WO2023221240A1

WO2023221240A1 - Composition and method for generating recombinant baculovirus in insect cell, and use

Info

Publication number: WO2023221240A1
Application number: PCT/CN2022/101538
Authority: WO
Inventors: 肖何; 何晓斌; 黄刚; 胡颖
Original assignee: 睿征医药科技(武汉)有限公司
Priority date: 2022-05-16
Filing date: 2022-06-27
Publication date: 2023-11-23
Also published as: CN114736929A; CN114736929B

Abstract

Provided are a composition and a method for generating a recombinant baculovirus in an insect cell, and use. The composition comprises a packaging-deficient baculovirus plasmid and a first rescue recombinant DNA. The packaging-deficient baculovirus plasmid lacks a CNE sequence and/or an NAE sequence in a baculovirus genome; the first rescue recombinant DNA comprises a first insertion sequence and first homologous arms located on both sides of the first insertion sequence; the first insertion sequence comprises a first functional fragment and a first complement sequence; the first complement sequence is at least one of the sequences deleted from the packaging-deficient baculovirus plasmid; and the composition can undergo homologous recombination in the insect cell to produce a recombinant baculovirus. The recombinant baculovirus can be obtained by one-step recombination of the composition in a host insect cell without screening and without the restriction of a single recombination site, so that the purity of the prepared recombinant baculovirus is relatively high.

Description

A composition, method and application for producing recombinant baculovirus in insect cells

【Technical field】

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

【Background technique】

Baculovirus is a type of enveloped circular double-stranded DNA virus. The most studied baculovirus strain at present is Autographa californica multinuclear polyhedrosis virus (MNPV), referred to as AcMNPV. Baculovirus expression system (BEVS) is a eukaryotic expression system that uses insect baculovirus vectors to infect host insect cells to produce foreign proteins.

Kitts et al. proposed the BacPAK baculovirus expression system, which inserts the lacZ gene into the polyhedrosis virus locus and constructs a Bsu36I enzyme-containing Bsu36I enzyme cleavage site by introducing a Bsu36I restriction site into the orf1629 and orf603 gene loci. Replication-deficient viruses that are linearized after cleavage. Due to the deletion of the essential gene orf1629, the linearized viruses cannot produce viruses with infectious activity even if they are self-ligated. The recombinant DNA transfer vector and the linearized virus will be co-transfected into the host. In insect cells, recombinant baculovirus is obtained after homologous recombination. However, since the Bsu36I enzyme cannot cleave 100% of the viral DNA to linearize it, the recombinant virus still needs to undergo plaque screening, which is time-consuming and labor-intensive.

Subsequently, Lee et al. proposed a Bac-to-Bac baculovirus expression system, which introduced a Bac artificial chromosome element into the polyhedron gene locus of AcMNPV. This element contained a mini-F replicon and a Tn7 transposon site, making Baculovirus DNA can replicate in bacteria, and foreign genes can be inserted into the viral genome through Tn7 transposition to obtain recombinant baculovirus. Although this system does not require plaque screening, it does require transposition recombination screening in bacteria, which is also time-consuming and labor-intensive; and the recombinant baculovirus obtained through this system is unstable during continuous passage.

In 2003, Zhao et al. proposed the flash-BAC system. This system introduced the mini-F replicon into the AcMNPV genome and at the same time inactivated the essential gene orf1629. The baculovirus in which the essential gene orf1629 was inactivated was unable to produce infection. Active virus unless homologous recombination occurs with the rescue recombinant DNA transfer vector. This system combines the advantages of BacPAK and Bac-to-Bac and requires only one step of recombination in host insect cells to obtain recombinant baculovirus without screening. However, since orf1629 is a trans-element protein related to replication, viral DNA that has undergone homologous recombination will provide the function of orf1629, and since viral DNA that has not undergone homologous recombination will also be packaged, resulting in the final Recombinant baculovirus is impure.

[Content of the invention]

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a composition, method and application for producing recombinant baculovirus in insect cells, aiming to solve the problem of using the flash-BAC system to produce recombinant baculovirus. Viral DNA that has not undergone homologous recombination will also be packaged, and viral DNA that has undergone homologous recombination will provide the function of orf1629, leading to the problem of impurity of the recombinant baculovirus.

In order to achieve the above object, the present invention provides a composition for producing recombinant baculovirus in insect cells, comprising a packaging-defective baculovirus plasmid and a first rescue recombinant DNA, the packaging-defective baculovirus plasmid is deleted CNE sequence and/or NAE sequence in the baculovirus genome, the first rescue recombinant DNA includes a first insertion sequence and a first homology arm located on both sides of the first insertion sequence, the first insertion sequence includes A first functional fragment and a first complement sequence, the first complement sequence is at least one of the sequences deleted from the packaging-deficient baculovirus plasmid, and the composition is capable of homologous recombination in insect cells. to produce recombinant baculovirus.

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

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

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

Preferably, the first complementing sequence is located between the cap gene expression cassette and the rep gene expression cassette, and its two ends are respectively close to the starting end of the cap gene expression cassette and the rep gene expression cassette. Starting end.

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

Preferably, the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.

Preferably, the first functional fragment is an AAV ITR core expression element carrying an exogenous gene.

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

Preferably, the first functional fragment is a reporter gene.

