrated into the insect cell genome but the vector can also be free. The vector does not need to be permanently present in the insect cell, and also includes transient free vectors. The vector can be introduced by any known method, such as by chemical treatment, electroporation or infection of the cell. In some embodiments, the vector is a baculovirus, a viral vector or a plasmid. For example, the vector can be a baculovirus, that is, the construct is a baculovirus vector.

In certain embodiments, the nucleic acid construct is a baculovirus vector.

In certain embodiments, the insect cell comprises a baculovirus vector described herein.

For example, the insect cells can contain two different baculoviruses, namely: ① Bac-Rep: containing Rep52 and Rep78 of rAAV, regulated by the polyhedrin promoter (Polyhedrin promotor) and the p10 promoter, respectively; ② Bac-Transgene: containing a transgene wrapped by the terminal repeat sequence of rAAV, regulated by the mammalian cytomegalovirus (CMV) promoter.

For another example, the insect cells may contain two different baculoviruses, namely: ① Bac-Rep: containing Rep52 and Rep78 of rAAV, regulated by the polyhedrin promoter (Polyhedrin promotor) and the p10 promoter, respectively; ② Bac-Transgene: containing a transgene wrapped by the terminal repeat sequence of rAAV, co-regulated by the insect p10 and mammalian cytomegalovirus (CMV) promoters.

In certain embodiments, the nucleotide sequence encoding the gene of interest is positioned so that it can be integrated into the neDNA replicated in insect cells. Any nucleotide sequence can be integrated for subsequent expression in mammalian cells transfected with the neDNA produced according to the invention. For example, a nucleotide sequence that can express an RNAi agent, i.e., an RNA molecule capable of RNA interference, such as shRNA (short hairpin RNA) or siRNA (short interfering RNA) can be encoded.

In certain embodiments, the nucleotide sequence encoding the target gene may encode a transposase or a defective transposon of a transposon system, and the transposon system includes but is not limited to the Sleeping Beauty transposon and the piggyBac transposon.

In certain embodiments, the nucleotide sequence encoding the target gene may encode a gene editor or a DNA template for gene editing-mediated homologous recombination, and the gene editing system includes but is not limited to CRISPR, TALEN, and various single-base gene editors.

The target product expressed in mammalian cells can be a therapeutic gene product. The therapeutic gene product can be a polypeptide or an RNA molecule (siRNA) or other gene products, and the other gene products can provide the desired therapeutic effect when expressed in the target cell, such as eliminating unwanted activity, such as removing infected cells or supplementing gene defects (such as defects that cause enzyme activity to be missing). Examples of therapeutic polypeptide gene products include CFTR, Factor IX, Factor VIII, PAH, lipoprotein lipase (LPL, preferably LPLS447X; see WO 01/00220), apolipoprotein A1, uridine diphosphate glucuronyl transferase (UGT), retinitis pigmentosa GTPase regulator interacting protein (RP-GRIP) and cytokines or interleukins such as IL-10. In some embodiments, examples of the therapeutic gene products include polypeptide gene therapy products encoding therapeutic antibodies. In some embodiments, examples of the therapeutic gene products include antigens that encode humoral immune responses or cellular immune responses in vivo for the treatment of infectious diseases and tumors.

In addition, the third nucleotide sequence can also contain the nucleotide sequence of the polypeptide encoding the marker protein to measure cell transformation and expression. Suitable marker proteins for this purpose are, for example, fluorescent protein GFP, luciferase, selective marker gene HSV thymidine kinase (for the selection on HAT culture medium), bacterial hygromycin B phosphotransferase (for the selection of hygromycin B), Tn5 aminoglycoside phosphotransferase (for the selection of G418) and dihydrofolate reductase (DHFR) (for the selection of methotrexate), CD20 (low-affinity nerve growth factor gene). The source of these marker genes and their use methods are shown in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

In addition, the third nucleotide sequence defined above herein may contain a nucleotide sequence encoding a polypeptide that can be used as a failsafe mechanism, which, when deemed necessary, allows the subject to be cured with cells transduced with the neDNA of the present invention. This nucleotide sequence, often referred to as a suicide gene, encodes a protein that can convert a prodrug into a toxic substance that can kill the transgenic cell in which the protein is expressed. Suitable examples of such suicide genes include, for example, the Escherichia coli (E. coli) cytosine deaminase gene or one of the thymidine kinase genes of herpes simplex virus, cytomegalovirus, and varicella zoster virus, in which instances ganciclovir can be used as a prodrug to kill transgenic cells in a subject (see, for example, Clair et al. 1987, Antimicrob. Agents Chemother. 31: 844-849).

In another embodiment, a target gene product can be an AAV protein. In particular, a Rep protein, such as Rep78 and/or Rep52, or a functional fragment thereof. Expression of Rep78 and/or Rep52 in mammalian cells transduced or infected with ne DNA can facilitate certain applications of the vector by allowing long-term or permanent expression of other target gene products introduced into the cell via the recombinant parvovirus (rAAV) vector.

use

On the other hand, the present application provides the use of the baculovirus expression system described in the present application or the insect cell described in the present application in preparing a target nucleic acid molecule.

In certain embodiments, the target nucleic acid molecule is a linear DNA molecule (neDNA) with covalently blocked ends.

On the other hand, the present application provides a method for preparing a target nucleic acid molecule, comprising culturing the insect cells described in the present application.

In certain embodiments, the preparation method comprises:

1) Providing the baculovirus expression system described in the present application;

2) inserting the target gene sequence into the second baculovirus vector;

3) co-transfecting the first baculovirus vector and the second baculovirus vector into insect cells;

4) growing the insect cells under conditions that allow replication and release of the DNA containing the gene of interest;

5) Collect the target nucleic acid molecules.

In certain embodiments, the method further provides for isolating the nucleic acid molecule of interest.

In the specific operation, the two baculoviruses were first used to infect Spodopterafrugiperda (Sf9) cells and amplified separately, and the insect production cells co-infected with the two purified baculoviruses were suspended and cultured. During the production process, parameters such as the amount of infected cells, survival rate, and ne DNA yield were regularly tested to optimize the production process. The cells were harvested at the optimal time period and ne DNA was extracted and purified, and then the yield and quality of ne DNA were tested.

In another aspect, the present application provides a kit comprising the isolated nucleic acid molecule described in the present application, the baculovirus expression system described in the present application and/or the insect cell described in the present application.

This application also provides the following specific implementation methods:

1. An isolated nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:12.

2. An isolated nucleic acid molecule according to embodiment 1, which encodes adeno-associated virus (AAV) Rep52 protein.

3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the adeno-associated virus (AAV) Rep78 protein and a nucleotide sequence encoding the Rep52 protein, wherein the nucleic acid molecule encoding the Rep52 protein comprises the nucleotide sequence shown in SEQ ID NO:12.

4. The isolated nucleic acid molecule according to embodiment 3, wherein the nucleotide sequence encoding the Rep78 protein is wild type.

5. The isolated nucleic acid molecule according to embodiment 3, wherein the nucleic acid molecule encoding the Rep78 protein comprises the nucleotide sequence shown in SEQ ID NO:11.

6. An isolated nucleic acid molecule according to any one of embodiments 3-5, which also includes a first promoter that initiates transcription of the nucleotide sequence encoding the AAVRep78 protein and a second promoter that initiates transcription of the nucleic acid molecule encoding the AAV Rep52 protein, wherein the first promoter is the same as or different from the second promoter.

7. According to the isolated nucleic acid molecule according to embodiment 6, the first promoter and the second promoter comprise insect cell promoters.

8. According to the isolated nucleic acid molecule according to any one of embodiments 6-7, the first promoter comprises a strong promoter.

9. According to the isolated nucleic acid molecule according to any one of embodiments 6-8, the first promoter has the same or higher transcription initiation ability as the second promoter.

10. The isolated nucleic acid molecule of any one of embodiments 6-9, wherein the first promoter and the second promoter are each independently selected from the group consisting of: p10 promoter, Polyhedrin (polh) promoter, and IE1 promoter.

11. An isolated nucleic acid molecule according to any one of embodiments 6-10, wherein the transcription direction of the first promoter and the second promoter is the same or opposite.

12. An isolated nucleic acid molecule according to any one of embodiments 6-11, wherein the first promoter is operably linked to the nucleotide sequence encoding the Rep78 protein, and the second promoter is operably linked to the nucleotide sequence encoding the Rep52 protein.

13. The isolated nucleic acid molecule according to any one of embodiments 11-12, when the transcription directions of the first promoter and the second promoter are the same, comprises, in sequence, a first promoter, a nucleotide sequence encoding a Rep78 protein, a second promoter and a nucleotide sequence encoding a Rep52 protein.

14. The isolated nucleic acid molecule according to embodiment 13, wherein the nucleotide sequence encoding the Rep78 protein and the nucleotide sequence encoding the Rep52 protein further comprise a nucleotide sequence encoding polyA (pA) downstream thereof.

15. According to the isolated nucleic acid molecule described in embodiment 14, when the transcription directions of the first promoter and the second promoter are the same, it sequentially comprises a first promoter, a nucleotide sequence encoding Rep78 protein, a first pA, a second promoter, a nucleotide sequence encoding Rep52 protein and a second pA.

16. According to any one of embodiments 11-12, the isolated nucleic acid molecule comprises, in sequence, a nucleotide sequence encoding Rep78, a first promoter, a second promoter, and a nucleotide sequence encoding Rep52 when the transcription directions of the first promoter and the second promoter are opposite, wherein the first promoter initiates the transcription of the nucleotide sequence encoding the Rep78 protein and the second promoter initiates the transcription of the nucleotide sequence encoding the Rep52 protein.

17. An isolated nucleic acid molecule according to embodiment 16, wherein the 5’ end of the first promoter is directly or indirectly connected to the 5’ end of the second promoter.

18. An isolated nucleic acid molecule according to embodiment 17, wherein the 3’ end of the first promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep78.

19. An isolated nucleic acid molecule according to embodiment 18, which further comprises a first pA, wherein the 3' end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly connected to the 5' end of the first pA.

20. An isolated nucleic acid molecule according to embodiment 16, wherein the 3’ end of the second promoter is directly or indirectly connected to the 5’ end of the nucleotide sequence encoding Rep52.

21. An isolated nucleic acid molecule according to embodiment 20, which further comprises a second pA, wherein the 3' end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly connected to the 5' end of the second pA.

22. The isolated nucleic acid molecule of any one of embodiments 14-21, wherein the pA is selected from any one of SV40 polyA and HSV TK polyA.

23. An isolated nucleic acid molecule according to any one of embodiments 3-22, comprising the nucleotide sequence shown in SEQ ID NO:8.

24. An isolated nucleic acid molecule, which comprises, in sequence, a first polyA (pA), a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a nucleotide sequence encoding a Rep52 protein, and a second polyA (pA), wherein the first promoter is a transcriptional promoter of the nucleotide sequence encoding the Rep78 protein and the first pA, and the second promoter is a transcriptional promoter of the nucleotide sequence encoding the Rep52 protein and the second polyA, wherein the sequences of the nucleotide sequence encoding the Rep52 protein and/or the nucleotide sequence encoding the Rep78 protein are codon-optimized to avoid homologous recombination, the first promoter and the second promoter comprise insect cell promoters, and the first promoter is a strong promoter.

25. The isolated nucleic acid molecule of embodiment 24, wherein the first promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

26. The isolated nucleic acid molecule of embodiment 25, wherein the p10 promoter comprises the nucleotide sequence shown in SEQ ID NO:9.

27. The isolated nucleic acid molecule of embodiment 24, wherein the second promoter comprises a p10 promoter, a polh promoter, or an IE1 promoter.

28. An isolated nucleic acid molecule according to embodiment 27, wherein the polh promoter comprises the nucleotide sequence shown in SEQ ID NO:10.

29. A vector comprising the isolated nucleic acid molecule of any one of embodiments 1-28.

30. The vector of embodiment 29, wherein the vector comprises a viral vector.

31. The vector of embodiment 29, wherein the vector comprises a baculovirus vector.

32. The vector of embodiment 29, comprising a pFastBac vector.

33. A vector according to any one of embodiments 29-32, comprising the nucleotide sequence shown in SEQ ID NO:14.

34. A cell comprising the isolated nucleic acid molecule of any one of embodiments 1-28 or the vector of any one of embodiments 29-33.

35. The cell of embodiment 34, wherein the cell comprises an insect cell.

36. The cell of embodiment 35, which comprises a Spodoptera frugiperda (Sf9) cell.

37. A baculovirus expression system, comprising a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a target gene, wherein the first baculovirus vector is the baculovirus vector of any one of embodiments 31-33.

38. The baculovirus expression system according to claim 37, wherein from the 5' end to the 3' end, the nucleic acid sequence encoding the target gene comprises, in sequence, a first parvovirus inverted terminal repeat (ITR), the target gene and a second ITR.

39. The baculovirus expression system according to any one of embodiment 38, wherein at least one promoter is further included between the first ITR and the target gene.

40. The baculovirus expression system according to any one of embodiments 38-39, wherein at least one eukaryotic promoter is further included between the first ITR and the target gene.

41. The baculovirus expression system according to any one of embodiments 38-40, wherein at least one mammalian cell promoter is further included between the first ITR and the target gene.

42. The baculovirus expression system according to any one of embodiments 38-41, wherein a mammalian cell promoter and an insect cell promoter are further included between the first ITR and the target gene.

43. The baculovirus expression system of any one of embodiments 42, wherein the mammalian cell promoter comprises a ubiquitous promoter and a tissue-specific promoter.

44. The baculovirus expression system of embodiment 43, wherein the ubiquitous promoter comprises a CMV, SV40, EF1a, CAG or UBC promoter.

45. The baculovirus expression system of embodiment 43, wherein the tissue-specific promoter comprises ALB, hAAT, TBG, TTR, GFAP, MHCK7 or hSyn promoter.

46. The baculovirus expression system of any one of embodiments 42-25, wherein the insect cell promoter comprises a p10 promoter.

47. The baculovirus expression system of any one of embodiments 42-26, wherein the promoter comprises CMV and p10 promoters.

48. An insect cell comprising a first nucleotide sequence encoding a first amino acid sequence and a second nucleotide sequence encoding a second amino acid sequence, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a Rep78 protein, and the second nucleotide sequence comprises a nucleotide sequence encoding a Rep52 protein, wherein the first nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:11, and the second nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:12.

49. An insect cell according to embodiment 48, wherein the first and second nucleotide sequences are part of a nucleic acid construct, wherein each of the first and second nucleotide sequences is operably linked to an expression control sequence for insect cell expression.

50. The insect cell of any one of embodiments 48-49, wherein the insect cell further comprises: a third nucleic acid sequence comprising at least one parvoviral inverted terminal repeat nucleotide sequence (ITR).

51. An insect cell according to embodiment 50, wherein the third nucleotide sequence further contains at least one nucleotide sequence encoding a gene of interest.

52. An insect cell according to any one of embodiments 50-51, wherein the third nucleotide sequence contains two parvoviral ITR nucleotide sequences, and wherein the at least one nucleotide sequence encoding a gene of interest is located between the two parvoviral ITR nucleotide sequences.

53. An insect cell according to embodiment 52, wherein the parvovirus comprises an adeno-associated virus.

54. An insect cell according to any one of embodiments 51-53, wherein the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression.

55. An insect cell according to any one of embodiments 49-54, wherein the nucleic acid construct is an insect cell compatible vector.

56. The insect cell of embodiment 55, wherein the nucleic acid construct is a baculovirus vector.

57. An insect cell according to any one of embodiments 48-56, comprising the baculovirus vector of any one of embodiments 31-33 or the baculovirus expression system of any one of embodiments 37-47.

58. Use of the baculovirus expression system described in any one of embodiments 37-47 or the insect cell described in any one of embodiments 48-57 in preparing a target nucleic acid molecule.

59. The use according to embodiment 58, wherein the target nucleic acid molecule is a linear DNA molecule (neDNA) with covalently closed ends.

60. A method for preparing a target nucleic acid molecule, comprising culturing the insect cell described in any one of embodiments 48-57.

61. The preparation method according to embodiment 60, comprising:

1) providing a baculovirus expression system according to any one of embodiments 37-47;

2) inserting the target gene sequence into the second baculovirus vector;

3) co-transfecting the first baculovirus vector and the second baculovirus vector into insect cells;

4) growing the insect cells under conditions that allow replication and release of the DNA containing the gene of interest;

5) Collect the target nucleic acid molecules.

62. According to the preparation method described in embodiment 62, the target nucleic acid molecule is also protected and separated.

63. A kit comprising the isolated nucleic acid molecule of any one of embodiments 1-28, the baculovirus expression system of any one of embodiments 37-47, and/or the insect cell of any one of embodiments 48-57.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the nucleic acid molecules, vectors, expression systems, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

1. Experimental Methods

The technology described herein involves the use of an insect cell-baculovirus system to prepare a linear double-stranded DNA (neDNA) expression vector containing AAV inverted terminal repeats (ITRs) and a gene expression cassette (the EGFP expression cassette is used as an example in this article). The exemplary synthesis method of the neDNA vector disclosed herein involves the following main steps:

1. Vector construction

1.1 Construction of pFastBac-ITR-EGFP donor vector

Using plasmid pAAV-CMV-p10-EGFP as a template, the CMV-p10-EGFP sequence was amplified by PCR, and the upstream and downstream primers were P1 and P2 (see Table 1), respectively. The gene was cloned into the vector pFastBac-AAV-MCS-PA through the restriction sites 5’BamHI and 3’SalI to construct the plasmid pFastBac-ITR-EGFP.

The pAAV-CMV-p10-EGFP vector was obtained by gene synthesis of the CMV-p10 sequence [SEQ ID No: 1], adding 5'KpnI and 3'NcoI restriction sites, and then inserting the restriction sites into the pAAV-EGFP vector [SEQ ID No: 2].

The pFastBac-AAV-MCS-PA vector was obtained by gene synthesis of the ITR-MCS-PA-ITR sequence [SEQ ID No:.3], adding 5’KpnI and 3’HindIII restriction sites, and then restriction digestion and ligation and insertion into the pFastBac dual vector [SEQ ID No:4].

1.2 Construction of pFastBac-ITR-Fluc donor plasmid

The CpGfreeFluc sequence [SEQ ID No: 5] was synthesized by gene synthesis, and 5'SalI and 3'PmlI restriction sites were added. The residues were then cut and ligated and inserted into pFastBac-AAV-MCS-PA to construct pFastBac-CpGfreeFluc.

Table 1: Primer sequences

Primer name Sequence (5'-3') SEQ ID NO: P1 GATCCGGTACCACGCGTCTAG 15 P2 CTCGACGTCGACTTTACTTGTACAGC 16 P3 GCGGGGTTTTACGAGATTGTG 17 P4 GGGGTGCCTGCTCAATCAGA 18 P5 GCAGCACACACTGACATCCA 19 P6 GATCACCGGCGCATCAGAATTG 20 P7 ACTTCAAGATCCGCCACAACAT twenty one P8 TCTCGTTGGGGTCTTGCTCAG twenty two M13F CCCAGTCACGACGTTGTAAAACG twenty three M13R AGCGGATAACAATTTCACACAGG twenty four

1.3 Construction of pFastBac-RepWT and pFastBac-inRep auxiliary vectors

The synthetic gene Rep52WT [SEQ ID No: 13] was cloned into the vector pFastBac-Rep via 5'XmaI and 3'NheI to construct the plasmid pFastBac-RepWT. The synthetic gene inRep [SEQ ID No: 6] was cloned into the vector pFastBac dual via 5'BstZ17I and 3'SphI to construct the plasmid pFastBac-inRep.