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

Preferably, the composition further comprises a second rescue recombinant DNA, the packaging-deficient baculovirus plasmid deletes the CNE sequence and the NAE sequence in the baculovirus genome, the second rescue recombinant DNA comprises a second insertion sequence and The second homology arms located on both sides of the second insertion sequence, the second insertion sequence includes a second complementation sequence, the first complementation sequence is a CNE sequence or an NAE sequence, and the second complementation sequence It is the sequence different from the first complement sequence in the CNE sequence and the NAE sequence.

Preferably, the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV, and the second insertion sequence also includes a second functional fragment, and the second functional fragment is a gene containing an exogenous gene. AAV ITR core expression element.

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

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

According to another aspect of the present invention, a method for growing or producing recombinant baculovirus in vitro is provided, comprising co-transfecting insect cells with any of the above compositions and culturing the insect cells.

According to another aspect of the present invention, a method for growing or producing recombinant adeno-associated viruses in vitro is provided, comprising co-transfecting insect cells with any of the above compositions and culturing the insect cells.

Generally speaking, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:

The present invention improves on the basis of the flash-BAC system to construct a packaging-deficient baculovirus plasmid, which lacks the CNE sequence and/or NAE sequence in the baculovirus genome and cannot package the baculovirus normally unless rescued. Homologous recombination occurs in recombinant DNA. This system only requires one step of recombination in host insect cells to obtain recombinant baculovirus without screening; at the same time, the insertion sequence can be inserted into any locus of the baculovirus genome, and there is no restriction on the insertion site, and it can be inserted into the baculovirus genome as needed. target site, and can express multiple foreign proteins from a single baculovirus with greater flexibility. In addition, when using this system to prepare recombinant baculovirus, only viral DNA that has undergone homologous recombination will be packaged into recombinant baculovirus, while viral DNA that has not undergone homologous recombination will not be packaged, so the final recombinant baculovirus obtained The virus is purer.

[Picture description]

Figure 1 is a schematic diagram of the first homologous recombination expression box targeting CNE sequence constructed in Example 1 of the present invention.

Figure 2 is a schematic diagram of the second homologous recombination expression box targeting the NAE sequence constructed in Example 1 of the present invention.

Figure 3 is a schematic diagram of the third homologous recombination expression box targeting NAE sequence constructed in Example 1 of the present invention.

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

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

Figure 6 is a schematic diagram of the recombinant DNA fragment Ac96-Rep-CNE-Cap containing AAV Cap and Rep expression cassette constructed in Example 2 of the present invention.

Figure 7 is a schematic diagram of the recombinant DNA fragment Ac96-Rep-NAE-Cap containing AAV Cap and Rep expression cassette constructed in Example 2 of the present invention.

Figure 8 is a green fluorescent plaque diagram produced after co-transfection of insect host cells using defective baculovirus vector ΔCNE-Bac and recombinant DNA fragment Ac96-CNE-GFP in Example 3 of the present invention.

Figure 9 shows the effect of expressing green fluorescence after the recombinant baculovirus infects the cells after co-transfecting insect host cells with the defective baculovirus vector ΔCNE-Bac and the recombinant DNA fragment Ac96-CNE-GFP in Example 3 of the present invention. picture.

Figure 10 is a red fluorescent plaque diagram produced after co-transfection of insect host cells with defective baculovirus vector ΔNAE-Bac and recombinant DNA fragment Ac96-NAE-mcherry in Example 3 of the present invention.

Figure 11 shows the effect of the recombinant baculovirus expressing red fluorescence after infecting the cells after co-transfecting insect host cells with the defective baculovirus vector ΔNAE-Bac and the recombinant DNA fragment Ac96-NAE-mcherry in Example 3 of the present invention. picture.

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

Figure 13 shows the results produced by co-transfecting insect host cells with the defective baculovirus vector ΔCNE-Bac and the recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP in Example 4 of the present invention. Green fluorescent plaque diagram. Figure 14 shows the results obtained after co-transfecting insect host cells with the defective baculovirus vector ΔCNE-Bac and the recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP in Example 4 of the present invention. The effect of expressing green fluorescence after the recombinant baculovirus infects cells.

Figure 15 shows the results produced by co-transfecting insect host cells with the defective baculovirus vector ΔNAE-Bac and the recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry in Example 4 of the present invention. Red fluorescent plaque diagram.

Figure 16 shows Example 4 of the present invention using defective baculovirus vector ΔNAE-Bac to co-transfect insect host cells with recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry. The effect of expressing red fluorescence after the recombinant baculovirus infects cells.

Figure 17 is a Western Blot detection of expression of VP capsid protein and Rep protein after co-transfection of insect host cells using defective baculovirus vector ΔCNE-Bac and recombinant DNA fragment Ac96-Rep-CNE-Cap in Example 5 of the present invention. picture.

Figure 18 is a Western Blot detection of expression of VP capsid protein and Rep protein after co-transfection of insect host cells using defective baculovirus vector △NAE-Bac and recombinant DNA fragment Ac96-Rep-NAE-Cap in Example 5 of the present invention. picture.

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

Figure 20 is a silver staining detection picture of the purified recombinant AAV virus particles subjected to SDS-PAGE after the three recombinant baculoviruses were transfected into host cells in Example 9 of the present invention, all showing the three capsid proteins VP1/VP2/VP3. .

【Detailed ways】

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

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

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited. In the description of the present invention, "several" means at least one, such as one, two, etc., unless otherwise expressly and specifically limited.

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

Cis-acting elements refer to specific DNA sequences connected in series with structural genes and are binding sites for transcription factors. They regulate the precise initiation and transcription efficiency of gene transcription by binding to transcription factors. Cis-acting elements include promoters, enhancers, regulatory sequences and inducible elements. Their role is to participate in the regulation of gene expression. They do not encode any protein themselves, but only provide an action site.

AAV is a single-stranded DNA virus with a simple genome structure and a full length of about 4.7kb. Its genome contains a rep gene expression cassette, a cap gene expression cassette and AAV inverted terminal repeats (ITR) located at both ends of the genome. These are the three elements necessary for packaging the AAV virus. The Cap gene encodes a structural VP capsid protein, which contains three overlapping open reading frames, encoding three types of subunits: VP1, VP2, and VP3 respectively. The Rep gene encodes four overlapping multifunctional proteins, Rep78, Rep68, Rep52 and Rep40, which are involved in AAV replication and integration. ITR is a 125-nucleotide palindromic structure at both ends of the genome, which can form a self-complementary inverted T-shaped hairpin structure. It is a cis-acting element required for the initiation of DNA replication and packaging of recombinant AAV genome into infectious virus particles. . As a defective virus, AAV cannot replicate independently in the absence of helper viruses. Therefore, AAV can only be integrated into the host cell chromosome at a specific site and remain in a latent state. In the presence of the helper virus, the increased expression of the rep gene can rescue the AAV genome integrated in the host cell chromosome, and a large amount of AAV DNA is obtained. The single-stranded rAAV genome is packaged into a complex structure under the action of the VP capsid protein. Infectious virus particles.

The conserved non-protein-coding element (CNE) was found to be present in all sequenced genomes of the genus Alphabaculovirus, with highly homologous sequences ranging from 154 to 157 bp. There are reports that speculate that an at-rich CNE sequence located in the ac152 region of the Autographa californica multiple nucleopolyhedro-virus (AcMNPV) genome is a cis-acting element necessary for virion production.

The NAE sequence was first discovered as an essential element for nucleocapsid assembly in the genus Alphabaculovirus, and it plays an essential role in the nucleocapsid assembly process. The natural NAE sequence is located in the ac83 gene and its homologous genes in the genus Alphabaculovirus, and is located at the near end (CN106566829A). ac83 is a core gene related to baculovirus nucleocapsid assembly. It has a full length of 2544bp, encoding 847 amino acids, and a predicted molecular weight of 96.2kDa. Knocking out ac83 does not affect the replication of the viral genome, but completely blocks the assembly of the viral nucleocapsid. A large number of hollow capsid precursors can be observed in the nucleus under an electron microscope.

The invention provides a composition for producing recombinant baculovirus in insect cells, which contains a packaging-deficient baculovirus plasmid and a first rescue recombinant DNA, and the packaging-deficient baculovirus plasmid lacks the baculovirus genome. The CNE sequence and/or NAE sequence in and a first complementing sequence, the first complementing sequence being at least one of the sequences deleted from the packaging-deficient baculovirus plasmid, and the composition is capable of homologous recombination in insect cells to produce recombinant rods virus.

It should be noted that the packaging-deficient baculovirus plasmid involved in the present invention lacks the CNE sequence and/or the NAE sequence in the baculovirus genome, resulting in the inability to normally package the recombinant baculovirus. The CNE sequence can be exactly the same as the wild-type AcMNPV CNE sequence, or it can be a CNE sequence from other baculoviruses, or it can share at least 50% sequence identity or at least 60% sequence identity with the wild-type AcMNPV CNE sequence. Artificial CNE sequences that are homogeneous, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or higher sequence identity. Likewise, the NAE sequence can be an identical NAE sequence to the wild-type AcMNPV NAE sequence, or it can be an NAE sequence from other baculoviruses, or it can share at least 50% sequence identity, at least 60% sequence identity, and at least 60% sequence identity with the wild-type AcMNPV NAE sequence. % sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or an artificial NAE sequence of higher sequence identity.

In addition, when the packaging-defective baculovirus plasmid lacks the CNE sequence in the baculovirus genome, correspondingly, the first complement sequence is the CNE sequence; when the packaging-defective baculovirus plasmid lacks the NAE sequence in the baculovirus genome When , correspondingly, the first complement sequence is the NAE sequence; when the packaging-defective baculovirus plasmid simultaneously lacks the CNE sequence and the NAE sequence in the baculovirus genome, correspondingly, the first complement sequence contains both the CNE sequence and the NAE sequence. NAE sequence. In this way, the recombinant bacmid after homologous recombination in insect cells contains normal CNE and NAE sequences, and can then package the recombinant baculovirus; but when homologous recombination does not occur, the CNE or NAE sequence is missing on the bacmid. This makes it impossible to package the recombinant baculovirus normally. In another embodiment, the second rescue recombinant DNA and the first rescue recombinant DNA can also be used to jointly compensate for the missing sequence, that is, when the packaging-deficient baculovirus plasmid simultaneously deletes the CNE sequence and the NAE sequence in the baculovirus genome, The second rescue recombinant DNA includes a second insertion sequence and a second homology arm located on both sides of the second insertion sequence. The second insertion sequence includes a second complement sequence. The first complement sequence is a CNE sequence or an NAE sequence. The second complement sequence is a CNE sequence or a NAE sequence. The complementing sequence is the sequence of the CNE sequence and the NAE sequence that is different from the first complementing sequence. It is worth noting that the first homology arm and the second homology arm here are homologous sequences corresponding to different loci on the baculovirus genome, so that the first insertion sequence and the second insertion sequence can be inserted into the baculovirus genome. in different loci.