1.4 Rep52 codon optimization and construction of pFastBac-CORep auxiliary vector

Extract Sf9 insect cell transcriptome sequencing data from the NCBI database, and capture the codon preference adaptation index and codon background parameters. After inputting the initial Rep52WT sequence, the codon optimization algorithm was used to randomly generate progeny sequences, and the results were iterated until convergence to obtain the candidate codon optimized gene sequence. To avoid homologous recombination, the candidate sequences with the same number of consecutive bases less than or equal to 30 and homology less than or equal to 85% were selected for subsequent experimental verification, and finally the Rep52 codon optimized sequence was obtained.

The codon-optimized sequence of the synthetic gene Rep52, Rep52-CO [SEQ ID No: 12], was cloned into the vector pFastBac-Rep via 5'XmaI and 3'NheI to construct the plasmid pFastBac-CORep.

1.5 Construction of pFastBac-p10Rep auxiliary vector

The conventional synthetic gene p10 [SEQ ID No: 9] was added with 5’BstZ17I and 3’NotI, and the gene was cloned into the vector pFastBac-CORep through 5’BstZ17I and 3’NotI to construct the plasmid pFastBac-p10Rep.

2. Plasmid transformation DH10 Bac

The donor plasmid pFastBac-ITR-EGFP or pFastBac-ITR-Fluc and the helper plasmid (pFastBac-RepWT, pFastBac-inRep, pFastBac-CORep or pFastBac-p10Rep plasmid) were transformed into DH10 Bac E. coli competent cells (Solabo, Cat. No. C1480), respectively. The plasmid in DH10 Bac cells was induced to recombine with the Bacmid bacmid shuttle plasmid to generate the recombinant Bacmid bacmid plasmid. The product of the Φ80dlacZΔM15 gene in DH10 Bac cells can achieve α-complementation of β-galactosidase, and is used for blue-white screening of recombinant Bacmid on LB solid medium (kanamycin (50 μg/ml), tetracycline (10 μg/ml), gentamicin (7 μg/ml), IPTG (40 μg/ml) and X-gal (100 μg/ml)); single white colonies caused by the translocation that destroys the β-galactosidase indicator gene are selected and cultured overnight at 37°C in LB medium (kanamycin (50 μg/ml), tetracycline (10 μg/ml) and gentamicin (7 μg/ml)); PureLink ^TM HiPure Plasimd DNA Miniprep Kit (Thermo Fisher, Cat. No.: K2100-02) is used to extract the recombinant Bacmid rod-shaped plasmid in Escherichia coli.

3. PCR identification of recombinant Bacmid plasmid

The universal primers M13F/R on Bacmid (see Table 1) were used to identify the recombinant Bacmid bacmid by PCR. The conditions for PCR amplification were: 98°C for 2 min; 98°C for 10 s, 60°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min. After the PCR was completed, an agarose gel electrophoresis experiment was performed to determine the size of the target band.

4. Obtaining P0 recombinant baculovirus

The correctly identified recombinant bacmid plasmids Bacmid-ITR-EGFP, Bacmid-ITR-Fluc, Bacmid-RepWT, Bacmid-inRep, Bacmid-CORep and Bacmid-p10Rep were transfected into cells using ExpiFectamine ^™ SfTransfection transfection reagent (Thermo Fisher, Catalog No.: A38915). Sf9 cells (Thermo Fisher, Catalog No.: 11496-015) were pre-plated in 6-well plates and cultured at 27°C without _CO2 . Each well contained 3 ml of Sf900 ^™ III SFM ^™ medium (Thermo Fisher, Catalog No.: 12658-019) containing 1*10 ⁶ Sf9 cells were cultured for 72-96 hours. When the cells showed vacuolar structures and tended to lyse, the cell culture supernatant was collected by centrifugation (500 g, 5 min) and filtered through a 0.22 μm filter to obtain the P0 generation baculovirus, which could be stored at 4°C in the dark.

The titer of recombinant baculovirus was determined by plaque assay, which relies on the replication of the virus in infected cells and the formation of focal lesions in infected surrounding cells. Sf9 cells were pre-plated in a 6-well cell plate at 1*10 ⁶ cells, and the P0 generation baculovirus stock solution was serially diluted with Sf900 ^TM IIISFM ^TM medium in a 10-fold ratio, with dilutions of 10 ^-1 to 10 ^-8 , respectively, and the volume of each dilution was 5 ml. Each 1 ml dilution was added to the above 6-well cell plate, and 2 replicate wells were set for each dilution, and incubated at 27 ° C for 1 hour. Prepare 10 ml of 4% agar solution and 30 ml of Sf-900 medium (1.3x) (Thermo Fisher, Cat. No.: 10967-032) and mix thoroughly, and place in a 40 ° C water bath for use. Completely remove the virus dilution in the 6-well plate, spread 2ml/well of the above agar solution, incubate at room temperature for 1h, and transfer the cell culture plate to a 27℃ incubator for further incubation after the agar is completely solidified. After 7-10 days, small white spots can be seen with the naked eye, which are virus plaques. You can also use 1mg/ml neutral red dye to stain the agar in the 6-well plate to count the number of plaques more clearly. Count the number of single visible plaques at each dilution and calculate the virus titer using the following formula:

At the same time, the SYBR dye method was used for real-time quantitative PCR detection to detect the number of recombinant baculovirus exogenous gene copies to determine the recombinant baculovirus titer. GeneJET Viral DNA and RNA Purification Kit (Thermo Fisher, Cat. No. K0821) was used to extract P0 generation baculovirus genomic DNA, and the viral DNA was dissolved with TE solution and stored at -80°C for later use. qPCR primers were designed with EGFP, RepWT, inRep, CORep and p10Rep sequences, respectively, among which RepWT, CORep and p10Rep sequences shared primers P3 and P4, inRep sequence primers were P5 and P6, and EGFP sequence primers were P7 and P8 (primer sequence information is shown in Table 1). The real-time quantitative PCR system was: 25 μl of SYBR dye premix, 2 μl of upstream and downstream primers (10 μm), 5 μl of sample solution, and 16 μl of distilled water. PCR reaction program: pre-denaturation 95℃60s, 95℃15s, 60℃15s, 72℃45s, 40 cycles; melting curve analysis. Three replicate test tubes are set for each sample. According to the corresponding relationship between the concentration of the standard curve and the Ct value (threshold cycle, Ct), the initial concentration of each sample to be tested can be determined.

5. Amplification of recombinant baculovirus

Usually, the P0 generation baculovirus is small in size and low in titer, and needs to continue to infect Sf9 cells to obtain high-titer baculovirus. The initial P0 generation baculovirus titer is 1*10 ⁶ to 1*10 ⁷ pfu/ml (plaque forming units, pfu), and the P1 generation baculovirus titer after amplification is 1*10 ⁷ to 1*10 ⁸ pfu/ml. Use a 125ml cell shaker containing 30ml Sf9 cells (27℃, 130rpm), with a cell density of 2*10 ⁶ cells/ml, take the P0 generation baculovirus and infect the cells at a multiplicity of infection MOI=0.1; continue to culture for 72-96h, when the number of dead cells reaches 60--80%, collect the cell culture supernatant by centrifugation (500g, 5min) and pass it through a 0.22μm filter, and the P1 generation baculovirus is obtained. Follow the same steps as above to obtain a high-titer P2 virus. The virus titer was determined using real-time quantitative fluorescence PCR and plaque assay (same as above).

6. Identification of baculovirus-expressed Bac-p10Rep protein

Western Blotting (WB) was used to detect the expression of Rep protein. P2-P5 viruses were used to infect Sf9 cells at MOI=3, and the cell samples were collected by centrifugation. The cells were lysed with 1x SDS solution to prepare the protein loading solution. SDS-PAGE was used for electrophoresis. After the electrophoresis, the protein samples were transferred to nitrocellulose membranes; the expression of Rep protein was detected with anti-AAVRep mouse monoclonal antibody (ARP, catalog number: 03-65171), and the stability of Rep protein expression was investigated, that is, the expression of Rep protein in P2, P3, P4 and P5 baculoviruses was observed.

7. Preparation of neDNA-ITR-EGFP and neDNA-ITR-Fluc expression vectors

P2 generation two recombinant baculovirus BacV-ITR-EGFP or BacV-ITR-Fluc and BacV-Rep at MOI = 1-5 (select appropriate MOI parameters), co-infect 2*10 ⁶ cells/ml Sf9 insect cells, continue to culture for 72-96h, when the cell diameter is 18-20μm and the cell viability is about 80%, collect the cells (500g, 5min). Use QIAGEN plasmid extraction kit (Cat. No.: 12163) to extract low molecular weight DNA in Sf9 cells.

8. Expression ability of neDNA in HEK293T cells

HEK293T cells were transfected with neDNA-ITR-EGFP using LipoFectamine 2000 transfection reagent (Thermo Fisher, Catalog No.: 11668-019) (using high-glucose DMEM medium (Gibco, Catalog No.: 11965-092) containing 10% fetal bovine serum and cultured at 37°C, 5% _CO2 ), and the expression of EGFP was observed using a fluorescence microscope after 72 hours.

9. Expression ability of lipid nanoparticles in delivering neDNA in C57BL/6 mice

1 mg of neDNA-ITR-Fluc was dissolved in sodium acetate-acetic acid buffer as the aqueous phase mixture; ionizable cationic lipid Dlin-MC3-DMA, dioleoylphosphatidylcholine DOPC, cholesterol and PEG lipid were dissolved in ethanol at a ratio of 50:10:38:2 as the lipid phase mixture; the aqueous phase and lipid phase were mixed using a Precision Nanosystems Ignite microfluidic chip, and then dialyzed with a neutral phosphate buffer to obtain a LNP-DNA complex suspension in a neutral buffer. C57BL/6 mice were injected into the tail vein at a dose of 2 mg/kg. When observing neDNA-ITR-Fluc-mediated gene expression, 150 mg/kg of luciferase substrate luciferin was injected intraperitoneally into the mice, and the fluorescence signal was observed on a Xenogen IVIS Spectrum small animal in vivo imager.

2. Experimental Results

1. Preparation of donor plasmid and baculovirus

The EGFP gene expression cassette containing ITR sequences at both ends was inserted into the plasmid pFastBac to obtain the recombinant plasmid pFastBac-ITR-EGFP (as shown in FIG1A ).

Auxiliary plasmid pFastBac-p10Rep (as shown in Figure 1B, SEQ ID NO: 14) was constructed, where p10 is the promoter of Rep78-WT and polh is the promoter of Rep52-CO. Auxiliary plasmid pFastBac-RepWT was constructed, where ΔIE1 is the promoter of Rep78 and polh is the promoter of Rep52WT. Auxiliary plasmid pFastBac-inRep was constructed, where p10 is the promoter of Rep78 and there is an artificially synthesized intron sequence (including polh promoter) between Rep78 and Rep52 in the same expression frame as the promoter of Rep52. This construction is based on the work of Haifeng Chen in 2008. Auxiliary plasmid pFastBac-CORep was constructed, where ΔIE1 is the promoter of Rep78 and polh is the promoter of Rep52-CO. FIG. 2 is a schematic diagram of the transcription of the p10Rep gene, the RepWT gene, the inRep gene, and the CORep gene.

The donor plasmid (pFastBac-ITR-EGFP) and different helper plasmids were transformed into DH10 Bac E. coli competent cells, and the bacmids Bacmid-ITR-EGFP and Bacmid-Rep were obtained by blue-white screening, and the recombinant Bacmid bacmids with correct sequences were further screened by PCR. The recombinant Bacmids were transfected into Sf9 cells to obtain recombinant baculoviruses BacV-ITR-EGFP and BacV-Rep.

2. Expression of Rep protein in insect cells Sf9

The Rep protein expressed in Sf9 cells infected with BacV-p10Rep was detected using a mouse monoclonal antibody against AAV Rep (ARP, Catalog No. 03-65171), and uninfected Sf9 cells were used as negative controls. BacV-RepWT ^[1] , BacV-inRep ^[2] , and BacV-CORep (optimized Rep52 sequence in RepWT) were used as controls. P1 generation BacV-Rep baculovirus was used to continuously infect Sf9 cells at an MOI of 0.1 to obtain P2, P3, P4, and P5 baculoviruses, respectively. P2-P5 generations of BacV-Rep were used to infect Sf9 cells at an MOI of 3, and cell samples were collected. The expression of Rep protein was detected by WB, and the stability of Rep protein expression in different generations of baculovirus was investigated, as shown in Figure 3.

3. Preparation of neDNA-ITR-EGFP gene expression vector

Two recombinant baculoviruses of the P2 generation, BacV-ITR-EGFP and BacV-Rep, were co-infected with Sf9 insect cells at an MOI of 3 and cultured for 72-96 hours. When the cell diameter was 18-20 μm and the cell viability was about 80%, the cells were harvested and the small molecular weight neDNA-ITR-EGFP gene expression vector was extracted. The band size of neDNA was identified by electrophoresis using 0.8% agarose gel. The main bands were at 2.7 kb and 5.4 kb, corresponding to the monomer and dimer of the neDNA-ITR-EGFP expression vector, respectively. The bands above the dimer can be inferred to be trimers and polymers according to their size, as shown in Figure 4.

The neDNA was digested with SalI restriction endonuclease to obtain 2kb and 0.7kb bands, which were consistent with the expected fragment size after monomer digestion, as shown in Figure 5A-B. 5A. The neDNA-EGFP monomer fragment is 2.7kb long, and the SalI restriction endonuclease can digest the monomer fragment into 2kb and 0.7kb fragments. 5B. The dimer has a "head-to-head, tail-to-tail" structure and can be digested into 2kb and 1.4kb fragments or 4kb and 0.7kb fragments. The dark blue rectangle indicates the 5'ITR sequence, and the light blue rectangle indicates the 3'ITR sequence.

The expression yields of neDNA-ITR-EGFP gene expression vectors driven by different Rep proteins were different. Compared with the expression yields of neDNA-ITR-EGFP gene expression vectors driven by RepWT, inRep and CORep, the expression yield of neDNA-ITR-EGFP gene expression vector driven by p10Rep was approximately: nearly 270 μg of neDNA gene expression vector could be expressed in every 6*10 ⁷ Sf9 cells, which was 2-3 times that of the other three groups, as shown in Table 2 for details.

Table 2: neDNA yield

4. Transfection of HEK293 cells with neDNA-ITR-EGFP gene expression vector

neDNA-ITR-EGFP was transfected into HEK293 cells using LipoFectamine 2000 transfection reagent, and the expression of EGFP was observed using a fluorescence microscope starting at 24 hours. 72 hours after transfection, the expression of the neDNA-ITR-EGFP gene expression vector in HEK293 cells is shown in FIG6 .

5. neDNA-ITR-Fluc gene expression vector expresses luciferase in mice

The donor plasmid pFastBac-ITR-Fluc and the auxiliary plasmid pFastBac-p10Rep were used to construct the bacmid plasmids Bacmid-ITR-Fluc and Bacmid-p10Rep, and the recombinant baculovirus BacV-ITR-Fluc and BacV-p10Rep were obtained to prepare the neDNA-ITR-Fluc gene expression vector. The neDNA-ITR-Fluc was delivered by nanolipid particles (LNP) and injected into the tail vein of C57BL/6 mice. From 24 hours on, the stable expression of luciferase in the mouse liver could be observed, as shown in Figure 7.

In summary,

The present application provides a method for producing neDNA using an insect cell-baculovirus expression system, wherein the neDNA produced by the method has a variety of configurations, such as monomers, dimers, trimers and polymers, etc. In mouse experiments, compared with plasmid DNA, neDNA can mediate long-term expression of the target gene expression frame in vivo (such as liver).

This method optimizes the Rep protein expression vector and improves the stability of Rep protein expression, thereby improving the yield of neDNA and the stability of the production system. Among them, p10Rep is an optimized expression frame and a complete p10 promoter, which increases the expression level of Rep78; after the Rep52 sequence codon is optimized, homologous recombination between Rep78 and Rep52 is avoided.

Compared with other Bac-Rep and Bac-EFGP co-infected Sf9 cells, Bac-p10Rep has the highest neDNA production, with an average yield increase of 2-3 times.

Compared with other Bac-Rep, Bac-p10Rep has better expression stability of Rep protein (Rep78) after three consecutive baculovirus passages.

references:

[1]Masashi Urabe, Chuantian Ding, Robert M Kotin. Insect cells as afactory to produce adeno-associated virus type 2 vectors. Hum Gene Ther, 2002, 13(16):1935-43.

[2]Haifeng Chen. Intron splicing-mediated expression of AAV Rep andCap genes and production of AAV vectors in insect cells. Mol Ther, 2008, 16(5):924-30.

Sequence Listing

<110> Shanghai Boyin Biotechnology Co., Ltd.

<120> Baculovirus vector and its use

<130> 0251-PA-002

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 1097

<212> DNA

<213> Artificial Sequence

<220>

<223> CMV-p10

<400> 1

ggtaccacgc gtctagttat taatagtaat caattacggg gtcattagtt catagcccat 60

atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 120

acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 180

tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 240

tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 300

attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag 360

tcatcgctat taccatgctg atgcggtttt ggcagtacac caatgggcgt ggatagcggt 420

ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 480

accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg 540

gcggtaggcg tgtacggtgg gaggtctata taagcagacg tcgtttagtg aaccgtcaga 600

tcactagatg ctttattgcg gtagtttatc acagttaaat tgctaacgcc agtctcgaac 660

ttaacgtgca gaagttggtc gtgaggcact gggcaggtaa gtatcgggcc ctttgtgcgg 720

ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg 780

cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg 840

ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt 900

ttggcaaaga attggatcgg accgaaatta atacgactca ctatagggga attgtgagcg 960

gataacaatt ccccggagtt aatccgggac ctttaattca acccaacaca atatattata 1020

gttaaataag aattattatc aaatcatttg tatattaatt aaaatactat actgtaaatt 1080

acattttatt tacaatc 1097

<210> 2

<211> 5547

<212> DNA

<213> Artificial Sequence

<220>

<223> pAAV-GFP

<400> 2

cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60

cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120

gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180

agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240

acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300

acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360

agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420

gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480

cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540

cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600

tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660

gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720

tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780

ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840

taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900

cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960

acgattaccg ttcatcgcct gcactgcgcg ctcgctcgct cactgaggcc gcccgggcaa 1020

agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 1080

agggagtgga attcacgcgt ggtacgatct gaattcggta caattcacgc gtgggtacca 1140

cgcgtctagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 1200

gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 1260

cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 1320

acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 1380

tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 1440

ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 1500

tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 1560

acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 1620

tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 1680

gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg 1740

gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctcca 1800

ccggttcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 1860

ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 1920

cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1980

gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc 2040

cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 2100

gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 2160

gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 2220

catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga 2280

caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag 2340

cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 2400

gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 2460

cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 2520

gctgtacaag taaagcggcc atcaagctta tcgataccgt cgactagagc tcgctgatca 2580

gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2640

ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2700

cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2760

gaggattggg aagacaatag caggcatgct ggggagagat cgatctgagg aacccctagt 2820

gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa 2880

ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga 2940

gggagtggcc aacccccccc cccccccccc tgcatgcagg cgattctctt gtttgctcca 3000

gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag ctaccctctc 3060

cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 3120

cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat 3180

atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 3240

attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 3300

gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg gaattcctga 3360

tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3420

gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 3480

acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3540

ccggggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3600

gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3660

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3720

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 3780

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 3840

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 3900

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 3960

gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 4020

cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactgagtga 4080

taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4140

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4200

agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4260

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4320

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4380

tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4440

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4500

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4560

agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4620

gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4680

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4740

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4800

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4860

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4920

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4980

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5040

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5100

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5160

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5220

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5280

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5340

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5400

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5460

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 5520

ccccgcgcgt tggccgattc attaatg 5547

<210> 3

<211> 925

<212> DNA

<213> Artificial Sequence

<220>

<223> ITR-MCS-PA-ITR

<400> 3

ggtaccacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 60

agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 120

agggagtggc caactccatc actaggggtt cctgcggccg cagatctacc ggtggcgcgc 180

cggatccgaa ttctctagag tcgacgtcga gctagcatcg atgtttaaac gagctcacta 240

gtctcgagac gcgtacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct 300

ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt 360

gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg 420

gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc 480

agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc 540

tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt 600

tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca 660

ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc 720

cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc 780

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 840

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 900

agctgcctgc aggggcgcca agctt 925

<210> 4

<211> 5238

<212> DNA

<213> Artificial Sequence

<220>

<223> pFastBac dual

<400> 4

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540

attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040

gggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100

acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160

ggtaagcggc agggtcggaa caggagagcg cacgaggggag cttccagggg gaaacgcctg 2220

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520

tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggctttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020

gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080

aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140

ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200

ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260

tcccccatct cccggtaccg catgctatgc atcagctgct agcaccatgg ctcgagatcc 4320

cgggtgatca agtcttcgtc gagtgattgt aaataaaatg taatttacag tatagtattt 4380

taattaatat acaaatgatt tgataataat tctttatttaa ctataatata ttgtgttggg 4440

ttgaattaaa ggtccgtata ctccggaata ttaatagatc atggagataa ttaaaatgat 4500

aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt aataaaaaaa 4560

cctataaata ttccggatta ttcataccgt cccaccatcg ggcgcggatc ccggtccgaa 4620

gcgcgcggaa ttcaaaggcc tacgtcgacg agctcactag tcgcggccgc tttcgaatct 4680

agagcctgca gtctcgacaa gcttgtcgag aagtactaga ggatcataat cagccatacc 4740

acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 4800

cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 4860

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 4920

ggtttgtcca aactcatcaa tgtatctttat catgtctgga tctgatcact gcttgagcct 4980

aggagatccg aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt 5040

cgtattagct tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa 5100

tccttaaaaa ctccatttcc acccctccca gttcccaact attttgtccg cccacagcgg 5160

ggcatttttc ttcctgttat gtttttaatc aaacatcctg ccaactccat gtgacaaacc 5220

gtcatcttcg gctacttt 5238

<210> 5

<211> 3129

<212> DNA

<213> Artificial Sequence

<220>

<223> CpGfreeFluc

<400> 5

tttagggtta gggttagggt tagggaaaaa tttagggtta gggttagggt tagggaaaaa 60

tttagggtta gggttagggt tagggaaaaa aagcttgagt caatgggaaa aacccattgg 120

agccaagtac actgactcaa tagggacttt ccattgggtt ttgcccagta cataaggtca 180

atagggggtg agtcaacagg aaagtcccat tggagccaag tacattgagt caatagggac 240

tttccaatgg gttttgccca gtacataagg tcaatgggag gtaagccaat gggtttttcc 300

cattactgac atgtatactg agtcattagg gactttccaa tgggttttgc ccagtacata 360

aggtcaatag gggtgaatca acaggaaagt cccattggag ccaagtacac tgagtcaata 420

gggactttcc attgggtttt gcccagtaca aaaggtcaat agggggtgag tcaatgggtt 480

tttcccatta ttggcacata cataaggtca ataggggtga ctagtggaga agagcatgct 540

tgagggctga gtgcccctca gtgggcagag agcacatggc ccacagtccc tgagaagttg 600

gggggagggg tgggcaattg aactggtgcc tagagaaggt ggggcttggg taaactggga 660

aagtgatgtg gtgtactggc tccacctttt tccccagggt gggggagaac catataag 720

tgcagtagtc tctgtgaaca ttcaagcttc tgccttctcc ctcctgtgag tttggtaagt 780

cactgactgt ctatgcctgg gaaagggtgg gcaggaggtg gggcagtgca ggaaaagtgg 840

cactgtgaac cctgcagccc tagacaattg tactaacctt cttctctttc ctctcctgac 900

aggttggtgt acagtagctt ccaccatgga ggatgccaag aatattaaga aaggccctgc 960

cccattctac cctctggaag atggcactgc tggtgagcaa ctgcacaagg ccatgaagag 1020

gtatgccctg gtccctggca ccattgcctt cactgatgct cacattgagg tggacatcac 1080

ctatgctgaa tactttgaga tgtctgtgag gctggcagaa gccatgaaaa gatatggact 1140

gaacaccaac cacaggattg tggtgtgctc tgagaactct ctccagttct tcatgcctgt 1200

gttaggagcc ctgttcattg gagtggctgt ggcccctgcc aatgacatct acaatgagag 1260

agagctcctg aacagcatgg gcatcagcca gccaactgtg gtctttgtga gcaagaaggg 1320

cctgcaaaag atcctgaatg tgcagaagaa gctgcccatc atccagaaga tcatcatcat 1380

ggacagcaag actgactacc agggcttcca gagcatgtat acctttgtga ccagccactt 1440

accccctggc ttcaatgagt atgactttgt gcctgagagc tttgacaggg acaagaccat 1500

tgctctgatt atgaacagct ctggctccac tggactgccc aaaggtgtgg ctctgcccca 1560

cagaactgct tgtgtgagat tcagccatgc cagagacccc atctttggca accagatcat 1620

ccctgacact gccatcctgt ctgtggttcc attccatcat ggctttggca tgttcacaac 1680

actggggtac ctgatctgtg gcttcagagt ggtgctgatg tataggtttg aggaggagct 1740

gtttctgagg agcctacaag actacaagat ccagtctgcc ctgctggtgc ccactctgtt 1800

cagcttcttt gccaagagca ccctcattga caagtatgac ctgagcaacc tgcatgagat 1860

tgcctctgga ggagcacccc tgagcaagga ggtgggtgag gctgtggcaa agaggttcca 1920

tctcccagga atcagacagg gctatggcct gactgagacc acctctgcca tcctcatcac 1980

ccctgaagga gatgacaagc ctggtgctgt gggcaaggtg gttccctttt ttgaggccaa 2040

ggtggtggac ctggacactg gcaagaccct gggagtgaac cagaggggtg agctgtgtgt 2100

gaggggtccc atgatcatgt ctggctatgt gaacaaccct gaggccacca atgccctgat 2160

tgacaaggat ggctggctgc actctggtga cattgcctac tgggatgagg atgagcactt 2220

tttcattgtg gacaggctga agagcctcat caagtacaaa ggctaccaag tggcacctgc 2280

tgagctagag agcatcctgc tccagcaccc caacatcttt gatgctggtg tggctggcct 2340

gcctgatgat gatgctggag agctgcctgc tgctgttgtg gttctggagc atggaaagac 2400

catgactgag aaggagattg tggactatgt ggccagtcag gtgaccactg ccaagaagct 2460

gaggggaggt gtggtgtttg tggatgaggt gccaaagggt ctgactggca agctggatgc 2520

cagaaagatc agagagatcc tgatcaaggc caagaagggt ggcaaacaat tgatctctgg 2580

agccaatgga gtctagctag ctggccagac atgataagat acattgatga gtttggacaa 2640

accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2700

ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2760

atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2820

tgtggtatgg aattcggatc cggtgtggaa agtccccagg ctccccagca ggcagaagta 2880

tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 2940

caggcagaag tatgcaaagc atgcatctca attagtcagc aaccagagct ctggggactt 3000

tccgctgggg actttccgct ggggactttc cgctggggac tttccgctgg ggactttccg 3060

catttaaatg gtacattttg ttctagaaca aaatgtaccg gtacattttg ttctggtaca 3120

ttttgttct 3129

<210> 6

<211> 2305

<212> DNA

<213> Artificial Sequence

<220>

<223> inRep

<400> 6

gacctttaat tcaacccaac acaatatatt atagttaaat aagaattatt atcaaatcat 60

ttgtatatta attaaaatac tatactgtaa attacatttt atttacaatc actcgacgaa 120

gacttgatca ccctacccgc catgccgggg ttttacgaga ttgtgattaa ggtccccagc 180

gaccttgacg agcatctgcc cggcatttct gacagctttg tgaactgggt ggccgagaag 240

gaatggggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca ggcacccctg 300

accgtggccg agaagctgca gcgcgacttt ctgacggaat ggcgccgtgt gagtaaggcc 360

ccggaggccc ttttctttgt gcaatttgag aagggagaga gctacttcca catgcacgtg 420

ctcgtggaaa ccaccggggt gaaatccatg gttttgggac gtttcctgag tcagattcgc 480

gaaaaactga ttcagagaat ttaccgcggg atcgagccga ctttgccaaa ctggttcgcg 540

gtcacaaaga ccagaaatgg cgccggaggc gggaacaagg tggtggatga gtgctacatc 600

cccaattact tgctccccaa aacccagcct gagctccagt gggcgtggac taatatggaa 660

cagtatttaa ggtaagtact ccctatcagt gatagagatc tatcatggag ataattaaaa 720

tgataaccat ctcgcaaata aataagtatt ttactgtttt cgtaacagtt ttgtaataaa 780

aaaacctata aatattccgg attattcata ccgtcccacc atcgggcgcg aagggggaga 840

cctgtagtca gagcccccgg gcagcacaca ctgacatcca ctcccttcct attgtttcag 900

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 960

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 1020

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 1080

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 1140

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 1200

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg 1260

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1320

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1380

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1440

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1500

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1560

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat 1620

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1680

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1740

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1800

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1860

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1920

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1980

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 2040

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 2100

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 2160

gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 2220

catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt 2280

ggctcgagga cactctctct gaagg 2305

<210> 7

<211> 3884

<212> DNA

<213> Artificial Sequence

<220>

<223> CORep

<400> 7

gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60

gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg 120

catgctatgc atcagctgct agcttactgc tcgaagatgc agtcgtccag atccacgttc 180

acgagatcgc aagcagtgca agcgtcgggc accttgccca tgatgtggtg gatgtagcac 240

agcttctggt aggccttctt gacgacggac acgggttggg actcggagac ggggaagcat 300

tccagacagt ctttctggcc gtgggtgaag cagatgttgg agttctggtt catgcgctcg 360

cactggcggc aagggaacag catcagattc atgcccacgt ggcggctgca cttattctgg 420

tagcggtcgg cgtagttgat ggaggcttca gcatcggagg tggaaggctg agcgacggac 480

tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg gagcggggcg cttcttagca 540