It should also be noted that the rescue recombinant DNA involved in the present invention can be either a linear DNA fragment or a baculovirus transfer vector, which is not limited here. In addition, the rescue recombinant DNA provided by the present invention has no restriction on the insertion site. When homologous recombination occurs between the rescue recombinant DNA and the packaging-defective baculovirus plasmid, the insertion sequence on the rescue recombinant DNA can be inserted into the baculovirus genome. Any locus, such as, but not limited to, Ac18, Ac83, Ac96, Ac126, Ac127, Ac130, Ac152.

The first functional fragment in the present invention can be the cap gene expression cassette of AAV and the rep gene expression cassette of AAV, or it can be the AAV ITR core expression element (i.e. ITR-GOI) with exogenous genes, which is used to prepare recombinant adenocarcinoma. Related viruses. The foreign gene can be at least one nucleotide sequence encoding a gene of interest (Gene of Interest, GOI) product. The gene product of interest can be a therapeutic gene product, specifically a polypeptide, an RNA molecule ( siRNA) or other gene products, such as but not limited to lipoprotein esterase, apolipoprotein, cytokines, interleukins or interferons; it can also be a reporter protein that evaluates vector transformation and expression, such as but not limited to fluorescent protein (green Fluorescent protein (GFP, red fluorescent protein (RFP)), chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, Renilla luciferase, firefly luciferase or alkaline phosphatase. In addition, the first functional fragment can also be a reporter gene, such as but not limited to expressing fluorescent proteins (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyltransferase, β-galactosidase, β-glucuronide The gene sequence of the enzyme, Renilla luciferase, firefly luciferase or alkaline phosphatase, is used to evaluate and verify whether homologous recombination has occurred to produce recombinant baculovirus. The first functional fragment can also be any nucleotide sequence encoding a therapeutic gene product, such as but not limited to encoding a drug polypeptide (such as interleukin, etc.) or a recombinant viral subunit protein.

It should be noted that in the embodiments of the present invention, the specific positions and directions of the cap gene expression cassette and rep gene expression cassette are not limited. The arrangement of the three parts of the cap gene expression cassette, rep gene expression cassette and the first complement sequence can be as follows: Six types: 1. Arranged from the 5' end to the 3' end are the cap gene expression cassette, rep gene expression cassette and the first complement sequence; 2. Arranged from the 5' end to the 3' end are the rep gene expression cassette, cap gene expression cassette and the first complement sequence; 3. Arranged from the 5' end to the 3' end are the cap gene expression cassette, the first complement sequence and the rep gene expression cassette; 4. From the 5' end to the 3' end Arranged in order are the rep gene expression cassette, the first complementation sequence and the cap gene expression cassette; 5. From the 5' end to the 3' end, the first complementation sequence, the cap gene expression cassette and the rep gene expression cassette are arranged in order; 6. Arranged from the 5' end to the 3' end are the first complement sequence, rep gene expression cassette and cap gene expression cassette. The cap gene expression cassette and the rep gene expression cassette can be in the same direction or in opposite directions. The sequence of the cap gene expression cassette in the recombinant expression cassette can be from the 5' end to the 3' end, or from the 3' end to the 5' end. Similarly, the sequence of the rep gene expression cassette in the recombinant expression cassette can be from the 5' end to the 3' end, or from the 3' end to the 5' end.

As a preferred embodiment, the first complementation sequence is located between the cap gene expression cassette and the rep gene expression cassette. Specifically, the first insertion sequence includes the cap gene expression cassette, CNE sequence and rep in order from 5' to 3'. The gene expression cassette, or the first insertion sequence contains the cap gene expression cassette, the NAE sequence and the rep gene expression cassette in the order from 5' to 3', or the first insertion sequence contains the rep gene expression in the order from 5' to 3' cassette, CNE sequence and cap gene expression cassette, or comprise rep gene expression cassette, NAE sequence and cap gene expression cassette in order from 5' to 3'. Further preferably, the two ends of the first complementation sequence are respectively close to the starting end of the cap gene expression cassette and the starting end of the rep gene expression cassette, that is, the cap gene expression cassette and the rep gene expression cassette are in opposite directions, and the starting ends of the two are respectively. Set oppositely and towards the first backfill sequence.

An insect cell provided by the invention includes any of the above compositions.

The invention provides a method for growing or producing recombinant baculovirus in vitro, which method includes co-transfecting insect cells with any of the above compositions and cultivating the insect cells. The recombinant baculovirus can then be recovered.