ccgcccttct tcacgtagaa ctcgtgctcc acctccacga cgtgatcctt ggcccaacgg 600

aagaagtcct tcacctcttg cttggtgact ttgccgaagt cgtggtccag acggcgggtg 660

agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat gttcgaaggt agtggagttg 720

ccgtcgatga cagcgcacat gttggtgttg gaagtcacga tgacgggggt ggggtcgatc 780

tgagcggagg acttgcactt ctggtcgaca cgcaccttgc taccacccag aatggccttg 840

gcggattcga ccaccttggc agtcatcttg ccctcttccc accagatgac catcttgtcg 900

acgcagtcgt tgaaggggaa gttctcgttg gtccagttga cgcagccgta aaagggcacg 960

gtatgggcga tggcttcggc gatgttggtc ttaccagtgg tagcgggacc gaagagccag 1020

atagtgttgc gcttgccgaa cttcttggta gcccaaccga ggaagacgga ggcggcatac 1080

tgggggtcgt agccgttgag ctccagaatc ttgtagatgc ggttggagga gatgtcctcc 1140

acgggctgtt gaccgaccag ataatcggga gcggtcttgg tgaggctcat gatcttacca 1200

gcgttgtcga gggcagcctt gatctgggaa cgggagttgc tggcagcatt gaagctgatg 1260

tagctggctt ggtcctcttg gatccactgc ttctcgctag tgatgccctt gtcgaccagc 1320

caaccgacca gttccatggt ggcccgggtt tcggaccgag atccgcgccc gatggtggga 1380

cggtatgaat aatccggaat atttataggt ttttttatta caaaactgtt acgaaaacag 1440

taaaatactt atttatttgc gagatggtta tcattttaat tatctccatg atctattaat 1500

attccggagt atacaataaa cgataacgcc gttggtggcg tgaggcatgt aaaaggttac 1560

atcattatct tgttcgccat ccggttggta taaatagacg ttcatgttgg tttttgtttc 1620

agttgcaagt tggctgcggc gcgcgcagca cctttgcggc cgccaccatg gcggggtttt 1680

acgagattgt gattaaggtc cccagcgacc ttgacgagca tctgcccggc atttctgaca 1740

gctttgtgaa ctgggtggcc gagaaggaat gggagttgcc gccagattct gacatggatc 1800

tgaatctgat tgagcaggca cccctgaccg tggccgagaa gctgcagcgc gactttctga 1860

cggaatggcg ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa tttgagaagg 1920

gagagagcta cttccacatg cacgtgctcg tggaaaccac cggggtgaaa tccatggttt 1980

tgggacgttt cctgagtcag attcgcgaaa aactgattca gagaatttac cgcggggatcg 2040

agccgacttt gccaaactgg ttcgcggtca caaagaccag aaatggcgcc ggaggcggga 2100

acaaggtggt ggatgagtgc tacatcccca attacttgct ccccaaaacc cagcctgagc 2160

tccagtgggc gtggactaat atggaacagt atttaagcgc ctgtttgaat ctcacggagc 2220

gtaaacggtt ggtggcgcag catctgacgc acgtgtcgca gacgcaggag cagaacaaag 2280

agaatcagaa tcccaattct gatgcgccgg tgatcagatc aaaaacttca gccaggtaca 2340

tggagctggt cgggtggctc gtggacaagg ggattacctc ggagaagcag tggatccagg 2400

aggaccaggc ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg 2460

ctgccttgga caatgcggga aagattatga gcctgactaa aaccgccccc gactacctgg 2520

tgggccagca gcccgtggag gacatttcca gcaatcggat ttataaaatt ttggaactaa 2580

acgggtacga tccccaatat gcggcttccg tctttctggg atgggccacg aaaaagttcg 2640

gcaagaggaa caccatctgg ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg 2700

aggccatagc ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat gagaactttc 2760

ccttcaacga ctgtgtcgac aagatggtga tctggtggga ggaggggaag atgaccgcca 2820

aggtcgtgga gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat 2880

gcaagtcctc ggcccagata gacccgactc ccgtgatcgt cacctccaac accaacatgt 2940

gcgccgtgat tgacgggaac tcaacgacct tcgaacacca gcagccgttg caagaccgga 3000

tgttcaaatt tgaactcacc cgccgtctgg atcatgactt tgggaaggtc accaagcagg 3060

aagtcaaaga ctttttccgg tgggcaaagg atcacgtggt tgaggtggag catgaattct 3120

acgtcaaaaa gggtggagcc aagaaaagac ccgcccccag tgacgcagat ataagtgagc 3180

ccaaacgggt gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa gcttcgatca 3240

actacgcaga caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt 3300

ttccctgcag acaatgcgag agaatgaatc agaattcaaa tatctgcttc actcacggac 3360

agaaagactg tttagagtgc tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa 3420

aggcgtatca gaaactgtgc tacattcatc atatcatggg aaaggtgcca gacgcttgca 3480

ctgcctgcga tctggtcaat gtggatttgg atgactgcat ctttgaacaa taaatgattt 3540

aaatcaggta tggctgccga tggttatctt ccagattggc tcgaggacac tctctctgat 3600

ctagagcctg cagtctcgac aagcttgtcg agaagtacta gaggatcata atcagccata 3660

ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 3720

aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 3780

aataaagcaa tagcatcaca aatttcacaa ataaagcatttttttcactg cattctagtt 3840

gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatc 3884

<210> 8

<211> 3874

<212> DNA

<213> Artificial Sequence

<220>

<223> p10Rep

<400> 8

gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60

gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg 120

catgctatgc atcagctgct agcttactgc tcgaagatgc agtcgtccag atccacgttc 180

acgagatcgc aagcagtgca agcgtcgggc accttgccca tgatgtggtg gatgtagcac 240

agcttctggt aggccttctt gacgacggac acgggttggg actcggagac ggggaagcat 300

tccagacagt ctttctggcc gtgggtgaag cagatgttgg agttctggtt catgcgctcg 360

cactggcggc aagggaacag catcagattc atgcccacgt ggcggctgca cttattctgg 420

tagcggtcgg cgtagttgat ggaggcttca gcatcggagg tggaaggctg agcgacggac 480

tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg gagcggggcg cttcttagca 540

ccgcccttct tcacgtagaa ctcgtgctcc acctccacga cgtgatcctt ggcccaacgg 600

aagaagtcct tcacctcttg cttggtgact ttgccgaagt cgtggtccag acggcgggtg 660

agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat gttcgaaggt agtggagttg 720

ccgtcgatga cagcgcacat gttggtgttg gaagtcacga tgacgggggt ggggtcgatc 780

tgagcggagg acttgcactt ctggtcgaca cgcaccttgc taccacccag aatggccttg 840

gcggattcga ccaccttggc agtcatcttg ccctcttccc accagatgac catcttgtcg 900

acgcagtcgt tgaaggggaa gttctcgttg gtccagttga cgcagccgta aaagggcacg 960

gtatgggcga tggcttcggc gatgttggtc ttaccagtgg tagcgggacc gaagagccag 1020

atagtgttgc gcttgccgaa cttcttggta gcccaaccga ggaagacgga ggcggcatac 1080

tgggggtcgt agccgttgag ctccagaatc ttgtagatgc ggttggagga gatgtcctcc 1140

acgggctgtt gaccgaccag ataatcggga gcggtcttgg tgaggctcat gatcttacca 1200

gcgttgtcga gggcagcctt gatctgggaa cgggagttgc tggcagcatt gaagctgatg 1260

tagctggctt ggtcctcttg gatccactgc ttctcgctag tgatgccctt gtcgaccagc 1320

caaccgacca gttccatggt ggcccgggtt tcggaccgag atccgcgccc gatggtggga 1380

cggtatgaat aatccggaat atttataggt ttttttatta caaaactgtt acgaaaacag 1440

taaaatactt atttatttgc gagatggtta tcattttaat tatctccatg atctattaat 1500

attccggagt atacggacct ttaattcaac ccaacacaat atattatagt taaataagaa 1560

ttattatcaa atcatttgta tattaattaa aatactatac tgtaaattac attttattta 1620

caatcactcg acgaagactt gatcagcggc cgccaccatg gcggggtttt acgagattgt 1680

gattaaggtc cccagcgacc ttgacgagca tctgcccggc atttctgaca gctttgtgaa 1740

ctgggtggcc gagaaggaat gggagttgcc gccagattct gacatggatc tgaatctgat 1800

tgagcaggca cccctgaccg tggccgagaa gctgcagcgc gactttctga cggaatggcg 1860

ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa tttgagaagg gagagagcta 1920

cttccacatg cacgtgctcg tggaaaccac cggggtgaaa tccatggttt tgggacgttt 1980

cctgagtcag attcgcgaaa aactgattca gagaatttac cgcggggatcg agccgacttt 2040

gccaaactgg ttcgcggtca caaagaccag aaatggcgcc ggaggcggga acaaggtggt 2100

ggatgagtgc tacatcccca attacttgct ccccaaaacc cagcctgagc tccagtgggc 2160

gtggactaat atggaacagt atttaagcgc ctgtttgaat ctcacggagc gtaaacggtt 2220

ggtggcgcag catctgacgc acgtgtcgca gacgcaggag cagaacaaag agaatcagaa 2280

tcccaattct gatgcgccgg tgatcagatc aaaaacttca gccaggtaca tggagctggt 2340

cgggtggctc gtggacaagg ggattacctc ggagaagcag tggatccagg aggaccaggc 2400

ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc caaatcaagg ctgccttgga 2460

caatgcggga aagattatga gcctgactaa aaccgccccc gactacctgg tgggccagca 2520

gcccgtggag gacatttcca gcaatcggat ttataaaatt ttggaactaa acgggtacga 2580

tccccaatat gcggcttccg tctttctggg atgggccacg aaaaagttcg gcaagaggaa 2640

caccatctgg ctgtttgggc ctgcaactac cgggaagacc aacatcgcgg aggccatagc 2700

ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat gagaactttc ccttcaacga 2760

ctgtgtcgac aagatggtga tctggtggga ggaggggaag atgaccgcca aggtcgtgga 2820

gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg gaccagaaat gcaagtcctc 2880

ggcccagata gacccgactc ccgtgatcgt cacctccaac accaacatgt gcgccgtgat 2940

tgacgggaac tcaacgacct tcgaacacca gcagccgttg caagaccgga tgttcaaatt 3000

tgaactcacc cgccgtctgg atcatgactt tgggaaggtc accaagcagg aagtcaaaga 3060

ctttttccgg tgggcaaagg atcacgtggt tgaggtggag catgaattct acgtcaaaaa 3120

gggtggagcc aagaaaagac ccgcccccag tgacgcagat ataagtgagc ccaaacgggt 3180

gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa gcttcgatca actacgcaga 3240

caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat ctgatgctgt ttccctgcag 3300

acaatgcgag agaatgaatc agaattcaaa tatctgcttc actcacggac agaaagactg 3360

tttagagtgc tttcccgtgt cagaatctca acccgtttct gtcgtcaaaa aggcgtatca 3420

gaaactgtgc tacattcatc atatcatggg aaaggtgcca gacgcttgca ctgcctgcga 3480

tctggtcaat gtggatttgg atgactgcat ctttgaacaa taaatgattt aaatcaggta 3540

tggctgccga tggttatctt ccagattggc tcgaggacac tctctctgat ctagagcctg 3600

cagtctcgac aagcttgtcg agaagtacta gaggatcata atcagccata ccacatttgt 3660

agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 3720

gaatgcaatt gttgttgtta acttgtttat tgcagctttat aatggttaca aataaagcaa 3780

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3840

caaactcatc aatgtatctt atcatgtctg gatc 3874

<210> 9

<211> 110

<212> DNA

<213> Artificial Sequence

<220>

<223> p10

<400> 9

gacctttaat tcaacccaac acaatatatt atagttaaat aagaattatt atcaaatcat 60

ttgtatatta attaaaatac tatactgtaa attacatttt atttacaatc 110

<210> 10

<211> 92

<212> DNA

<213> Artificial Sequence

<220>

<223> polh

<400> 10

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa at 92

<210> 11

<211> 1866

<212> DNA

<213> Artificial Sequence

<220>

<223> Rep78-WT

<400> 11

atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg 240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc 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> 12

<211> 1194

<212> DNA

<213> Artificial Sequence

<220>

<223> Rep52-CO

<400> 12

atggaactgg tcggttggct ggtcgacaag ggcatcacta gcgagaagca gtggatccaa 60

gaggaccaag ccagctacat cagcttcaat gctgccagca actcccgttc ccagatcaag 120

gctgccctcg acaacgctgg taagatcatg agcctcacca agaccgctcc cgattatctg 180

gtcggtcaac agcccgtgga ggacatctcc tccaaccgca tctacaagat tctggagctc 240

aacggctacg acccccagta tgccgcctcc gtcttcctcg gttgggctac caagaagttc 300

ggcaagcgca acactatctg gctcttcggt cccgctacca ctggtaagac caacatcgcc 360

gaagccatcg cccataccgt gcccttttac ggctgcgtca actggaccaa cgagaacttc 420

cccttcaacg actgcgtcga caagatggtc atctggtggg aagagggcaa gatgactgcc 480

aaggtggtcg aatccgccaa ggccattctg ggtggtagca aggtgcgtgt cgaccagaag 540

tgcaagtcct ccgctcagat cgaccccacc cccgtcatcg tgacttccaa caccaacatg 600

tgcgctgtca tcgacggcaa ctccactacc ttcgaacatc agcaacctct gcaagaccgc 660

atgttcaaat tcgagctcac ccgccgtctg gaccacgact tcggcaaagt caccaagcaa 720

gaggtgaagg acttcttccg ttgggccaag gatcacgtcg tggaggtgga gcacgagttc 780

tacgtgaaga agggcggtgc taagaagcgc cccgctccca gcgacgctga tatcagcgag 840

cctaagcgcg tgcgtgagtc cgtcgctcag ccttccacct ccgatgctga agcctccatc 900

aactacgccg accgctacca gaataagtgc agccgccacg tgggcatgaa tctgatgctg 960

ttcccttgcc gccagtgcga gcgcatgaac cagaactcca acatctgctt cacccacggc 1020

cagaaagact gtctggaatg cttccccgtc tccgagtccc aacccgtgtc cgtcgtcaag 1080

aaggcctacc agaagctgtg ctacatccac cacatcatgg gcaaggtgcc cgacgcttgc 1140

actgcttgcg atctcgtgaa cgtggatctg gacgactgca tcttcgagca gtaa 1194

<210> 13

<211> 1194

<212> DNA

<213> Artificial Sequence

<220>

<223> Rep52WT

<400> 13

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag 120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg 180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta 240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc 300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg 360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt 420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc 480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa 540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg 600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg 660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag 720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc 780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag 840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc 900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg 960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga 1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa 1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc 1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa 1194

<210> 14

<211> 8310

<212> DNA

<213> Artificial Sequence

<220>

<223> pFastBac-p10Rep

<400> 14

ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60

ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540

attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200

accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040

gggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100

acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160

ggtaagcggc agggtcggaa caggagagcg cacgaggggag cttccagggg gaaacgcctg 2220

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520

tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580

agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640

caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700

aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760

cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820

gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880

gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940

ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000

ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060

cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120

ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180

ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240

atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300

atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360

gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420

gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480

cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540

aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600

agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggctttatgtc aactgggttc 3660

gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720

aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840

aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900

tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960

atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020

gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080

aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140

ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200

ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260

tcccccatct cccggtaccg catgctatgc atcagctgct agcttactgc tcgaagatgc 4320

agtcgtccag atccacgttc acgagatcgc aagcagtgca agcgtcgggc accttgccca 4380

tgatgtggtg gatgtagcac agcttctggt aggccttctt gacgacggac acgggttggg 4440

actcggagac ggggaagcat tccagacagt ctttctggcc gtgggtgaag cagatgttgg 4500

agttctggtt catgcgctcg cactggcggc aagggaacag catcagattc atgcccacgt 4560

ggcggctgca cttattctgg tagcggtcgg cgtagttgat ggaggcttca gcatcggagg 4620

tggaaggctg agcgacggac tcacgcacgc gcttaggctc gctgatatca gcgtcgctgg 4680

gagcggggcg cttcttagca ccgcccttct tcacgtagaa ctcgtgctcc acctccacga 4740

cgtgatcctt ggcccaacgg aagaagtcct tcacctcttg cttggtgact ttgccgaagt 4800

cgtggtccag acggcgggtg agctcgaatt tgaacatgcg gtcttgcaga ggttgctgat 4860

gttcgaaggt agtggagttg ccgtcgatga cagcgcacat gttggtgttg gaagtcacga 4920

tgacgggggt ggggtcgatc tgagcggagg acttgcactt ctggtcgaca cgcaccttgc 4980

taccacccag aatggccttg gcggattcga ccaccttggc agtcatcttg ccctcttccc 5040

accagatgac catcttgtcg acgcagtcgt tgaaggggaa gttctcgttg gtccagttga 5100

cgcagccgta aaagggcacg gtatgggcga tggcttcggc gatgttggtc ttaccagtgg 5160

tagcgggacc gaagagccag atagtgttgc gcttgccgaa cttcttggta gcccaaccga 5220

ggaagacgga ggcggcatac tggggggtcgt agccgttgag ctccagaatc ttgtagatgc 5280

ggttggagga gatgtcctcc acgggctgtt gaccgaccag ataatcggga gcggtcttgg 5340

tgaggctcat gatcttacca gcgttgtcga gggcagcctt gatctgggaa cgggagttgc 5400

tggcagcatt gaagctgatg tagctggctt ggtcctcttg gatccactgc ttctcgctag 5460

tgatgccctt gtcgaccagc caaccgacca gttccatggt ggcccgggtt tcggaccgag 5520

atccgcgccc gatggtggga cggtatgaat aatccggaat atttataggt ttttttatta 5580

caaaactgtt acgaaaacag taaaatactt atttatttgc gagatggtta tcattttaat 5640

tatctccatg atctattaat attccggagt atacggacct ttaattcaac ccaacacaat 5700

atattatagt taaataagaa ttattatcaa atcatttgta tattaattaa aatactatac 5760

tgtaaattac attttattta caatcactcg acgaagactt gatcagcggc cgccaccatg 5820

gcggggtttt acgagattgt gattaaggtc cccagcgacc ttgacgagca tctgcccggc 5880

atttctgaca gctttgtgaa ctgggtggcc gagaaggaat gggagttgcc gccagattct 5940

gacatggatc tgaatctgat tgagcaggca cccctgaccg tggccgagaa gctgcagcgc 6000

gactttctga cggaatggcg ccgtgtgagt aaggccccgg aggccctttt ctttgtgcaa 6060

tttgagaagg gagagagcta cttccacatg cacgtgctcg tggaaaccac cggggtgaaa 6120

tccatggttt tgggacgttt cctgagtcag attcgcgaaa aactgattca gagaatttac 6180

cgcgggatcg agccgacttt gccaaactgg ttcgcggtca caaagaccag aaatggcgcc 6240

ggaggcggga acaaggtggt ggatgagtgc tacatcccca attacttgct ccccaaaacc 6300

cagcctgagc tccagtgggc gtggactaat atggaacagt atttaagcgc ctgtttgaat 6360

ctcacggagc gtaaacggtt ggtggcgcag catctgacgc acgtgtcgca gacgcaggag 6420

cagaacaaag agaatcagaa tcccaattct gatgcgccgg tgatcagatc aaaaacttca 6480

gccaggtaca tggagctggt cgggtggctc gtggacaagg ggattacctc ggagaagcag 6540

tggatccagg aggaccaggc ctcatacatc tccttcaatg cggcctccaa ctcgcggtcc 6600

caaatcaagg ctgccttgga caatgcggga aagattatga gcctgactaa aaccgccccc 6660

gactacctgg tgggccagca gcccgtggag gacatttcca gcaatcggat ttataaaatt 6720

ttggaactaa acgggtacga tccccaatat gcggcttccg tctttctggg atgggccacg 6780

aaaaagttcg gcaagaggaa caccatctgg ctgtttgggc ctgcaactac cgggaagacc 6840

aacatcgcgg aggccatagc ccacactgtg cccttctacg ggtgcgtaaa ctggaccaat 6900

gagaactttc ccttcaacga ctgtgtcgac aagatggtga tctggtggga ggaggggaag 6960

atgaccgcca aggtcgtgga gtcggccaaa gccattctcg gaggaagcaa ggtgcgcgtg 7020

gaccagaaat gcaagtcctc ggcccagata gacccgactc ccgtgatcgt cacctccaac 7080

accaacatgt gcgccgtgat tgacgggaac tcaacgacct tcgaacacca gcagccgttg 7140

caagaccgga tgttcaaatt tgaactcacc cgccgtctgg atcatgactt tgggaaggtc 7200

accaagcagg aagtcaaaga ctttttccgg tgggcaaagg atcacgtggt tgaggtggag 7260

catgaattct acgtcaaaaa gggtggagcc aagaaaagac ccgcccccag tgacgcagat 7320

ataagtgagc ccaaacgggt gcgcgagtca gttgcgcagc catcgacgtc agacgcggaa 7380

gcttcgatca actacgcaga caggtaccaa aacaaatgtt ctcgtcacgt gggcatgaat 7440

ctgatgctgtttccctgcag acaatgcgag agaatgaatc agaattcaaa tatctgcttc 7500

actcacggac agaaagactg tttagagtgc tttcccgtgt cagaatctca acccgtttct 7560

gtcgtcaaaa aggcgtatca gaaactgtgc tacattcatc atatcatggg aaaggtgcca 7620

gacgcttgca ctgcctgcga tctggtcaat gtggatttgg atgactgcat ctttgaacaa 7680

taaatgattt aaatcaggta tggctgccga tggttatctt ccagatggc tcgaggacac 7740

tctctctgat ctagagcctg cagtctcgac aagcttgtcg agaagtacta gaggatcata 7800

atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 7860

ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagctttat 7920

aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatttttttcactg 7980

cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatctgatca 8040

ctgcttgagc ctaggagatc cgaaccagat aagtgaaatc tagttccaaa ctattttgtc 8100

atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat tttgtcactc 8160

ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa ctattttgtc 8220

cgcccacagc ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc tgccaactcc 8280

atgtgacaaa ccgtcatctt cggctacttt 8310

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P1

<400> 15

gatccggtac cacgcgtcta g 21

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> P2

<400> 16

ctcgacgtcg actttacttg tacagc 26

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P3

<400> 17

gcggggtttt acgagattgt g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> P4

<400> 18

ggggtgcctg ctcaatcaga 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> P5

<400> 19

gcagcacaca ctgacatcca 20

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P6

<400> 20

gatcaccggc gcatcagaat tg 22

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> P7

<400> 21

acttcaagat ccgccacaac at 22

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> P8

<400> 22

tctcgttggg gtcttgctca g 21

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> M13 F

<400> 23

cccagtcacg acgttgtaaa acg 23

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> M13 R

<400> 24

agcggataac aatttcacac agg 23

Claims (41)

1. An isolated nucleic acid molecule, which comprises, in sequence, a first polyA, a nucleotide sequence encoding a Rep78 protein, a first promoter, a second promoter, a nucleotide sequence encoding a Rep52 protein, and a second polyA, wherein the first promoter is a transcriptional promoter of the nucleotide sequence encoding the Rep78 protein and the first polyA, and the second promoter is a transcriptional promoter of the nucleotide sequence encoding the Rep52 protein and the second polyA, wherein the sequence of the nucleotide sequence encoding the Rep52 protein and/or the nucleotide sequence encoding the Rep78 protein is codon-optimized to avoid homologous recombination, the first promoter is a p10 promoter, and the second promoter is a polh promoter, the nucleotide sequence encoding the Rep78 protein is as shown in SEQ ID NO: 11, and the nucleotide sequence encoding the Rep52 protein is as shown in SEQ ID NO: 12. 2. The isolated nucleic acid molecule according to claim 1, wherein the p10 promoter comprises the nucleotide sequence shown in SEQ ID NO: 9. 3. The isolated nucleic acid molecule according to claim 1, wherein the polh promoter comprises the nucleotide sequence shown in SEQ ID NO: 10. 4. An isolated nucleic acid molecule according to claim 1, wherein the 5' end of the first promoter is directly or indirectly linked to the 5' end of the second promoter. 5. An isolated nucleic acid molecule according to claim 1, wherein the 3' end of the first promoter is directly or indirectly connected to the 5' end of the nucleotide sequence encoding Rep78. 6. An isolated nucleic acid molecule according to claim 1, wherein the 3' end of the nucleotide sequence encoding the Rep78 protein is directly or indirectly connected to the 5' end of the first polyA. 7. An isolated nucleic acid molecule according to claim 1, wherein the 3' end of the second promoter is directly or indirectly connected to the 5' end of the nucleotide sequence encoding Rep52. 8. An isolated nucleic acid molecule according to claim 1, wherein the 3' end of the nucleotide sequence encoding the Rep52 protein is directly or indirectly connected to the 5' end encoding the second polyA. 9. The isolated nucleic acid molecule according to claim 1, wherein the polyA is selected from any one of SV40 polyA and HSV TK polyA. 10. The isolated nucleic acid molecule according to claim 1, comprising the nucleotide sequence shown in SEQ ID NO: 8. 11. A vector comprising the isolated nucleic acid molecule of any one of claims 1-10. The vector according to claim 11 , which is a viral vector. The vector according to claim 11 , which is a baculovirus vector. The vector according to claim 11 , which is a pFastBac vector. 15. The vector according to any one of claims 11-14, comprising the nucleotide sequence shown in SEQ ID NO: 14. 16. A cell comprising the isolated nucleic acid molecule of any one of claims 1-10 or the vector of any one of claims 11-15, wherein the cell is of a non-plant or animal species. 17. The cell according to claim 16, which is an insect cell. 18. The cell of claim 16, which is a Spodoptera frugiperda cell. 19. A baculovirus expression system, comprising a first baculovirus vector and a second baculovirus vector comprising a nucleic acid sequence encoding a target gene, wherein the first baculovirus vector is the baculovirus vector according to any one of claims 13 to 14. 20. The baculovirus expression system according to claim 19, wherein from the 5' end to the 3' end, the nucleic acid sequence encoding the target gene comprises, in sequence, a first parvovirus inverted terminal repeat (ITR), the target gene, and a second ITR. 21 . The baculovirus expression system according to claim 20 , wherein at least one promoter is further included between the first ITR and the target gene. 22. The baculovirus expression system according to claim 20, wherein at least one eukaryotic promoter is further included between the first ITR and the target gene. 23. The baculovirus expression system according to claim 20, wherein at least one mammalian cell promoter is further included between the first ITR and the target gene. 24. The baculovirus expression system according to claim 20, wherein a mammalian cell promoter and an insect cell promoter are further included between the first ITR and the target gene. 25. The baculovirus expression system according to claim 24, wherein the mammalian cell promoter is selected from a ubiquitous promoter and a tissue-specific promoter. 26. The baculovirus expression system according to claim 25, wherein the ubiquitous promoter is CMV, SV40, EF1a, CAG or UBC promoter. 27. The baculovirus expression system according to claim 25, wherein the tissue-specific promoter is ALB, hAAT, TBG, TTR, GFAP, MHCK7 or hSyn promoter. 28. The baculovirus expression system of claim 24, wherein the insect cell promoter is the p10 promoter. 29. The baculovirus expression system according to claim 24, wherein the mammalian promoter and the insect cell promoter are CMV and p10 promoters. 30. An insect cell comprising the baculovirus expression system of any one of claims 19-29. 31. The insect cell according to claim 30, further comprising a third nucleotide sequence, wherein the third nucleotide sequence contains two parvovirus ITR nucleotide sequences and at least one nucleotide sequence encoding a target gene, and wherein the at least one nucleotide sequence encoding a target gene is located between the two parvovirus ITR nucleotide sequences. 32. The insect cell of claim 31, wherein the parvovirus is an adeno-associated virus. 33. The insect cell according to claim 31, wherein the third nucleotide sequence is part of another nucleic acid construct, wherein each nucleotide sequence encoding a gene of interest is operably linked to an expression control sequence for mammalian expression. 34. The insect cell according to claim 33, wherein the nucleic acid construct is an insect cell compatible vector. 35. The insect cell of claim 33, wherein the nucleic acid construct is a baculovirus vector. 36. Use of the baculovirus expression system according to any one of claims 19 to 29 or the insect cell according to any one of claims 30 to 35 in preparing a target nucleic acid molecule. 37. The use according to claim 36, wherein the target nucleic acid molecule is a linear DNA molecule (neDNA) with covalently blocked ends. 38. A method for preparing a target nucleic acid molecule, comprising culturing the insect cell according to any one of claims 30-35. 39. The preparation method according to claim 38, comprising: a) providing the baculovirus expression system according to any one of claims 19 to 29; b) inserting the target gene sequence into the second baculovirus vector; c) co-transfecting the first baculovirus vector and the second baculovirus vector into insect cells; d) growing the insect cells under conditions that allow replication and release of the DNA comprising the gene of interest; e) Collecting target nucleic acid molecules. 40. The preparation method according to claim 39, further comprising protecting and isolating the target nucleic acid molecule. 41. A kit comprising the isolated nucleic acid molecule of any one of claims 1-10, the baculovirus expression system of any one of claims 19-29, or the insect cell of any one of claims 30-35.