The present invention also provides a method for growing or producing recombinant adeno-associated viruses in vitro, which method includes co-transfecting insect cells with the above composition, and cultivating the insect cells to produce recombinant adeno-associated viruses. It should be emphasized that the composition required to prepare the recombinant adeno-associated virus needs to contain the AAV cap gene, rep gene and ITR core expression element necessary for the production of rAAV. Therefore, if the first rescue recombinant DNA in the composition contains AAV's cap gene, rep gene and ITR core expression element, cap gene expression cassette and AAV rep gene expression cassette, you also need to insert the AAV ITR core expression element with foreign genes into the packaging-deficient baculovirus plasmid. This can be done through Tn7 transfer mediated by baculovirus transfer vector. Seat is obtained. If the first rescue recombinant DNA in the composition contains the AAV ITR core expression element with foreign genes, it is also necessary to insert the AAV cap gene expression cassette and the AAV rep gene expression cassette into the packaging-defective baculovirus plasmid. Can be obtained by baculovirus transfer vector-mediated Tn7 transposition. In addition, the purpose of obtaining rAAV can also be achieved through two rescue recombinant DNAs. The packaging-deficient baculovirus plasmid simultaneously deletes the CNE sequence and NAE sequence in the baculovirus genome. For example, the first rescue recombinant DNA contains the expression of the cap gene of AAV. cassette, AAV rep gene expression cassette and CNE sequence, and the second rescue recombinant DNA contains the AAV ITR core expression element and NAE sequence with foreign genes.

The above technical solution will be described in detail below with reference to specific embodiments.

Example 1 Construction of bacmids lacking CNE sequence △CNE-Bac, bacmids lacking NAE sequences △NAE-Bac, and bacmids △CNE-△NAE-Bac lacking both CNE and NAE sequences

Red recombination is a highly efficient recombination method at the bacterial level that can be used to rapidly transform recombinant baculovirus genomes in Escherichia coli (DH10Bac). Red recombination uses lambda phage Red recombinase (composed of three proteins: Exo, Beta and Gam) to perform homologous recombination between linear DNA fragments carrying homology arms introduced into cells and specific target sequences of the genome, thereby achieving the target gene. Replacement (Doublet et al., 2008, J Microbiol Methods, 75(2): 359-361).

First, the first homologous recombination expression cassette (SEQ ID No. 1) targeting the CNE sequence is constructed. As shown in Figure 1, the expression cassette includes the CNE upstream homologous sequence and chloramphenicol (Chol) from 5' to 3'. Resistance gene expression cassette and CNE downstream homologous sequence; then Red recombination technology was used to replace the expression cassette with the CNE sequence on the bacmid, thereby obtaining a bacmid △CNE-Bac lacking the CNE sequence.

In the same way, first construct the second homologous recombination expression cassette (SEQ ID No. 2) targeting the NAE sequence, as shown in Figure 2. The expression cassette includes the NAE upstream homologous sequence, chloramphenicol, and (Chol) resistance gene expression cassette and NAE downstream homologous sequence; then Red recombination technology was used to replace the expression cassette with the NAE sequence on the bacmid, thereby obtaining a bacmid △NAE-Bac lacking the NAE sequence.

Similarly, the third homologous recombination expression cassette (SEQ ID No. 3) targeting the NAE sequence is first constructed, as shown in Figure 3. The expression cassette includes the NAE upstream homologous sequence, Gentamella sp. Gene (GM) resistance gene expression cassette and NAE downstream homologous sequence; then Red recombination technology was used to replace the expression cassette with the NAE sequence on the bacmid △CNE-Bac, thus obtaining a gene lacking both CNE and NAE sequences. Bacmid ΔCNE-ΔNAE-Bac.

Example 2 Construction of recombinant DNA transfer vector containing CNE or NAE sequences

In various embodiments of the present invention, green fluorescent protein (GFP) or red fluorescent protein (mcherry) is used as an exogenous gene to be inserted into the AcMNPV genome. Construct the first recombinant DNA fragment with the foreign gene insertion site at the AcMNPV orf96 locus: See Figure 4. The recombinant DNA fragment includes the orf96 upstream homologous sequence and the CNE sequence (SEQ ID No. 4) from 5' to 3'. , GFP expression cassette and orf96 downstream homologous sequence, the above sequences were connected through artificial direct synthesis or overlap extension PCR amplification to obtain the construct Ac96-CNE-GFP respectively. Similarly, the construct Ac96-NAE-mcherry containing the NAE sequence (SEQ ID No. 5) was constructed (Figure 5). The nucleotide sequences of construct Ac96-CNE-GFP and construct Ac96-NAE-mcherry are shown in SEQ ID No. 6 and SEQ ID No. 7 respectively.

Construct the second recombinant DNA fragment with the insertion site of the AAV rep and cap gene expression cassette at the AcMNPV orf96 locus: See Figure 6. This recombinant DNA fragment includes the orf96 upstream homologous sequence and the Rep gene expression cassette from 5' to 3'. (SEQ ID No.8), CNE sequence, Cap gene expression cassette (SEQ ID No.9) and orf96 downstream homologous sequence, the above sequences are connected through artificial direct synthesis or overlap extension PCR amplification to obtain the construct Ac96- Rep-CNE-Cap. Similarly, the construct Ac96-Rep-NAE-Cap containing the NAE sequence was constructed (Fig. 7). The nucleotide sequences of construct Ac96-Rep-CNE-Cap and construct Ac96-Rep-NAE-Cap are shown in SEQ ID No. 10 and SEQ ID No. 11 respectively.

Example 3 Verification of homologous recombination to produce recombinant baculovirus after co-transfection of packaging-deficient baculovirus vector and recombinant DNA fragment into insect host cells

In this example, the packaging-deficient baculovirus vectors △CNE-Bac and △NAE-Bac constructed in Example 1 were co-constructed with the corresponding recombinant DNA fragments Ac96-CNE-GFP and Ac96-NAE-mcherry constructed in Example 2, respectively. Transfect Sf9 insect host cells, and 96 hours after co-transfection, use a fluorescence microscope to observe whether the host cells can produce green fluorescent plaques or red fluorescent plaques to determine the production of recombinant baculovirus. In order to further confirm that co-transfection indeed produced recombinant baculovirus, 120 h after co-transfection, the Sf9 cell culture supernatant was collected and transferred to the host cells cultured in suspension. After 72 h of infection, green fluorescence or red fluorescence was observed with a fluorescence microscope. Luminous condition.

The observation results are shown in Figures 8 to 11: baculovirus vector △CNE-Bac and recombinant DNA fragment Ac96-CNE-GFP were co-transfected into Sf9 cells for 96 hours. Green fluorescent plaques were produced (Figure 8). After 120 hours, the Sf9 cell culture supernatant was collected and transferred to the host cells cultured in suspension. A large amount of green fluorescence was produced after 72 hours of infection (Figure 9); the baculovirus vector △NAE-Bac and the recombinant DNA fragment Ac96-NAE-mcherry were co-infected. Red fluorescent plaques were produced 96 hours after transfection of Sf9 cells (Figure 10). After 120 hours of co-transfection, the Sf9 cell culture supernatant was collected and transfected with suspension-cultured host cells. A large amount of red fluorescence was produced 72 hours after infection (Figure 10). 11). This example shows that after the packaging-deficient baculovirus vector ΔCNE-Bac or ΔNAE-Bac and the recombinant DNA fragment are co-transfected into insect host cells, homologous recombination can occur to produce recombinant baculovirus, and protein expression of foreign genes can be performed. .

Example 4 Foreign genes can be inserted into any locus of the AcMNPV genome. There is no restriction on the insertion site and can be inserted into the target site as needed.

First, refer to Example 2 to construct recombinant DNA fragments with insertion sites at orf83, orf126 and orf152 respectively, as shown in Figures 12A-F: Among them, the constructs Ac83-CNE-GFP (Figure 12A) and Ac126-CNE-GFP (Figure 12B ) and Ac152-CNE-GFP ((Figure 12C) are recombinant DNA fragments with CNE complement sequence insertion sites at orf83, orf126 and orf152 respectively; the constructs Ac83-NAE-mcherry (Figure 12D), Ac126-NAE -mcherry (Figure 12E) and Ac152-NAE-mcherry (Figure 12F) are recombinant DNA fragments with NAE complement sequence insertion sites at orf83, orf126 and orf152 respectively;

In this example, the packaging-deficient baculovirus vectors ΔCNE-Bac and ΔNAE-Bac in Example 1 were co-transfected into Sf9 insect host cells with the recombinant DNA fragment in this example. After co-transfection for 96 hours, using Use a fluorescence microscope to observe whether the host cells can produce green fluorescent plaques or red fluorescent plaques to determine the production of recombinant baculovirus. In order to further confirm that co-transfection indeed produced recombinant baculovirus, 120 h after co-transfection, the Sf9 cell culture supernatant was collected and transferred to the host cells cultured in suspension. After 72 h of infection, green fluorescence or red fluorescence was observed with a fluorescence microscope. Luminous condition.

The observation results are shown in Figure 13 to Figure 16: The baculovirus vector △CNE-Bac was co-transfected with the recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP for 96 hours in Sf9 cells. For green fluorescent plaques (Figure 13), 120h after co-transfection, the Sf9 cell culture supernatant was collected and transferred to the host cells cultured in suspension. A large amount of green fluorescence was produced after 72h of infection (Figure 14); Baculovirus vector △ NAE-Bac was co-transfected with the recombinant DNA fragments Ac83-NAE-mcherry, Ac126-NAE-mcherry and Ac152-NAE-mcherry in Sf9 cells for 96 hours, respectively, and red fluorescent plaques were produced (Figure 15). After 120 hours of co-transfection, The Sf9 cell culture supernatant was collected and transferred to the host cells cultured in suspension. A large amount of red fluorescence was produced 72 hours after infection (Figure 16). This example shows that foreign genes can be inserted into any locus of the AcMNPV genome, and there is no restriction on the insertion site. They can be inserted into the target site as needed, and the foreign genes can be expressed.

Example 5 Obtaining recombinant baculovirus containing Cap and Rep, essential elements for AAV packaging, and detecting the expression of Cap and Rep

The packaging-deficient baculovirus vector ΔCNE-Bac in Example 1 and the recombinant DNA fragment Ac96-Rep-CNE-Cap in Example 2 were co-transfected into Sf9 insect cells, and the packaging-deficient rod in Example 1 The ravivirus vector ΔNAE-Bac and the recombinant DNA fragment Ac96-Rep-NAE-Cap in Example 2 were co-transfected into Sf9 insect cells to prepare recombinant baculovirus BEV. The transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicating and proliferating BEV caused obvious cytopathic effect (CPE) in Sf9 cells. The culture supernatant of Sf9 cells that developed CPE was collected, which contains a large amount of BEV, which is the 0th generation BEV (BEV-P0). At the same time, the Sf9 cells containing a large amount of rAAV were collected. The prepared BEV-P0 was infected into suspension-cultured Sf9 cells at a multiplicity of infection (MOI=1). After 72 hours of infection, the cell activity dropped below 50%. The cell culture medium was centrifuged at 1000g for 5 minutes, and the culture supernatant and cell pellet were collected respectively. The supernatant was labeled as passage 1 BEV (BEV-P1). Continue to expand the culture, and infect the Sf9 cells in suspension culture with the prepared BEV-P1 at a multiplicity of infection (MOI=1). After 72 hours of infection, the cell activity drops below 50%. The cell culture medium is centrifuged at 1000g for 5 minutes, and the cell pellet is collected. Western Blot detects the expression of VP proteins (VP1, VP2, VP3) and Rep proteins (Rep78, Rep52).

The results are shown in Figure 17 and Figure 18: The recombinant baculovirus was produced after co-transfection of Sf9 insect cells with the baculovirus vector △CNE-Bac and the recombinant DNA fragment Ac96-Rep-CNE-Cap, and successfully expressed the VP protein and Rep protein (Figure 17); the baculovirus vector ΔNAE-Bac and the recombinant DNA fragment Ac96-Rep-NAE-Cap were co-transfected into Sf9 insect cells to produce a recombinant baculovirus, and successfully expressed the VP protein and Rep protein ( Figure 18).

Example 6 Construction of recombinant baculovirus vector containing AAV core expression element (ITR-GOI)

The method of constructing a recombinant AAV bacmid for producing AAV viruses in insect cells in this example refers to Example 1 in the applicant's previous patent application CN112553257A, which includes the following steps:

(1) Construct a recombinant transfer vector containing the ITR core element (ITR-GOI). The nucleotide sequence of ITR-GOI is shown in SEQ ID No. 12. In this example, the GOI in the ITR core element uses the red fluorescent protein mcherry gene expression cassette, that is, the miniEf1a promoter controls mcherry expression, which facilitates the detection of rAAV activity. The ITR and red fluorescent protein expression cassettes are constructed into the transfer vector pFastDual.

(2) Use the recombinant transfer vector constructed in step (1) above to transform competent cells containing △CNE-Bac or △NAE-Bac bacmid, and use Tn7 recombination to insert ITR-GOI into △CNE-Bac or △NAE- On the Tn7 site of the Bac bacmid, recombinant baculovirus plasmids containing the ITR core elements necessary for rAAV production were finally obtained, numbered △CNE-Bac-Tn7-ITR and △NAE-Bac-Tn7-ITR respectively.

Example 7 Using △CNE-Bac-Tn7-ITR or △NAE-Bac-Tn7-ITR deficient bacmid to prepare AAV recombinant baculovirus containing the essential elements Cap, Rep and ITR-GOI for AAV packaging

The recombinant bacmid ΔCNE-Bac-Tn7-ITR prepared in Example 6 was combined with the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in Example 2, and the recombinant bacmid ΔNAE-Bac prepared in Example 6 -Tn7-ITR and the recombinant DNA fragment Ac97-Rep-NAE-Cap prepared in Example 2 were co-transfected into the host insect cell line and cultured to obtain AAV recombinant baculovirus, numbered respectively △CNE-Bac-AAV and △NAE- Bac-AAV, the specific steps are as follows:

The above recombinant bacmid and transfer vector DNA were extracted and co-transfected into Sf9 insect cells to prepare recombinant baculovirus BEV and rAAV. The co-transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicated and proliferated BEV caused obvious cytopathic effects (CPE) in Sf9 cells. The culture supernatant of Sf9 cells that developed CPE was collected, which contains a large amount of BEV, which is the 0th generation BEV (BEV-P0). At the same time, the Sf9 cells containing a large amount of rAAV were collected. The prepared BEV-P0 was infected into suspension-cultured Sf9 cells at a multiplicity of infection (MOI=1). After 72 hours of infection, the cell activity dropped below 50%. The cell culture medium was centrifuged at 1000g for 5 minutes, and the culture supernatant and cell pellet were collected respectively. The supernatant was labeled with passage 1 BEV (BEV-P1), and the cells were labeled with rAAV packaged with BEV-P0.

Example 8 Preparation of AAV recombinant baculovirus containing the essential elements Cap, Rep and ITR-GOI for AAV packaging using △CNE-△NAE-Bac deficient bacmid

First, a recombinant DNA fragment with the insertion site of the AAV ITR core expression element (ITR-GOI) at the AcMNPV orf83 locus was constructed: see Figure 19. The recombinant DNA fragment includes orf83 upstream homologous sequence, ITR- GOI sequence, NAE sequence and orf83 downstream homologous sequence, the above sequences are connected through artificial direct synthesis or overlap extension PCR amplification to obtain the construct Ac83-ITR-NAE.

Then, the defective bacmid ΔCNE-ΔNAE-Bac prepared in Example 1 was combined with the recombinant DNA fragment Ac97-Rep-CNE-Cap prepared in Example 2, and the recombinant DNA fragment Ac83-ITR prepared in this example. -NAE was co-transfected into the host insect cell line and cultured to obtain an AAV recombinant baculovirus, numbered ΔCNE-ΔNAE-Bac-AAV. Please refer to Example 7 for specific steps.

Example 9 Purification of recombinant AAV virus particles and detection of packaging efficiency

According to the operation of Example 5, the recombinant baculovirus ΔCNE-Bac-AAV, ΔNAE-Bac-AAV and ΔCNE-ΔNAE-Bac-AAV in Example 7 and Example 8 are continued to be expanded and cultured until BEV is used. -P2 seed virus is used to infect suspension-cultured Sf9 cells at a multiplicity of infection (MOI=1) for rAAV packaging, with a packaging volume of 300 mL to 400 mL. Monitor cell activity 3 days after infection. The activity is lower than 50%. Harvest the cell pellet and supernatant by centrifugation. Purify the harvested cell pellet and supernatant respectively. Cells are lysed by freezing and thawing three times. Centrifuge at 5000 rpm for 10 minutes to collect the supernatant. Add nuclease (Benzonase) to the clear water and treat it in a 37°C water bath for 60 minutes. After treatment, centrifuge at 5000 rpm for 10 minutes. The collected cell lysate and supernatant were PEG precipitated, resuspended and purified by iodixanol density gradient centrifugation (for methods, see Aslanidi et al., 2009, Proc. Natl. Acad. Sci. USA, 206:5059-5064 ). The final purified virus was resuspended in 80 μL to 190 μL PBS, and 10 μL of the purified virus was run on SDS-PAGE gel and stained with silver.

The SDS-PAGE gel picture is shown in Figure 20: Lane 1 is the silver staining detection picture of the purified recombinant AAV virus particles subjected to SDS-PAGE after the recombinant baculovirus △CNE-Bac-AAV infected the host cells, showing three Capsid proteins VP1/VP2/VP3; lane 2 is the silver staining detection picture of the purified recombinant AAV virions subjected to SDS-PAGE after the recombinant baculovirus △NAE-Bac-AAV infected the host cells, showing the three capsids. Protein VP1/VP2/VP3; Lane 3 is the silver staining detection picture of SDS-PAGE of purified recombinant AAV virions after infecting host cells with recombinant baculovirus △CNE-△NAE-Bac-AAV, showing three types of capsids. Proteins VP1/VP2/VP3.

This embodiment also uses Q-PCR to detect the packaging rate of the harvested rAAV virus. The detection of the rAAV packaging rate uses a pair of primers targeting the ITR sequence (Q-ITR-F: GGAACCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA). The test results are shown in Table 1.

Table 1 Packaging rate test results of rAAV virions produced by recombinant baculovirus

重组杆状病毒编号Recombinant baculovirus number	细胞包装率(VG/cell)Cell packaging rate (VG/cell)
△CNE-Bac-AAV△CNE-Bac-AAV	4.83E+054.83E+05
△NAE-Bac-AAV△NAE-Bac-AAV	5.26E+055.26E+05
△CNE-△NAE-Bac-AAV△CNE-△NAE-Bac-AAV	4.85E+054.85E+05

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

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements, etc., made within the spirit and principles of the present invention, All should be included in the protection scope of the present invention.

Claims

A composition for producing recombinant baculovirus in insect cells, characterized by: comprising a packaging-deficient baculovirus plasmid and a first rescue recombinant DNA, the packaging-deficient baculovirus plasmid is missing from the baculovirus genome CNE sequence and/or NAE sequence, the first rescue recombinant DNA includes a first insertion sequence and first homology arms located on both sides of the first insertion sequence, the first insertion sequence includes a first functional fragment and A first complementing sequence, the first complementing sequence is at least one of the sequences deleted from the packaging-deficient baculovirus plasmid, and the composition is capable of homologous recombination in insect cells to produce recombinant rods Virus.
The composition according to claim 1, characterized in that: the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV.
The composition according to claim 2, characterized in that: the first insertion sequence includes the cap gene expression cassette, the first complement sequence and the rep gene expression in order from 5' to 3' box.
The composition according to claim 2, characterized in that: the first insertion sequence includes the rep gene expression cassette, the first complement sequence and the cap gene expression in order from 5' to 3' box.
The composition according to claim 2, characterized in that: the first complementation sequence is located between the cap gene expression cassette and the rep gene expression cassette, and its two ends are respectively close to the cap gene expression cassette. The starting end and the starting end of the rep gene expression cassette.
The composition according to claim 2, characterized in that: the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV ITR core expression element with an exogenous gene.
The composition according to claim 6, wherein the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
The composition according to claim 1, characterized in that: the first functional fragment is an AAV ITR core expression element with an exogenous gene.
The composition according to claim 8, characterized in that: the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV cap gene expression cassette and an AAV rep gene expression cassette.
The composition according to claim 9, wherein the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
The composition according to claim 1, wherein the first functional fragment is a reporter gene.
The composition according to claim 1, wherein the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.
The composition according to claim 1, further comprising: a second rescue recombinant DNA, the packaging-deficient baculovirus plasmid lacks the CNE sequence and the NAE sequence in the baculovirus genome, and the second rescue recombinant DNA DNA includes a second insertion sequence and second homology arms located on both sides of the second insertion sequence, the second insertion sequence includes a second complement sequence, and the first complement sequence is a CNE sequence or an NAE sequence, The second complementing sequence is a sequence different from the first complementing sequence among the CNE sequence and the NAE sequence.
The composition according to claim 13, characterized in that: the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV, and the second insertion sequence further includes a second functional fragment, The second functional fragment is the AAV ITR core expression element with foreign genes.
An application of the composition according to any one of claims 1 to 14 in preparing recombinant baculovirus and/or recombinant adeno-associated virus in insect cells.
An insect cell, characterized in that it contains the composition according to any one of claims 1-14.
A method for growing or producing recombinant baculovirus in vitro, characterized by: co-transfecting insect cells with the composition of any one of claims 1 to 14, and cultivating the insect cells.
A method for growing or producing recombinant adeno-associated viruses in vitro, characterized by: co-transfecting insect cells with the composition of any one of claims 6, 7, 9, 10 and 14, and cultivating the insect cells .